Method for producing total water-soluble proteolytic enzymes

FIELD: medicine.

SUBSTANCE: method for producing total water-soluble proteolytic enzymes from dried milky sap of green papaya fruits provides water extraction of a raw material at room temperature and with mixing, separation of solid and liquid phases, and recovery of a purified product. An aqueous extract produced after separation of the solid and liquid phases is post-filtered through a filter of pore size (0.45-2.0) mcm. The product is dried by a spraying technique to prepare a purified product in the form of a dry extract of proteolytic activity not less than 3.5 PU/mg of the preparation and the protein content 0.43-0.50 mg/mg of the preparation. The prepared dry extract is water-dissolved that is followed by sterilisation filtration and lyophilisation of the solution.

EFFECT: method allows preparing a product of high proteolytic activity and protein content.

2 cl, 1 ex

 

The invention relates to chemical-pharmaceutical industry, namely the method of obtaining the amount of water-soluble proteolytic enzymes used in medical practice as proteolytic tools.

The amount of water-soluble proteolytic enzymes get in the form of dry extract and freeze-dried from the dried milky juice (latex) of immature fruits of papaya tree - Carica papaya L., family papaiya - Caricaceae. The biologically active substances are: papain, chymopapain a and b, peptidases and (Kurkin V.A. Pharmacognosy, Samara, LLC "Etching", 2004, str-201).

Group products obtained from the milky juice of papaya (including the juice, food products and pharmaceuticals, as well as papain in a partially purified or crystalline product), known under the commercial name "papain". Papain is a proteolytic enzyme (cysteine-proteinase), i.e. the enzyme that breaks down protein. Its proteolytic activity is expressed in units of proteases (PE).

Based on the dried milky juice of papaya in the former USSR, in Russia and abroad (Georgia, Slovenia, and others) were developed and became widely known drugs such as "caritatem" and "Lekozim". These drugs have been suggested for medical use in the form of a lyophilisate d the I solution for outdoor applications (State register of medicines. M., 2004, str; same, 2008, str; Register of medicines of Russia, M., 1993, str).

A method of obtaining purified amount of a complex of proteolytic enzymes from the dried milky juice of the papaya tree (papaya). This method involves extraction of the raw water in the ratio of 1:25 at room temperature and stirring, filtering, extracting, placing the filtrate in an ice bath and the deposition amount of the enzymes from the filtrate chilled 95% ethanol until complete precipitation, maintaining the resulting suspension in an ice bath for completeness of precipitation, separation of the enzyme complex by centrifugation and dried in vacuum (proceedings of the I Congress of pharmacists of Georgia. Article Dgentry and other "Proteolytic enzyme papain papaya tree, introduced in Georgia". Institute of pharmacochemistry of Georgian Academy of Sciences, Ed. Tbilisi, 1978, page 172-175).

A method of obtaining the amount of water-soluble proteolytic enzymes and drugs based on it, including extraction of the dried milky juice of the papaya water at room temperature and stirring, separating the solid and liquid phases and isolation of the pure product (Program and abstracts of the XXIX scientific conference-competition of young scientists. Article Gstreamermm and other "Medicines from domestic papain and Art. Dgentry and others, "the Research process is and obtain drugs from papaya enzymes", Tbilisi, 1986, p.20-22).

In this way, as in the previous one, from the resulting aqueous extract precipitated enzymes 96% ethanol, the suspension of the enzymes survive, filtered and dried to obtain the finished product.

The disadvantages of these methods are: incomplete extraction of water-soluble enzymes of water due to insufficient quantity, the duration of the technological process (and consequently the possible reduction in the enzyme activity), the use of toxic and explosive ethyl alcohol, the need for subsequent regeneration of its mother liquor, it is possible to inactivate enzymes by ethanol, the use of low temperatures for the direct selection of the product. In addition, these methods are proposed for laboratory research.

Later on the basis of water-soluble complex of proteolytic enzymes was created for the treatment of burns and wounds - caritatem in sterile dosage form: lyophilized for solution for outdoor applications 350 PE (VFS 42-1749-87).

As you know, commercial papain, supplied to Russia from India, China, Vietnam and other tropical countries, as a rule:

contains different amount of ballast substances (up to 40% of the dried milky juice of extraction of water can not dissolve);

p> - microbiologically often very heavily polluted;

- has a different degree of reduction (up to "dust").

As a result, when the scaling process, these factors can lead to a significant increase in the duration of the process, reduced activity and non-sterile finished product.

It also helps that the extractant proteolytic enzymes is water, which quickly obamanauts and water extraction itself can quickly turn sour.

In connection with the foregoing, the process of obtaining water-soluble proteolytic enzymes from the dried latex by known methods requires continuous and expeditious conduct of the entire multi-stage process - the slightest failure of the process (normal and slow sterilizarea filtering, bottling or freeze drying) can produce non-standard finished product - caripazym.

In the preparation of production scale batches of the drug are required simultaneously or many units of expensive freeze-drying, or one big performance, which is economically disadvantageous.

In addition, upon receipt of the lyophilisate of proteolytic enzymes, the stage of purification of raw materials by extraction with water followed by filtering ballast substances and stage prepara the value of finished dosage forms caripazym freeze-dried sterilizarea filtering the resulting aqueous filtrate with subsequent freeze drying sterile solution is combined, i.e. the purified water-soluble complex of proteolytic enzymes in the dry state is not highlighted that, in our opinion, ethnologica.

In addition, sterilizers filtration and subsequent drying sent a very diluted solution, which is also irrational.

In the specified way the original product - a native of the dried milky juice of unripe fruit of the papaya cannot be used without additional purification for the preparation of other, for example soft, dosage forms containing the amount of water-soluble proteolytic enzymes, and to use lyophilized product unprofitable.

According to these authors the task, aimed at improving the workability and efficiency of the method with a high quality product due to the receipt of the trade purified papain amount of water-soluble proteolytic enzymes in the form of a dry extract, which is further used for preparation of various dosage forms, which significantly extends the capabilities of this method.

To solve this problem in the way of getting the amount of water-soluble proteolyticus is their enzymes and medicines on the basis of the dried milky juice of unripe fruits of papaya tree, including extraction of raw water at room temperature and stirring, separating the solid and liquid phases and isolation of the pure product, a water extract obtained after separation of the solid and liquid phases is additionally filtered through a filter with pore size of 0.45 to 2.0 μm, dried by a spray method to obtain the pure product in the form of a dry extract with a proteolytic activity of at least 3.5 PE/mg of the drug and the protein content from 0.43-0.50 mg/mg product.

Thus obtained dry extract in powder form can be used for solution preparation for external use or soft dosage forms (ointments, gels).

In the method of obtaining medicines on the basis of the obtained dry extract also proposed dry extract with a proteolytic activity of at least 3.5 PE/mg of the drug and the protein content from 0.43-0.50 mg/mg of the drug dissolved in the water, then hold sterilizers filtration and freeze drying of the solution.

The method is implemented as follows.

The dried milky juice of the papaya is extracted once with purified water at room temperature and constant stirring, the ratio of the raw water (kg/l) is 1:33-37, which allows the most complete proektirovanii the whole complex of water-soluble proteolytic EN zymes the s from the source of raw materials and to avoid the extraction of other substances, contained in it. Then the extraction mass, which is a suspension that was filtered from ballast substances that are not soluble in water, optionally filtered the resulting aqueous extract through a filter with pore size of 0.45 to 2.0 μm for increasing the degree of microbiological purity and dried in the spray dryer. The dry product is sifted through a sieve No. 23 in order to remove lumps in it and obtain a powder with the same particle size. Get dry extract - treated amount of water-soluble proteolytic enzymes with proteolytic activity at least 3.5 PE/mg of the drug and the protein content from 0.43 to 0.50 mg/mg of the drug, i.e. with the same rules as in finished dosage form - Cariprazine the lyophilisate.

Next, the dry extract was dissolved in water for injection, the resulting solution was filtered through stabiliziruyushchii filter with pore size 0.22 μm, poured into sterile solution of 2.0 ml ± 8% (350 PE) into vials and freeze-dried. Get drug "caritatem lyophilisate for preparation of solution for topical application 350 PE, corresponding normative documents.

In addition, use dry extract in the form of a packaged powder for solution preparation for external use or for the preparation of soft medicinal forms.

Offer posabilities by the following example.

Example. 1.0 kg of the dried latex (particle size 0.2 mm, the loss in weight on drying 7.4%) from proteolytic activity of 3.0 PE/mg of the drug and the protein content of 0.32 mg/mg of the drug suspended in 3 l of purified water to obtain a homogeneous suspension, after which the concentrated suspension of papain in water add 34 l of purified water and lead extraction amount of water-soluble proteolytic enzymes water for 30 min at room temperature and constant stirring. The ratio of raw material to water is 1:37. After the specified time extraction mass, which is a suspension that was filtered from ballast substances that are not soluble in water, through a filter filled with a filter cardboard T-grade. The filter cake is thoroughly drained, washed with 1.5 liters of purified water, after which the resulting aqueous extract of proteolytic enzymes (34 l) optionally filtered through a filter with pore size (of 0.45 to 2.0 microns). Get 33 l filtered water extraction, which is dried by the method of sputtering under the following process parameters:

the feed rate of extraction of 20-25 l/h;

- compressed air pressure of 5.0-5.5 kgf/cm2;

- the temperature of the inlet air 120-140°C;

- the temperature of the air exiting from 60-75°C.

The dried product is sieved by a nylon sieve No. 23. Get 074 kg Procarbazine dry extract, the relevant Fund 42-9123-08, including microbiological purity (category 1.2 (B), with access 74,0% by weight of the feedstock.

Prokaryotes (0,74 kg) is dissolved with stirring in 4 l of water for injection, add water up to 14 l, the resulting solution was stirred for 20-30 min, and then filtered through stabiliziruyushchii membrane filter with a pore size of 0.22 μm. Sterile solution of 13.5 l poured into 2.0 ml ± 8% (350 PE) into vials and freeze-dried in the freeze dryer with the following parameters:

- freeze solution to a temperature of minus 35°C and holding at this temperature for two hours;

- drying the frozen solution in a high vacuum (15 PA) with a gradual increase in temperature to 0°C for 7 hours;

- heat drying in high vacuum at temperatures above 0°C for 4-5 hours;

the final drying at a temperature of 25-27°C for 5-6 hours.

The total duration of drying is 30-32 hours.

Receive 664 vial 0.1 g of a medicinal product "caritatem lyophilisate for preparation of solution for topical application 350 PE corresponding to the Fund 42-9225-08, with access 89,73% by weight of dry extract. The total yield of Karipazim freeze-dried from the beginning of the process is 66,4% by weight of the feedstock.

The use of the proposed methods in comparison with the known charge of the AET following benefits:

is quite high and stable yield of purified amounts of proteolytic enzymes (as dry extract), and hence caripazym freeze-dried;

- obtaining purified amounts of proteolytic enzymes with high proteolytic activity and high protein content, i.e. with the same rules as karipazim freeze-dried;

- purified amount of proteolytic enzymes and caritatem lyophilisate in contrast to known methods contain the same complex of proteolytic enzymes that water extraction, since drying is sent to all water extraction, not the product obtained from him, precipitation with ethanol;

- stable output and quality caripazym freeze-dried in the processing of various quantities of papain, since its manufacturing have been used for purified amount of proteolytic enzymes - Prokaryotes dry extract with stable quality and not trading papain (dried milky juice of the papaya), containing up to 40% ballast substances and microbiologically contaminated;

- reduction in the total duration of the process of preparation of a sterile solution of karipazim due to the dissolution and sterilizarea filter already cleaned product and by reducing the volume of the solution to sterile filtration, because you can use twice more concentrated solutions cleaned up the th amount of proteolytic enzymes;

as a result, rapid acceleration of the process of manufacturing caripazym freeze-dried, depending on the needs of the market;

schema obtain purified amounts of proteolytic enzymes and caripazym freeze-dried reconstituted on the premises of a different class of purity that is necessary from the point of view of carrying out different types of technological processes and relevant in light of the requirements of GMP; as a result, the schemes become more compact.

- get Prokaryotes dry extract (purified amount of water-soluble enzymes) can be used not only for cooking caripazym freeze-dried, but in the form of a powder for solution preparation for external use or for the preparation of soft medicinal forms (gels, ointments).

The developed method was verified in an industrial environment when processing different batches of raw papain and confirmed the above results. The quality of the products obtained is stable and corresponds to the normative documentation on drugs.

1. The method of obtaining the amount of water-soluble proteolytic enzymes from the dried milky juice of unripe fruits of papaya tree, including extraction of raw water at room temperature and stirring, separating the solid and liquid phases and isolation of the pure product, characterized in that the water WPI is Uchenie, obtained after separation of the solid and liquid phases, optionally filtered through a filter with pore size of 0.45 to 2.0 μm, dried by a spray method to obtain the pure product in the form of a dry extract with a proteolytic activity of at least 3.5 PE/mg of the drug and the protein content of 0.43-0.50 mg/mg of the drug, dissolved in water, then spend sterilizers filtration and freeze drying of the solution.

2. The method according to claim 1, characterized in that the dry extract used for the manufacture of soft medicinal forms.



 

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