Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
  • A61K36/537—Salvia (sage)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/35—Caprifoliaceae (Honeysuckle family)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
  • A61K36/734—Crataegus (hawthorn)

Definitions

• the present invention relates to a bioactive complex of triterpene acids, as such or in the form of Na, K, or NH 4 salts, to its production process from the plants Salvia species, Lavandula species, Samhucus nigra and Crataegus species and to medicinal products based on it, applicable in therapeutical areas of large social incidence, for both human and veterinary use.
• the bioactive complex of triterpene acids produced according to the invention has a high purity (at least 90%), being standardized in the content of triterpene acids and characterized by pharmacotoxicologic screening, in point of specific activity and safety in administration, which allows its use as a basic bioactive component of some medicinal products for internal or external use, in human and veterinary therapeutics.
• triterpene acids particularly the ursolic and oleanolic ones
• these acids are known that, though isomers, show some structural differences that make a differentiation of pharmacological effects which, although similar to a large extent, they are not identical. That is why, especially when a mixture of triterpene acids is recommended to be used in therapeutics, it is highly important and essential to accurately know their proportions, since a content variability in the mixture may cause a variability in product pharmacological activity and bioavailability (D. BARICEVIC - Journal of Ethnopharmacology, 72(2001), 125-132; Paik KEE-JOO et al.
• TLC thin layer chromatography
• Patent SU 18186346/3/27.09.1995 entitled "Production method for ursolic acid” relates to a method for preparing ursolic acid from fruit residues of Oxycoccus q ⁇ adripetelus G, by isopropyl alcohol extraction, at ambient temperature, for 24 hours, vacuum concentration of alcohol extract up to residue, that is purified by petroleum ether extractions and successive changes into Na salt and purified ursolic acid. An increase in processing efficiency and quality of substance produced is mentioned, without specifying its purity, too.
• Patent RU 2151139/20.06.2000 entitled: "Method for the preparation of a biologically active total content of triterpene acids” relates to a method for extracting triterpene acids from fresh white fir tree needles or bark, steam treated beforehand, then extracted with organic solvents (methyl-buthyl ether; mixture: benzene - ethyl acetate; mixture: petroleum ether - ethyl acetate), followed by separation of the total content of triterpene acids by successive treatment of organic extract with alkaline and acid aqueous solutions.
• organic solvents methyl-buthyl ether; mixture: benzene - ethyl acetate; mixture: petroleum ether - ethyl acetate
• the quantity of the product obtained is mentioned, without specifying chemical denomination of triterpene acids and product purity.
• a group of patents belonging to UNILEVER company presents different production methods for some fruit residues concentrates containing a mixture of ursolic, oleanolic acids and other components, being intended to be used as nutritional supplements.
• 2002037882/28.03.2002 relates to two laboratory and pilot processes - that recommend acetone reflux extraction (50...58 0 C) of dry apple peels, extractive solution concentration up to residue and its crystallization from 50/50 acetone - water mixture up to 95/5 (laboratory process) or from hexane (pilot process).
• a mixture of ursolic and oleanolic acids is obtained in variable proportions, that also contains other polar and non-polar components typical of apple peels.
• a conditioning process is also shown for triterpene mixture by association with mono-, di-, triglycerides and with oils containing unsaturated fatty acids, with a view to being used as nutritional supplements.
• H 3 PO 4 acid aqueous solutions
• Na 2 CO 3 alkaline aqueous solutions
• Soxhlet acetone extraction and extractive solution concentration up to wet residue (sice) constituting the ursolic and oleanolic acid concentrate with a content of 30...65% in a
• Patent CN 1358733/17.07.2002 relates to a process for extracting ursolic acid from Ligustrum lucid ⁇ m leaves, that includes the following phases: leaves counter-flow extraction with reflux ethyl alcohol, extract concentration up to residue, that is washed with water, dried and the dry extract is dissolved in ethyl alcohol and decolourized, then concentrated and diluted in water, a precipitate being resulted; suspension pH is adjusted to 2...2.5, after then precipitate is separated, washed with neutral pH water and vacuum dried.
• a crude extract is mentioned to be produced, having ursolic acid as a main component, without indicating product quantitative determination and purity, as well as the other existing compounds beside ursolic acid.
• fruit residues are known to have a low content in triterpene acids ( ⁇ 0.5%) which negatively influences profitability of industrial-scale technologies in comparison with processing technologies for usual plant raw materials (medicinal and aromatic herbs);
• J.Liu in Journal of Ethnopharmacology shows the following pharmacological effects revealed in oleanolic and ursolic acids: antiinflammatory, hepatoprotective, antihyperlipidemic, antiulcerous, hypoglycemic, antitumorigenic, antiHIV, antimicrobial.
• Patent US 4752606/1988 relates to a pharmaceutical composition based on oleanolic acid, as such or as physiologically acceptable salts, conditioned as tablets and utilizable for prophylactic or curative treatment of different ulcer disfunctions in the stomach or bowels.
• Patent application US 10/488682/2002 published with no. US 2004/0235785 A1 relates to a pharmaceutical composition utilizable as dietetic supplement in cancer treatment or prevention, having the role of potentiator of antitumorigenic agents, used in immunotherapy and oncology.
• composition relies on compounds of terpene class (mono-, di-, tri-, sequiterpene).
• Patent application JP 05-211065 published under no.07-048260/1995 relates to a bioproduct utilizable as a growing agent for blood cells (erythrocytes) based on ursolic acid, administered in daily doses of 1-1000 mg and having an effect of life prolonging in the X-ray treatment of different cancer forms.
• erythrocytes blood cells
• ursolic acid ursolic acid
• A2 published on 22.09.1999, relate to a method and a composition based on derivatives of betulinic acid applicable to prevent or inhibit growth of malignant tumours.
• a method for producing betulinic acid from the stem bark of Ziziphus mauhtiana (Rhamnacae family), " in vitro” tests on cytotoxicity of betulinic acid as against different cancerigenic cell lines (melanoma, fibrosarcoma, breast and colon cancer, epidermoid carcinoma) are shown, and results of "in vivo" tests in mouse, as well, confirming the quality of betulinic acid as antineoplastic agent.
• Patent US 5985924/1999 relates to a new suppressing agent for metastases, with a low toxicity degree, based on ursolic acid, as such or in the form of salts, that can be orally or injection administered, in the postoperative treatment of various cancer forms.
• Patent application JP 63166195 published under no.02017121 A/1990 relates to a medicinal drug for external use, utilizable to prevent skin epithelial cells to grow cancerigenic, based on ursolic or oleanolic acid, as such or mixtured.
• Patent application JP 09-199323 published under no.JP 11-029467/1999 relates to a product having inhibitory effects on protease, based on ursolic acid or salts thereof, applicable for the treatment or prevention of different skin disfunctions (dermatitides) and conditioned in the form of ointment, cream, lotion and emulsion. Dermatoprotective effects of product are specified: antioxidating, hydrating and epidermis whitening.
• Patent US 4857554/1989 relates to a method for the treatment of psoriasis based on daily use of an ointment containing ursolic and oleanolic acids in a 3:1 ratio, dispersed in a fatty base of lanoline and vaseline. Product action is shown by a clinical trial for 3 weeks, which revealed that severe forms of psoriasis were cured in a 60...70% percentage.
• the technical problem solved by the present invention is the production of a bioactive complex of triterpene acids containing ursolic, oleanolic acids and other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulilnic, as such or in the form of Na, K or NH 4 salts, by an industrially applicable technological process that carries out extraction of all structural types of triterpene acids existing in plant raw materials, and that, for increasing selectivity and efficiencies of processes for extraction, purification and crystallization, uses structured waters ("I" structured water with acid pH and "S” structured water with alkaline pH) in composition of solvents used to perform these technological stages, which lead to a finished product of a high purity (minimum 90%) and a standardized composition.
• Bioactive complex of triterpene acids consists in that it has a minimum 90% content, represented by: minimum 75% ursolic acid, 10...15% oleanolic acid and 4...10% other triterpene acids such as: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic obtained in the form of free acids or as Na, K, NH 4 salts, by extraction from the following plant species: Salvia species, Lavandula species, Sambucus nigra and Crataegus species, showing the following pharmacological effects:
• the medicinal products which contain as pharmaceutically active substance the bioactive complex of triterpene acids as such or in the form of salts, are conditioned as: injectable solution, perfusable emulsion, buvable solution, tablets, hard and/or soft jelly , eyewash, ophthalmic ointment, gel, cream, ointment, solution for external use or spray, suppositories, ovules, being applicable in human and veterinary therapy.
• plant raw materials consisting of Salvia species herba, Lavandula species herba, Sambucus nigra - flowers or leaves or Crataegus species herba
• plant raw materials consisting of Salvia species herba, Lavandula species herba, Sambucus nigra - flowers or leaves or Crataegus species herba
• one of the extraction solvents represented by mixtures consisting of: 5...15% "I" structured water with 85....95% acetone or with 85...95% ethyl alcohol 95 C , for a period of 8...24h, at 15...80 0 C
• the resulted extractive solution being then purified by passage on a chromatographic column with acid active granulated carbon, next it is vacuum concentrated in a 1:10...1 :20 v/v ratio of the initial volume; the resulted suspension is vacuum filtered, the after drying precipitate constituting the crude bioactive complex of triterpene acids with a minimum 70% content.
• the resulted solution is purified by adsorption on acid active carbon, afterwards it is vacuum concentrated up to 1/10...1/20 of the initial volume, and the microcrystalline suspension is vacuum filtered, the after drying substance constituting the crystallized bioactive complex of triterpene acids, produced by 1...2 successive crystallizations, depending on the type of plant raw material processed, at the same parameters, excepting the second concentration carried out for 1/5 of the initial volume, with a minimum 90% content standardized in the following components: minimum 75% ursolic acid, 10...15% oleanolic acid and 4...10% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic.
• the crystallized product with a minimum 90% content is dissolved in the 1/100...1/200 m/v ratio in the mixture of solvents with alkaline pH: 90...95% ethyl alcohol 95 C with 5...10% "S" structured water, that also contains 1% NaOH or 1% KOH or 5% NH 4 OH 25%, followed by concentration of resulted solution and crystallization of bioactive complex of triterpene acids as Na, K or NH 4 salt, with a minimum 90% content.
• the technological process making the subject matter of the invention utilizes, concomitantly with ursolic and oleanolic acids existing in most plant raw materials as components in a majority, and other triterpene acids too, present in extractive solutions, as the following ones: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, that increase bioavailability of the entire complex, by step absorption, due to the difference in polarity and solubility of chemical structures of these acids.
• the bioactive complex of triterpene acids has a minimum 90% content, being standardized in the following components: minimum 75% ursolic acid, 10...15% oleanolic acid and 4...10% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, having a considerable and diversified pharmacological activity, exerted on some vital functions, namely: - bioregulation of proteinic, glucidic and lipidic metabolism, especially on aged organisms, with new applications in preventive and curative therapy of metabolic and cardiovascular diseases of atherosclerotic etiology, particularly in geriatric therapy;
• the bioactive complex of triterpene acids produced according to the invention, was conditioned as new medicinal products for human and/or veterinary internal use, such as: capsules, tablets, injectable solutions, buvable solutions, and for external use: eye washes, spray, gel, cream, ointment, suppositories, ovules, intended for new therapeutic applications, as compared to the already known ones.
• the production process consists in that the dried and pulverized plant raw material, with 2...4% triterpene acids, consisting of Salvia species herba, Lavandula species herba - not only the herba plant but also the residue resulted from producing essential oil by steaming, Sambucus nigra - flowers or leaves or Crataegus species herba, is extracted by dynamic maceration in the 1/15...1/30 m/v ratio, plant mass/solvent, with one of the extraction solvents represented by mixtures consisting of: 5...15% "I" structured water with 85...95% acetone or with 85...95% ethyl alcohol 95°, for 8...24 h, at 15...80 0 C, and the resulted extractive solution, in order to be purified, is passed on a chromatographic column with active granulated carbon with acid pH, then it is vacuum concentrated, at temperature of 35...40 0 C, up to 1/10...1/20 of the initial volume, a suspension being obtained, that
• the crude product is reflux dissolved at 80 0 C in the 1/100...1/200 m/v ratio in one of the solvents represented by mixtures consisting of: 5...10% "I” or "S” structured water with 90...95% ethyl alcohol 95 C or with 90...95% acetone.
• the obtained solution is purified by adsorption on acid pH active carbon, added in a 0.1...0.3% m/v ratio, is filtered, vacuum concentrated at 35...40 0 C up to 1/10...1/20 of the initial volume, the resulted microcrystalline suspension is kept for 12...24 h at 5...10 0 C for finishing crystallization, then it is vacuum filtered, the obtained precipitate is washed with 0.3...0.5 I distilled water up to neutral pH, is dried at 105 0 C for at least 3 h and pulverized, resulting in crystallized bioactive complex of triterpene acids, obtained through 1...2 successive crystallizations at the same parameters, depending on the type of plant raw material processed, excepting the second concentration made for 1/5 of the initial volume, with a minimum 90% standardized content, composed of the following components: minimum 75% ursolic acid, 10...15% oleanolic acid and 4...10% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxy
• the crystallized product is reflux dissolved at 80 0 C in a 1/100...1/200 m/v ratio in the alkaline pH solvent mixture: 90...95% ethyl alcohol 95 C with 5...10% "S" structured water, that contains 1% NaOH or 1% KOH or 5% NH 4 OH
• the resulted suspension is kept for 12...24 h at 5...10 0 C for finishing crystallization, followed by vacuum filtration, the precipitate is washed with 0.1...0.3 I distilled water up to neutral pH, dried at 105 0 C for 3 h and pulverized.
• the crystallized bioactive complex of triterpene acids is produced, as Na, K or NH 4 SaIt, with minimum 90% estimated content, and a 95% phase efficiency.
• Minimum 630 g microcrystalline, white powder are resulted, composed of crystallized bioactive complex of triterpene acids, with a 96.6% content formed of: 80.9% ursolic acid, 10.2% oleanolic acid and 5.5% other triterpene acids, consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic which represents a final efficiency of at least 50% compared with 4% triterpene acids content of the processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 1200 g slightly greenish yellow powder are obtained, constituting the crude complex of triterpene acids, with a minimum 70% content.
• Minimum 640 g microcrystalline, white powder are resulted composed of crystallized bioactive complex of triterpene acids, with a 96.7% content, consisting of: 80.4% ursolic acid, 11.7 oleanolic acid and 4.6% other triterpene acids formed of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a final efficiency of minimum 50% compared with the 4% content of triterpene acids of the processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 900 greenish powder are produced which constitute the crude complex of triterpene acids, with a minimum 70% content.
• Minimum 490 g white, microcrystalline powder are produced, constituted of crystallized bioactive complex of triterpene acids, with a 93.7% content formed of: 77.5% ursolic acid, 10.1% oleanolic acid and 6.1% other triterpene acids, consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 50% final efficiency, compared with 3% content of triterpene acids in the processed plant raw material.
• This bioproduct is used for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 900 g greenish powder are obtained which constitute the crude bioactive complex of triterpene acids with a minimum 70% content.
• the suspension is heated at 80 0 C for 1 h until full substance dissolution, and the resulted yellow solution is treated with 0.1...0.3% m/v acid active carbon; suspension is filtered, the obtained solution is vacuum concentrated, at 35...40 0 C, up to 1/10 of the initial volume (9 I), when a microcrystalline suspension is obtained, which is kept for 24 h at 5...10 0 C for finishing crystallization, then it is vacuum filtered, the precipitate is washed with 0.4 I distilled water, up to neutral pH, dried at 105 0 C for 3 h and pulverized.
• Minimum 480 g white, microcrystalline powder consisting of crystallized bioactive complex of triterpene acids, with a 94.2% content formed of: 78.1% ursolic acid, 11.2% oleanolic acid and 4.9% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 50% final efficiency compared with the 3% content of triterpene acids of processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• acid pH consisting of 90 volumes (54 I) ethyl alcohol 95 C + 10
• Minimum 330 g white, microcrystalline powder consisting of: crystallized bioactive complex of triterpene acids, with a 91.8% content formed of: 76.2% ursolic acid, 11.1% oleanolic acid and 4.5% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 50% final efficiency, compared with the 2% triterpene acids content of processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Variant B 30 kg dried and pulverized plant raw material, consisting of flowers or leaves of
• Minimum 325 g white, microcrystalline powder are produced, consisting of crystallized bioactive complex of triterpene acids, with a 91.5% content formed of: 77.1% ursolic acid, 10.3% oleanolic acid and 4.1% other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 50% final efficiency as compared with the 2% triterpene acids content of processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 450 g white, microcrystalline powder are produced, composed of crystallized bioactive complex of triterpene acids with a 92.6% content, consisting of: 76.5% ursolic acid, 12% oleanolic acid and 4.1% other triterpene acids formed of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 55% final efficiency, as compared with the 2.5% triterpene acids content of processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• 500 I extraction solvent consisting of: 90 volumes (450 I) ethyl alcohol 95 C + 10
• Minimum 430 g white, microcrystalline powder consisting of crystallized bioactive complex of triterpene acids, with a 93% content formed of: 77.8% ursolic acid, 10.7% oleanolic acid and 4.5% other triterpene acids consisting of one or more of the following acids: hydroxyrusolic, hydroxyoleanolic, betulinic, dehydrobetulinic, which represents a minimum 53% final efficiency as compared with the 2.5% triterpene acids content of processed plant raw material.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 19O g white, microcrystalline powder consisting of crystallized bioactive complex of triterpene acids, in the form of Na salt of ursolic, oleanolic acids and other triterpene acids formed of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, with a 90% content, which represents a 95% phase efficiency.
• This bioproduct is usable for conditioning some medicinal drugs for internal use (tablets, capsules, injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 19O g white, microcrystalline powder consisting of bioactive complex of triterpene acids, in the form of K salt of ursolic, oleanolic acids and other triterpene acids composed of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic with a 90% content, which represents a 95% phase efficiency.
• the bioproduct is usable for conditioning some medicinal drugs for internal use (injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Minimum 19O g white, microcrystalline powder are produced, composed of bioactive complex of triterpene acids, in the form of NH 4 salt of ursolic, oleanolic acids and other triterpene acids consisting of one or more of the following acids: hydroxyursolic, hydroxyoleanolic, betulinic, dehydrobetulinic, with a minimum 90% content, which represents a 95% phase efficiency.
• the bioproduct is usable for conditioning some medicinal drugs for internal use (injectable and buvable solutions) or for external use (eye washes, ophthalmic gel, spray).
• Test solution 0.1 g bioactive complex of triterpene acids - test sample in 100 ml methyl alcohol.
• Test no. 2 Quantitative determination of bioactive complex of triterpene acids by
• bioactive complex of triterpene acids - test sample is dissolved in 100 ml methyl alcohol. Out of the solution obtained, 5 ml are taken that are dissolved in 50 ml graduated flask with methyl alcohol (solution A).
• solution A 5 ml of solution A are diluted in 50 ml graduated flask with methyl alcohol (solution B).
• 1 ml of solution B is evaporated on water bath at a temperature of 70 0 C up to sice residue, over which there are added: 0.2 ml of 5% vanillin solution in acetic acid and 1 ml perchloric acid and they are heated on water bath at 60 0 C for 15 minutes; after cooling at room temperature, acetic acid is added up to 5 ml, then homogenized and absorbance is read at 550 nm as against a control solution prepared in the same way but containing 1 ml methyl alcohol instead of sample.
• Bioactive complex of triterpene acids content is determined by means of a standard curve of ursolic acid - reference substance, as 0.1% solution in methyl alcohol, the following calculating formula being applied:
• Bioactive complex of triterpene acids content % — xlO '2
• Test no. 3 Quantitative determination of bioactive complex of triterpene acids, by HPLC Method:
• CBAT bioactive complex of triterpene acids
• Table no.1 Effect of treatment with bioactive complex of triterpene acids (CBAT) on blood and hide proteins in young and old Wistar rats, under conditions of stimulating ageing processes with DL-ethionine. Significance of media difference by Student T- test at p ⁇ 0.05
• Induction of streptozotocin diabetes was carried out by classical method used in diabetes research, that provides intravenous administration in white Wistar rats deprived of feed for 18 hours, of a streptozotocin solution in 0.01 M citrate buffer, in a dose of 50 mg/100 g b.w.; after 1 h, 1 ml 30% glucose aqueous solution was intraperitonealiy given, and in 24 h after injecting streptozotocin, the experimentally induced diabetes was examined.
• the group of experimentally induced diabetes animals was treated with bioactive complex of triterpene acids in a dose of 50 mg/100 g b.w., for 10 days, and then the glucidic metabolism parameters were investigated: glycemia, liver glycogen and histologic aspects of pancreas, thyroid, spleen and thymus, as main endocrine glands 3 involved in' glucidic homeostasis.
• SOD superoxide - dismutase
• LM normal control group
• LS control group with streptozotocin - induced diabetes 50 mg/100 g.b.w.
• LSCBAT group with CBAT treated diabetes 50 mg/10Og b.w./day, 10 days a p ⁇ 0.001 vs normal control group; b p ⁇ 0.05 and c p ⁇ 0.001 vs control group with diabetes
• LM normal control group
• LP control group with diabetes induced by prednisone with 3mg/100 g b.w/day, 10 days
• LPCBAT group with CBAT-treated diabetes with 50 mg/100 g b.w./day, 10 days.
• LMr control group - standard diet, 42 days;
• LNA control group with atheromatosis - atherogenic diet, 42 days;
• LCBAT group with simultaneous administration of atherogenic diet + 50 mg CBAT/kg b.w. per oral/day.
• Atherogenic diet daily administered for 42 days in rabbits from atheromatosis control group induces an advanced atheromatosis process at aorta level that is correlated with a significant increase of triglycerides, cholesterol, lipoproteins, LDL and dimension of cholesterol/HDL ratio;
• bioactive complex of triterpene acids administered for 42 days in rabbits in a dose of 50 mg/kg b.w. per oral shows a significant antiatheromatous action, evidenced by the size of score/group as compared with values corresponding to LMA atheromatosis control group;
• GLUCOCORTICOSTEROIDS Having in view that both the glucocorticosteroids therapy and a number of serious diseases (cancer, viroses) are characterized by a drastic lymphocyte depression, followed by lowering of body immune response, the influence of administration of bioactive complex of triterpene acids (CBAT) was followed on an experimental model adequate for immunobiological studies; the model consists in experimental induction of lymphocyte depression by administering a single dose of cortisone, 6 mg/100 g b.w., in white Wistar rats.
• CBAT bioactive complex of triterpene acids
• Cortisone - acetate causes a decrease in liver proteins which is restored by treatment with bioactrive complex of triterpene acids, so that both groups record an increase of liver proteins by over 6% as against control group value and by 11.6%, as against the group treated with Cortisone-acetate.
• hematopoiesis depression leukopenia, thrombocytopenia; - thymus involution, atrophy of adrenal glands and spleen;
• cyclophosphamide has carcinogenic properties, with risk of developing a secondary tumour or acute leukaemia.
• LM control group that was given physiological serum
• LCf group with tumour inoculum of 10 5 cells/animal that was given cyclophosphamide in a dose of 15 mg/kg b.w. per oral/day;
• LCfCBAT group with tumour inoculum of 10 5 cells/ animal that was given cyclophosphamide in a dose of 15 mg/kg b.w. per oral/day and 10O g bioactive complex of triterpene acids/kg b.w. per oral/day.
• Hepatoprotective activity evidenced by experimental biochemical data from table no.12 is in concordance with results of ultrastructural histopathological investigations, achieved within evaluation of influence of bioactive complex of triterpene acids on adverse hepatotoxic effects of cyclophosphamide, namely: - following treatment of Wistar rats with cyclophosphamide, hepatocytes show a lipid metabolism disturbance, the liver cells nuclei undergoing an increase to a double volume against control group and nucleols ultrastructure is altered, swowing affection of nucleic acids synthesis and of cell proteins biosynthesis;
• bioactive complex of triterpene acids reduces alterations induced by cyclophosphamide in structure of hepatocytes and in their metabolic activity, thus the cyclophosphamide effect of lipid accumulation in hepatocytes is annihilated, the protective action of bioactive complex of triterpene acids determining cyclophosphamide metabolization without producing structural alterations; - bioactive complex of triterpene acids also counteracts cyclophosphamide effect of inhibiting nucleic acids synthesis, the presence of polyribosomes showing an increase in structural protein synthesis.
• LMT control group with tumour
• LTCBAT group with tumour + CBAT 100 mg/kg b.w.
• bioactive complex of triterpene acids in a dose of 100 mg kg/b.w. per oral in Wistar rats inoculated with 10 5 cells/animal produced significant extensions of latency period from inoculation until cancerous tumour occurrence and of average survival time, compared with latency period from inoculation 5 until cancerous tumour occurrence and average survival time in control group, inoculated under the same conditions.
• CBAT bioactive complex of triterpene acids
• LM absolute control group
• LTC1 group with TPA + CBAT 0.4 ⁇ moli/cm 2 ear
• bioactive complex of triterpene acids as such or in the form of Na, K, or NH 4 salts can be conditioned in the form of medicinal products for internal or external use, meant for clinical fields with large social incidence belonging to human or veterinary therapeutics.
• Medicinal products described further, according to invention are administered in human or veterinary therapeutics, by injection, orally or locally, for 1 day - 24 months, depending on disease progression, in daily doses of 10-1000 mg/kg b.w. divided in 1-6 intakes.
• Formulation examples shown further on do not preclude possible development of other conditioning the formulas, considering diversity and multitude of excipients known and used in pharmaceutical industry.
• the product is curatively given, in doses of 1...3 ampoules/day, for 1...6 months, having the following pharmacological effects: antitumorigenic, immunoprotective, hepatoprotective, antiulcerous, hypoglycemic, hypolipidemic, antiatherosclerotic, antiinflammatory, vascular tonific, especially at coronary level, in the following diseases: neoplasm, acute and chronic hepatites, liver cirrhosis, immune system depletions occurred after treatment with glucocorticosteroids, cytostatics.
• soy bean oil is heated at 70 0 C, in which the bioactive complex of triterpene acids - Na salt is dissolved, and egg phospholipids, vitamin E and natrium oleate are added in the resulted solution.
• alkalinized with 0.003 g NaOH at pH 8, anhydrous glycerine is added; the two phases- hydrophilic and lipophilic - are homogenized in a homogenizer, at 80...90 0 C at a pressure of 125...220 kg/cm 2 , under nitrogen atmosphere until dispersion of lipophilic phase, whose drops should reach an average diameter of 0.5 ⁇ m.
• Emulsion viscosity is ranged between 80...125 mPa x s and it is aseptically conditioned in sterilized glass vials under nitrogen atmosphere.
• Perfusable emulsion such prepared contains 10 mg/ml bioactive complex of triterpene acids and is administered in doses of 60...100 ml/day, having the following pharmacological effects: antitumorigenic, immunoprotective, hepatoprotective, antiulcerous, hypoglycemic, hypolipidemic, antiatherosclerotic, antiinflammatory, vascular tonic particularly at coronary level with therapeutical uses, especially in clinical cases where there are not possible oral administration and enteric absorption, as follows: neoplasm, lll-rd - IV-th stages, severe depletions of immune system, shocking conditions: serious burns on large areas of the body, hepatic or postoperative coma.
• Example no.10 MEDICINAL PRODUCT CONDITIONED IN THE FORM OF BUYABLE SOLUTION CON
• Method for preparation a mixture of 200 ml ethyl alcohol 95 C , 300 ml glycerin and 200 ml propylene glycol is prepared, in which 21 g bioactive complex of triterpene acids - Na salt are hot dissolved (60...70 0 C) under stirring.
• 200 ml phosphate buffer aqueous solution with pH 8 and the remaining propylene glycol, in which the two preservatives provided in the formula were dissolved, are added, then stirred up for homogenization, then filtered, and the resulted clear solution is conditioned in vials of 100 ml each.
• the product contains 20 mg/ml bioactive complex of triterpene acids in the form of Na salt and is administered in doses of 15...20 ml/day, for 1...6 months, for preventive or curative purpose, especially in pediatrics, having the following pharmacological effects: antitumorigenic, antiimmunosuppressive, hepatoprotective, antiulcerous, hypoglycemic, hypolipidemic, antiatherosclerotic, antiinflammatory, vascular tonic, especially at coronary level with therapeutical uses in the following diseases: neoplasm, acute and chronic hepatites, immune system depletions occurred following treatment with glucocorticosteroids and cytostatics, postoperative and posttraumatic conditions.
• Example no.11 Example no.11
• bioactive complex of triterpene acids is sieved with the excipients: lactose monohydrate and aerosil on a ll-sized sieve.
• a solution made by dissolving polyvinylpyrolidone K30 in ethyl alcohol is poured over the obtained mixture and then it is homogenized.
• Granules are produced that are dried at temperature of 35...45 0 C and then are ground to the size V given by sieve; starch sodium glycolate, sodium lauryl sulphate, talc and magnesium stearate are then added, homogenized and the powder produced is placed in 1 sized capsules.
• Product is administered in doses of 3...6 capsules/day, for 1...24 months, having the following pharmacological effects: antitumorigenic, immunoprotective, hepatoprotective, antiulcerous, hypoglycemic, hypolipidemic, antiatherosclerotic, antiinflammatory, vascular tonic especially at coronary level, with therapeutic uses in the following diseases: neoplasm, immune system depletions, acute and chronic hepatites, hepatic cirrhosis, ulcerous disease, diabetes, cardiovascular disorders of atherosclerotic etiology, vascular, tissular and dermic collagen degenerations: psoriasis, neurodermatitides, radiodermatitides, varicous syndrome and different symptoms of chronic venous insufficiency, vascular retinal affections, applicable in geriatrics too, for preventing and slowing down ageing processes.
• diseases neoplasm, immune system depletions, acute and chronic hepatites, hepatic cirrhosis, ulcer
• Method for preparation the usual known conditioning procedures are applied for pharmaceuticals for internal use in the form of tablets on industrial facilities, complying with good manufacturing practice regulations for medicinal drugs stipulated by the European legislation.
• the product is administered in doses of 3...6 tablets/day for 1...24 months, having the following pharmacological effects: antitumorigenic, immunoprotective, hepatoprotective, antiulcerous, hypoglycemic, hypolipidemic, antiatherosclerotic, antiinflammatory, vascular tonic, especially at coronary level, with therapeutic uses in the following diseases: neoplasm, immune system depletions, acute and chronic hepatites, hepatic cirrhosis, ulcerous disease, diabetes, cardiovascular disorders of atherosclerotic etiology, vascular, articular and dermic collagen degenerations: psoriasis, neurodermatitides, radiodermatitides, varicous syndrome and different symptoms of chronic venous insufficiency, vascular retinal affections, applicable in g
• Method for preparation 0.1g benzalkonium chloride and 10.5 bioactive complex of triterpene acids - Na salt are. aseptically dissolved by heating at 70 0 C, and the resulted clear solution is aseptically filtered and conditioned in vials of 10 ml each, with eye dropper.
• the product contains 10 mg/ml bioactive complex of triterpene acids in the form of Na salt and is administered for preventive or curative purpose, in doses of 0.2...0.5 ml/day, for 1...6 months, having the following pharmacological effects: trophic and regenerative for retinal vascular and tissular collagen, antiinflammatory, with ophthalmological therapeutic uses: diabetic retinopathy, cataract, postoperative treatment of cataract, glaucoma and cornea transplant.
• Method for preparation 0.1 g bezalkonium chloride and 10 g bioactive complex of triterpene acids - Na salt are aseptically dissolved by heating at 70 0 C 1 resulting in a slightly opalescent solution, which is incorporated into the mixture melted at 60 0 C, consisting of: 300 g emulsifying cetyl stearyl alcohol, 350 g white vaseline with the remaining paraffin oil, and then it is triturated until cooling.
• An ophthalmic ointment is obtained, in the form of a buttery, homogeneous, white mass, containing 1% bioactive complex of triterpene acids - Na salt, which is aseptically conditioned in tubes of 10 g each.
• vascular and dermic collagen Due to its pharmacological restoring effects on vascular and dermic collagen, it is used in local ophthalmologic therapy, in doses of 10...20 mg/day, for 1...6 months, having the following pharmacological effects: trophic and regenerative for retinal vascular and tissular collagen, antiinflammatory, with therapeutical ophthalmologic uses: diabetic retinopathy, cataract, postoperative treatment of cataract, glaucoma and cornea transplant Example no.15
• gel mass is prepared by dispersion of 20 g Carbopol 940 in the mixture consisting of 60 g glycerin with 837 g T structured water in which the mixture of preservatives mentioned in the formula is dissolved. It is gently stirred and left at rest at 15...25 0 C, afterwards the mixture composed of 10.5 g bioactive complex of triterpene acids, 60 g propylene glycol and 10 g of 15% sodium hydroxide solution is added under slow stirring, pH is adjusted to nearly 7.0 and stirring is continued for 30 minutes in order to become well homogenized and then it is packaged in tubes of 50 g each.
• the product contains 1% bioactive complex of triterpene acids - Na salt and is administered in doses of 10...30 mg/day, for 1...6 months, having the following pharmacological effects: trophic and regenerative for vascular, tissular, articular and dermic collagen, antiinflammatory, antihyaluronidase and antielastase, with therapeutic uses in the following diseases: burns, post-burn and postoperative cheloids, eschars, atonic wounds of different etiologies, degenerations of dermic, vascular, tissular and articular collagen such as: skin benign and malignant diseases, psoriasis, inflammatory and pruriginous dermatoses, neurodermatitides, radiodermatitides, variceal and post- thrombotic syndrome, chronic venous insufficiency, vascular fragility, rheumatic inflammatory conditions, and diabetic or endocrine obesity as well.
• Example no.16 Example no.16
• the product is in the form of a white, homogeneous, unctuous, rapidly absorbable cream, containing 1% bioactive complex of triterpene acids as Na salt, and it is administered in doses of 10...30 mg/day, for 1...6 months, having the following pharmacological effects: trophic and regenerative for vascular, tissular, articular and dermic collagen, antiinflammatory, antihyaluronidase and antielastase, with therapeutic uses in the following diseases: burns, post-burn and postoperative cheloids, eschars, atonic wounds of different etiologies, degenerations of dermic, vascular, tissular and articular collagen such as: skin benign and malignant affections, psoriasis, inflammatory and pruriginous dermatoses, neurodermatitides, radiodermatitides, variceal and post- thrombotic syndrome, chronic venous insufficiency, vascular fragility, rheumatic inflammatory conditions and diabetic or
• the product is administered in doses of 10...30 mg/day for 1...6 months, having the following pharmacological effects: trophic and regenerative for vascular, articular and dermic collagen, antiinflammatory, antihyaluronidase and antielastase, with therapeutic uses in the following diseases: burns, post-burn and postoperative cheloids, eschars, atonic wounds of different etiologies, degenerations of dermic, vascular, tissular and articular collagen such as: benign and malignant skin affections, psoriasis, inflammatory and pruriginous dermatoses, neurodermatitides, radiodermatitides, varicous and post-thrombotic syndrome, chronic venous insufficiency, vascular fragility, rheumatic inflammatory conditions, and diabetic or endocrine obesity as well.
• diseases burns, post-burn and postoperative cheloids, eschars, atonic wounds of different etiologies, degenerations of der
• Method for preparation the mixture of isopropyl alcohol and propylene glycol is prepared in which the bioactive complex of triterpene acids is dissolved by heating at 70...80 0 C and the resulted solution is packaged in spray vials of 100 ml each or in Freon pressurized vials.
• the product is administered in doses of 1...2 g/day, for 1...3 months, having the following pharmacological effects: trophic and regenerative for dermic, vascular and tissular collagen, membrane fluidity modulator, with therapeutic uses in the following diseases: burns, eschars, atonic wounds of different etiologies, allergic, inflammatory and pruriginous dermatoses, including psoriasis and radiodermatitides.
• Example no.19 the mixture of isopropyl alcohol and propylene glycol is prepared in which the bioactive complex of triterpene acids is dissolved by heating at 70...80 0 C and the resulted solution is packaged in spray vials of 100
• the product contains 25 mg bioactive complex of triterpene acids/suppository and it may be administered in doses of 50...100 mg/day for 1...6 months having the following pharmacological effects: antitumorigenic, immunoprotective, anti-inflammatory, OO trophic and regenerative for vascular, tissular and articular collagen, with pharmacological uses in the following diseases: neoplasm particularly in the colon, immunosuppressive conditions especially hepatotoxic ones, occurred after the treatment with glucocorticosteroids, cytostatics, rheumatic inflammatory diseases, haemorrhoidal syndrome.
• the product contains 25 mg bioactive complex of triterpene acids/ovule and may be given in doses of 25...100mg / day for 1...6 months, having the following pharmacological effects: antitumorigenic, antiinflammatory, trophic and regenerative for dermic and tissular collagen, with the following therapeutic uses: benign or malignant tumorigenic diseases at the level of vagina and uterine cervix, trophic vulvovaginal disorders and cervicites, particularly in the menopause. "I” - inhibitively activated and "S” - stimulatively activated structured waters are produced (Patents of loan MANZATU et al. nos.
• RO 109835/1996, US 5846397/1998, EP 0777631/1999, CN/ZL 951914722.4/2003 by passing a demineralised or distilled water, enriched - under control - in mineral salts, in order to reach conductivity values of 300...500 ⁇ S, in a chemically neutral, parallelipipedal column, with several structuring cells consisting of activators with two lamellar electrodes each, one negative and the other one positive, made up of stainless steel net, placed on one and the other side of a sandwich of porous, chemically inert membranes and, following the interaction processes between the water dipolar molecular structures and electrostatic field generated between electrodes, a process occurs by which there are produced arrangements, polarizations and energy needed to bind by hydrogen bridges the water molecules and negative radicals [R " ], resulting in T'-inhibitively activated structured water, with pH of 1.8...2.6 and conductivity of 1200...2500 ⁇ S, discharged from the left- hand and respectively, right-hand
• Patent application EP 1495754 A2 25.
• Patent application EP 0943620 A2 26.

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