GB1594199A

GB1594199A - Biological active substance

Info

Publication number: GB1594199A
Application number: GB23549/78A
Authority: GB
Original assignee: JINTAN DOLPH CO Ltd
Current assignee: JINTAN DOLPH CO Ltd
Priority date: 1978-05-26
Filing date: 1978-05-26
Publication date: 1981-07-30

Description

(54) A BIOLOGICAL ACTIVE SUBSTANCE (71) We, JINTAN DOLPH CO. LTD., a Joint Stock Company organised and existing under the Laws of Japan of: 543 Tamahori-cho, Higashi-ku, Osaka City, Japan, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to a biologically active substance which is not a protease but which has casein aggregating activity, to processes for preparing this substance and to compositions containing the substance.

In the study of the activities of protease enzyme preparations from various sources, it has now been found that these enzyme preparations all contain a minor proportion of a specific organic substance of low molecular weight in free state or various kinds of combined states, and that this organic substance shows notable effects of activating, promoting or adjusting various kinds of enzymatic reactions, e.g. protease activity in and outside a living body.

Further, it has been discovered that this organic substance has a common origin at least with protease and can be obtained from the same kind of raw material, and also is very useful as an anti-inflammatory agent, a medicine for the stomach and bowels or other medicines or promoting or adjusting agents for various kinds of enzymatic reactions in organisms.

Accordingly, the present invention provides a biologically active substance which is not a protease but which has casein aggregating activity, having the following properties: (a) it is readily soluble in water and hydrophilic and lipophilic solvents, (b) it is capable of being steam distilled, (c) it can be extracted from an alkaline, aqueous solution using an organic solvent, (d) its ultra-violet spectrum (determined on an aqeuous solution of concentration 2 CAU/cc and pH 9.5) shows a single sharp peak at about 253 nm, which is replaced by a strong peak at about 280 nm when said solution is treated with acid to adjust its pH to 2.0, (e) it prevents the aggregation of ADP-induced blood platelets, and (f) it promotes casein decomposition such that when an aqueous solution of refined active substance having a CAU activity of 25 is added to 100 cc's of an aqueous solution of 1% Hammersten casein at a temperature of 37"C, the optical density of the solution is substantially as follows: time after addition of solution of active substance to casein solution (in minutes) optical density 0 0 4 5 90 20 150 60 170 40 210 40 The present invention also provides a process for the preparation of this biologically active substance, which process comprises treating pineapple roots with water at a temperature of at least 50"C to extract the biologically active substance therefrom, and recovering said substance from the resulting aqueous solution.

The present invention provides another process for the preparation of the biologically active substance of the invention. which process comprises contacting pineapple stem or fruit juice with an organic solvent to precipitate the biologically active substance together with peptides and carbohydrate therein. and recovering said substance from the resulting precipitate.

An additional process for the preparation of the biologically active substance of the invention comprises treating acrude protease preparation so as to remove the protease component therefrom. and recovering the biologically active substance from the resulting solution.

The invention also provides a pharmaceutical composition comprising the biologically active substance of the present invention together with a pharmaceutically acceptable carrier or diluent; a cosmetic composition comprising the biologically active substance of the present invention with a cosmetically acceptable carrier or diluent; and a composition for activating, promoting or adjusting enzymatic processes in organisms comprising the biologically active substance of the present invention together with a biologically acceptable carrier or diluent.

For brevity, the biologically active substance of the invention will simply be referred to as the active substance hereinafter.

Properties of the active substance As mentioned above, the active substance is readily soluble in various kinds of hydrophilic and lipophilic solvents and water, is capable of being steam-distilled, and is extractable with an organic solvent from a water solution in the alkaline region. The aqueous solution is relatively stable to heating up to 1000C and shows similar liquid property to that of primary amine solution. The UV adsorption spectrum as determined on an aqueous solution of the active substance of concentration 2 CAU/cc at pH 9.5 (having a tendency of monomer), shows a very simple spectrum having a single sharp peak at 253 nm (see Figure 3). Mild acid treatment to adjust the pH to 2.0 shifts the end UV absorption to a strong 280 nm peak.

As far as the biochemical properties of the active substance are concerned, it shows no protease activity but has a notable casein aggregation reaction. Therefore, the active substance can be determined by determining its casein aggregation activity. A method of determining casein aggregation activity adopted in this Specification is as follows: 5 g of Hammersten casein is dissolved in 500 ml of distilled water. The pH of the solution is raised to 9 to 10 with aqueous ammonia. Then the solution is left to stand till all of the casein has completely dissolved. Further, 10 g of "Difco" (Registered Trade Mark) skim milk is added thereto, and 0.5 N acetic acid buffering solution of pH 4.7 is carefully added to adjust the solution to pH 5.4. The solution thus obtained is made 1 t in volume with distilled water, and filtered to obtain the reagent.

To 1 ml of active substance solution (pH 5.4, ().005 M acetic acid buffering solution which has been made 0.002 M with respect to cystein, 5 ml of the abovementioned reagent is added and mixed. and matured at 45"C for 1 hour, diluted with 5 ml of 0.4 M NaCI solution containing 0.05% H2O2, and subjected to color-imetric determination at the wavelength of 580 nm by a chromometer.

1 casein aggregation unit (CAU) is defined as equivalent to 0.10 D (optical density).

With regard to methods of preparing the active substance, it has been discovered that the active substance has a common origin with at least the proteases pepsin, trypsin, chymotrypsin, cathepsin, papain, bromelain, ficin, rennin and proteases from any bacteria or fungus. Most of the active substance present in such a raw material of protease origin is in a state of weak combination with high molecular substance e.g. peptide and acidic carbohydrate.

Examples of suitable raw materials from which crude protease preparations can be obtained are: Plant material from any member of the Bromeliaceae, and Ficus; animal or fish tissue of Stomach tissue, Pancreas; or appropriate concentrates or extracts thereof. Commercial preparations of proteases such as bromelain, ficin, rennin and pepsin fall within the category of crude protease preparations from which the active substance of the invention can be obtained.

The active substance in free state is present in a rather large amount in aerial roots and ground roots of pineapple (up to 120 CAU/g). Further, in pineapple stem juice fruit and leaf, the active substance is present in a rather large amount both in free state and state of weak combination with a high molecular substance e.g. acidic carbohydrate or protein.

Therefore, the conventional crude bromelain precipitated with acetone from a pineapple plant or pineapple stem juice is very useful raw material for preparing the active substance according to the present invention.

Advantageous methods for extracting the active substance from the original raw material and refining the same are described in the Examples below. More generally, this can be achieved by many separating and refining methods widely used in this art e.g. column chromatography, dialysis, electromigration, gel filtration, ion exchange resin method, precipitation or extraction with a suitable solvent, or a combination of these techniques.

Therefore, the present invention is not limited to any particular extraction or refining methods.

The processes for the preparation of the active substance according to the present invention can be easily carried out by combining suitably the above-mentioned various conventional methods depending upon the original raw material and the existing condition of the active substance.

Considering further the processes of the present invention: I. Pineapple roots are typical raw material containing the active substance in free state, from which the active substance is extracted with hot water.

The hot water extract from pineapple roots contains a minor proportion of the active substance and a major proportion of other substances such as tannin, organic acids, sugar, inorganic salt, purines and pyrimidines. From the extract, the active substance is obtained by separating and refining by ion exchange column chromatography or extraction with solvent.

II. Juice from pineapple stem, which is also the raw material of bromelain, is a typical raw material containing the active substance both in free and combined state. The active substance in the combined state is absorbed on or weakly associated with carbohydrate colloid and peptides of enzymes, which can provide the active substance in free state by hydrolysis by, for example, adjusting the pH and temperature or adding a hydrolyzing enzyme.

On the other hand, it has been found that peptides or acidic carbohydrate polymers in the stem juice can very effectively absorb the active substance in free state to be precipitated together with the latter by adding a precipitating agent e.g. acetone (at least two time volume) or methanol (at least three time volume) in a specific pH region (pH 4.2 to 5.8).

As is apparent from the abovementioned findings on pineapple plants, similar techniques can be applied in the case of other protease containing raw materials.

An example of an industrially useful preparatory method may be a method of obtaining the active substance as a by-product by modification of a process of manufacturing a protease enzyme preparation.

Pharmacological actions of the active substance Summary: The active substance according to the present invention is effective as an antiinflammatory agent, a medicine and digestive for the stomach and bowels. It is administered according to conventional methods e.g. oral administration, intravenous injection, abdominal cavity injection or topical application. It has been discovered that an effective dosage is 10 to 1000 CAU per 1 body (oral administration), and 0.1 to 1() CAU per 1 body (intravenous injection). An example of pharmacological test is as follows.

Anti-inflammatory effect: Rats were fasted one day and night and then given either an aqueous solution of the refined active substance (20 CAU/ml: containing 0.005 M cysteine) or physiological saline (containing 0.005 M cysteine) by oral administration. One hour later each rat was injected with 0.05 ml of an anti-rat serum. Two hours later the rats were killed and the extent of the edima was measured by punch method (see for example Ungar et al, Arch. int. pharmacodyn. 1959, CXXIII, No 1-2, pages 71-77). The results are shown in Table 1.

TABLE 1 Control Sample 117.2 86.1 133.1 114.8 117.1 110.1 127.1 77.7 133.0 115.1 Average 125.5 100.8 Mouse-tail bleeding test: The conventional method was modified so as to include induced stress. Namely, each mouse had been administered with 0.1 mg adrenalin per kg (body weight) one hour before the intravenous injection of the specimen. Each mouse was intravenously injected with 1/4 ml of a refined aqueous solution of the active substance (20 CAU/ml; containing 0.005 M cysteine), and 5 minutes later it was bled. The results are in Table 2.

TABLE 2 Bleeding Significant Treatment (min). time difference 0 60 (sec.) AS BS AB BB AS Adrenalin specimen 202.7 - - - - BS --- Specimen 150.5 AB Adrenalin --- 129.1 * y no BB --- --- 122.6 no no no Notes *: Differences significant at the 5% level Differences significant at the 1% level Test of ADP induced aggregation of blood platelets: Platelet-rich plasma was previously prepared from the blood of a person extraordinarily sensitive to ADP.

One drop of a refined aqueous water of the active agent (20 CAU/ml; containing 0.005 M cysteine) was mixed with one drop of suitably diluted aqueous ADP solution in a 5 ml plastic test tube. Then two drops of platelet-rich plasma was added thereto, and the mixture was shaken by a vortex shaker for 1 minute. The mixture was placed on silicon coated slides and covered with a plastic cover slip, and the degree of aggregation estimated under a microscope of 60 magnifications. The estimation was by 6 grade from 0 to 5 in with grade 0 corresponds to no aggregation and grade 5 to large mass aggregation accompanied with the dissolution of blood platelet membrane.

In this test, in order to indicate that the active substance is contained in commercial enzyme preparations such as bromelain and papain, the commercial enzyme preparations and the refined product obtained by refining through two time precipitation of the commercial enzyme preparations with 2 time volume acetone below pH 3.5 were also tested. All enzyme preparations were tested at a level of 1 GDU (gelatine digesting unit) enzyme unit per ml.

In this connection, it was thought heretofore that proteases e.g. bromelain and papain themselves have the ability of prohibiting the aggregation of ADP induced blood platelets.

The results are shown in Table 3.

TABLE 3 ADP Bromelain Papain Sample Control of Commercial Refined Commercial Refined Active [2X-1mg/ml] (1400GDU) (1000GDU) (3000GDU) (1800GDU) Substance 8 0 5 0 5 0 5 9 0 5 0 4 0 5 10 0 4- 0 2 0 3 11 0 3 0 1 0 2 12 0 1- 0 0 1 Action on fiber dissolving system: The active substance (20 CAU/ml, a water solution containing 0.005 M cysteine) was tested on its action on each of two fiber dissolving systems i.e. fibrinogen and fibrin dissolving systems.

In the test on fibrinogen dissolving system, a mixed solution consisting of 1 ml of human blood plasma, 1 ml of the sample and 0.1 ml of physiological saline was maintained at 37"C for 20 hours.

Then, 0.1 ml of thrombin (500 units per 10 ml) was added to the solution to convert the remaining fibrinogen to fibrin. 30 minutes after the addition of thrombin, the solution was centrifuged (3000 r.p.m. for 5 min.) to provide precipitate. The FDP (fibrinogen decomposing product) in the supernatant liquid was estimated by red cell aggregation inhibiting reaction.

On the other hand, in the test on fibrin decomposing system, to a mixture of 1 ml of human blood plasma and 1 ml of the sample 0.1 ml thrombin (500 unit/l0 ml) was added to aggregate the blood plasma. After the mixture was maintained at 370C for 20 hours, 0.1 ml of physiological saline was added thereto. The solution was centrifuged (3000 r.p.m., for 5 min.), and FDP in the supernatant liquid was estimated as abovementioned.

Blood plasma used in this test was prepared by mixing blood plasma from several patients. Thrombin was from human blood plasma. The treatments were all carried out by using plastic tubes.

The results are shown in Table 4.

TABLE 4 Fibrinogen Fibrin FDP (g/ml) FDP ( > g/ml) No.1 No.2 No.1 No.2 Sample 80 160 40 40 U.K* 640 < 640 80 160 Control 10 20 10 10 Notes: *U.K: Urokinase standard Two kinds of blood blasma tested Polyamylase activating test: 100 ml of 0.1% aqueous solution of Takadiastase made by Miles Co., Ltd. was mixed with 2 CAU of refined aqueous solution of the active substance (20 CAU/ml, containing 0.005 M cysteine), and the amount of amylase was estimated with time lapse.

The method for estimating amylase comprises measuring the amount of reducing material (mainly maltose) freed by the enzymes from 1% aqueous solution of soluble starch (pH 6.9) containing 0.07 M NaCI. The reducing material is estimated by the dinitrosalicylic acid method (see Summer "Enzymes" Academic Press 1944).

The result was summed in Table 5.

TABLE 5 Maturation pH 0 15 30 45 60 (min.) 4.5 0.36 0.4 0.48 0.345 0.225 7.0 0.36 0.475 0.435 0.350 0.260 Amylase activity of the aqueous solution of Takadiastase not mixed with the active substance (control): 0.025.

Similar test was carried out on bacterial amylase. The result is shown in Table 6.

TABLE 6 Maturation pH 0 15 30 45 60 (min.) 4.5 0.705 1.18 1.14 1.35 0.900 7.0 0.685 0.690 0.73 0.765 0.800 Control* 0.52 0.53 0.545 0.62 0.695 * Amylase tested shows self activating property in about 2 hours.

Burn eschar removing test: A polyethylene-base ointment was prepared which contains 50 CAU/g of the active substance.

This ointment was applied to remove the burn eschars of a rabbit 3 to 5 days after the burn. The removing ratio was about 8%.

It is presumed that this is due to the active substance acting on the enzymes in vivo to activate the same during the decomposition and removing of the modified protein in vivo e.g. dead tissue.

Further it has been confirmed that the active substance is effective as a medicine for external application for curing acnes, removing horny layer, smoothing rough skin and other purposes.

Determination of the dosage: Using a rat, the action of the active substance to restrain the albumen edema was estimated by measuring the thickness, thereby determining the dosage.

Result: Restraining ratio above 50% with 10 CAU/kg (i.p.).

Acute toxity: dd type male mouse 15 + ig were used. 0.2 ml of each concentration of the sample per 1 body was intravenously injected, 0.5 ml of the same per 1 body abdominally injected and 0.5 ml per 1 body orally administered. LD 50 was calculated after 24 hours by Litchfield-Wilcoxon method.

The result was summed in Table 7.

TABLE 7 Used amount Dead number LD50 Administration CAU/kg used number CAU/kg Intravenous 65 10/10 injection 50 9/10 39 6/10 41.0 30 2/10 23 0/10 Abdominal 69 10/10 injection 53 9/10 41 6/10 43.5 39 1/10 30 0/10 Oral 10,000 0/10 Other biological actions 5,000 0/10 > 10,000 Action on casein: To 100 ml of aqueous solution of 1% Hammersten casein, 25 CAU of an aqueous solution of refined active substance was added at 370C, and the biuret-colored product was colorimetrically determined with time lapse (Table 8).

In the process, it is presumed that the active substance coacts with the HIS-SER region of k-casein, to contribute to cleave the Phe.AI-MET (phenylalanine-methionine) bond thereof and modify the K-casein into para Kappa-Casein. It is also presumed that cleavage of the Phe.Al-MET bond provides a peptide comprising 66 amino acids, which shows biuret color.

This casein decomposition is now widely confirmed to be one of essential function for casein aggregation reaction, but it has ordinarily been postulated to be a reaction specific to rennin only.

TABLE 8 0 4 90 150 170 210 (min.) Optical density 0 5 20 60 40 40 (OD) Cereal powder solubilization promoting effect: 9 g of barley powder was suspended in 100 ml of water containing 2 CAU of the active substance, and incubated for 30 minutes, and then heated at 700C for 1 minute and immediately centrifuged to measure the amount of free water (Table 9).

TABLE 9 Free water (ml) Viscosity of free water Control 51 Fluid Sample 43 Coagulated gel Fish meat solubillzanon promoting effect: Fish body was ground by a meat chopper and matured under the following conditions A to E. Then, after the centrifugation (5000 r.p.m.) of the fish meat paste, 15 ml of INNAOH was added to 1 ml of the obtained supernatant solution, and then 1 ml of Benedict's solution also added thereto. The mixture was colorimetrically determined at 560 nm (Table 10).

A. Fish meat paste was matured at 45"C for 1 hour (control).

B. Fish meat paste was heated to 95"C and then matured at 450C for 1 hour (control).

C. The same as B with the exception that 2 CAU of the active substance per 100 g of the fish meat paste was added.

D. The same as A with the exception that 2 CAU/100g (paste) was added.

E. The same as D with the exception that the active substance was added during the grinding of fish body.

TABLE 10 Condition Biuret color degree (OD) A 0.300 B 0.235 C 0.253 D 0.452 E 0.512 Softening of chicken meat: 1 ml of physiological saline containing each of 0, 1/4 and 1 CAU of the active substance was injected in the intravene of the wing of an 18 month old cock.

5 minutes later the cock was killed, cooked and subjected to Functional panel test (5 grade evaluation).

The result is shown in Table 11.

TABLE 11 CAU Evaluation* 0 2 1/4 3 1 5 Refined bromelain 1 (20 mg/kg weight) * 1 = very tough 5 = very tender Summarization of the industrial application: As apparent from the above tests, the active substance according to the present invention has an effect of activating certain kinds of proenzymes in and outside living bodies.

According to this finding, the active substance may be extremely widely utilized for industrial purposes, some of which are as follows.

A) Food industry a) Improvement of the quality of stored cereals: By treating cereals e.g. peas, corn, rice, wheat (powder, seeds, etc.,) tea or other farm products with the active substance, the proenzymes which they contain are activated, and the quality of the products are improved to provide sweeter, softer and soluble food. b) Promotion of the fermentation of food. of farm products: By treating for example, coffee beans, cacao beans or vanilla beans with the active substance food of a better flavor can be provided. c) By treating the cereal powder used for manufacturuing, for example cakes or bread with the active substance, powder is provided which permits good finishing of products and short time of kneading. d) Meat is softened and its quality is improved by in vivo or in vitro administration of the active substance. e) By adding the active substance, the stickiness of fish meat paste e.g. for fish cake is increased. f) Promoting of the maturation of seeds.

B) Food waste treating industry a) Solubilizing waste fish meat (suitable for animal foods) b) Decomposing starch gluten residue from starch industry to obtain high quality starch and useful gluten concentrate.

C) Application as the ingredient of cosmetics Each kind of cosmetic comprises water and oil soluble base material to which medicine and flavor may be added for various purposes. The active substance is also a new factor which can decompose the base protein and therefore is applicable as an ingredient of cosmetics never used heretofore. The main ingredient of the horny layer is pseudokeratin which is apt to be influenced by enzymes and has a high metabolism. It is known that by artificially adding natural protein decomposing enzyme, the decomposition rate of the horny layer is increased to smooth the skin. However, protein decomposing enzyme of high activity does damage to the skin and in some cases the intended purpose is not achieved due to the activity being lost. In this connection, the active substance helps the natural metabolism of the horny layer as well as normally control the metabolism of the skin in an abnormal state. Therefore, the active substance is characterized by its very little side-effects but proper effects. Further, since it can decompose abnormal secretion from the skin, its advantages can be expected that acnes and spots can be removed and the skin can be smoothed thereby.

The following Examples illustrate the present invention. As elsewhere in the Specification, percentages are by weight. Dole bromelain is bromelain produced in Hawaii by the Dole Company.

Example 1 0 70g of Formosan pineapple roots were boiled in 800 f of water and extracted for 30 minutes. The extraction process was repeated with two additional batches of water (the extracts having a pH of 4.8). The extract still hot was filtered and was run over a strong cation resin column in the H form (the effluent having a pH of 3.0), and then over a weak anion resin column in the basic form (the effluent having a pH of 7.5).

The effluent from the anion column did not show the effect of casein aggregation.

The anion column was eluted with 1 e of 1N-NH4OH. The eluate was fractioned into 100 ml fractions and assayed. The first 300 ml of the eluate was inactive and the whole of the remaining 700 ml active. The pH of the active eluate was adjusted to 3.0 and run over a XAD-2 column. The colorless effluent was concentrated to 100 ml and the activity thereof estimated (Sample A).

The cation column was eluted with lN-HCl and the activity of the obtained colorless eluate (Sample B) was estimated.

The activity of each sample was shown in Table 12.

TABLE 12 Sample A 10,325 (CAU) Sample B 2,600 (CAU) o Each of Samples A and B was run over a "Sephadex" (Registered Trade Mark) G-10 column (0.005 M cystein chloride - 0.01 M NaCl developing solution (pH 4.8)), and the effluent was taken every 10 ml, and estimated on its activity, sugar and organic phosphorus.

The active part are obtained in the tubes Nos. 10 to 13, and the mean molecular weight of this complex was presumed to be about 3,000 by comparison with Indigo Carmine (M.W.

466) and chlorophyllin B (M.W. about 1200) On the other hand, a considerable amount of active part having a molecular weight of about less than 800, which was caught in gel pores together with Indigo Camine, was found.

The result is shown in the accompanying Figures 1 and 2.

Referring to Figure 2, it is observed that the active substance eluated from the cation column is present mainly as a complex salt. o Sample A solution from which salts had been deposited and removed was adjusted to have a pH of 8.5 and extracted with ethyl acetate.

The solvent was removed from the extract and the slight volume of residue was extracted with chloroform.

The chloroform extract was fractioned by a silicic acid column chromatography (CHCl3 and then CHCl, - MeOH was used as the developing solution). Three clearly distinguishable fractions were obtained respectively. The activity was shown only in the intermediate fraction. which showed strong ninhydrin color reaction. UV spectrum of this active fraction (Figure 3 of the accompanying drawings) provided a single band having a sharp peak at 253 nm.

Example 2 o 20 g of Dole bromelain (E-X-1) was dissolved in 1 e of 0.9% NaCl aqueous solution and adjusted to have a pH of 3.2. Then the same amount of acetone was added thereto and the mixture was centrifuged to provide precipitate.

The supernatant solution was run over a XAD-2 column, and three fractions i.e. the first effluent, 2% formic acid - 50% acetone eluate, and pH 11 (NH4OH) - 50% acetone eluate were obtained respectively.

The first fraction was extracted with CHCl3:MeOH (1:1) (Sample A). And the other fractions were concentrated and left to stand in a refrigerator; then deposited protein precipitate was removed. and the organic acids were precipitated with CaCI2 (Sample B and Sample C). The activities of each of the Samples were as shown in Table 13.

TABLE 13 Sample A 200 (CAU) Sample B 5,600 (CAU) Sample C 0 (CAU) o Sample B was adjusted to have a pH of 2, heated at 900C for 10 minutes, then adjusted to have a pH of 9.5, boiled for 5 minutes and suitably diluted and the UV spectrum was measured.

The result was as shown in the accompanying Figure 4, in which a single peak was formed at about 250 nm, and the product is regarded as the derivative of substance in the form of a monomer.

The spectrum of Sample B solution before the heating treatment with an acid and alkali showed negligible absorption except end UV absorption.

Example 3 o 10 g of Dole bromelain (E-Xl-1) was dissolved in 500 ml of water, adjusted to have a pH of 10.7, and heated at 1000C for 20 minutes. After cooling, the aqueous solution was adjusted to have a pH of 3.3 and centrifuged to remove a precipitate. The supernatant solution was extracted at pH 3.3 with chloroform so as to remove organic acids.

The activity of the aqueous solution was 8.2 CAU/ml (5,000 CAU in amount).

(2) 10 g of Dole bromelain (N-1V-1) was dissolved in 200

4) If the enzyme solution is left to stand at pH 7 for 1 hour, the best pH value for the precipitation step to be used to recover the active substance in the supernatant solution; a) pH 6.0, the addition of cystein is used in the tests or in the applications. b) pH 8, at this pH value, cystein is not necessarily added. c) pH 3.8. o 100 ml of 1% aqueous solution of Dole X-XI-I bromelain was adjusted to have a pH of 8.0, and various additives (reducing agent or SH protecting agent) were added thereto. The solution was left to stand for 1 hour, made to form precipitate by adding 150 ml acetone and centrifuged to form precipitate. After removing acetone from the supernatant solution, the whole volume was made to be 100 ml and pH was adjusted to 5.4.

Then the solution was boiled. and extracted at pH 10 with chloroform. The obtained chloroform extract and the residual aqueous solution were estimated on their activities (CAU) respectively. The result was summarized in Table 14.

TABLE 14 20% FeSO4 0.1% 1% ascorbic Aqueous Chloroform (ml) CPC* acid solution extract 0 0 0 57 30 0 0 + 300 56 0 + 0 160 40 0 + + 284 36 4 0 0 320 53 4 0 + 280 56 4 + 0 52 18 4 + + 268 42 *) Cetylpyridinium O 1:1 acetone supernatant solution of 5% bromelain solution precipitated at pH 3.2 was run over a XAD-2 column, and the column was eluted with 50% acetone containing NH4OH and adjusted to have a pH of 11. This fraction contained most of the casein aggregating activity.

Then the column was eluted with IN NaOH, and the eluate was extracted with chloroform and further extracted with acid aqueous solution. This fraction did not show the casein aggregating activity in the absence of cysteine but with the addition of 0.002 M cysteine, it showed 8 CAU/ml of activity.

Example 4 o Pineapple roots and peelings were extracted with boiling water, concentrated and freeze-dried.

The freeze-dried powder is dissolved in water and adjusted to have a pH of 8, and extracted with ethyl acetate. The extract was washed with pH 4 buffer solution, concentrated and dried. The dry powder was dissolved in water and estimated on its organic phosphorus content and CAU activity.

Organic phosphorus /ml 1.9 FM CAU/ml 79 .CAUlg (powder) 117 o 10 g of ficin (commercial preparation) was dispersed in 200 ml of water, and its pH value was lowered from 5 to 3.5. The organic acid was extracted with chloroform and removed.

Then the solution was adjusted to pH 5 and left to stand for 30 minutes. Then the solution was further adjusted to pH 9.7 and extracted with chloroform, thereby obtaining a clear chloroform layer, an emulsion layer and a water layer. To the emulsion phase 4 times its volume of methanol was added, and the precipitate was removed by centrifugation, and the solution was filtered. Chloroform and methanol was distilled away from the filtrate, and the activity was estimated.

NO RSH* +RSH*

Acetone supernatant solution from colloid precipitate 2 4 in water phase Emulsion precipitate Emulsion supernatant 1456 2626/g solution (preparation) Clear chloroform trace phase *) Presence or absence of cysteine activation &num;) CAU estimation impossible due to high protease activity. o 4 g of Nakarai rennin was dispersed in 150 ml of water adjusted to pH 5.7 and left to stand for 1 hour.

Then the dispersion was adjusted to pH 10 and extracted with 50 ml of chloroform containing 1% methanol. This extraction was repeated two more times. The emulsion phase was taken out and the 5 time volume of methanol was added thereto to remove precipitate. The solvent was removed from all fractions and the activities were estimated.

CAU/g (rennin preparation)

Water phase 0 Emulsion precipitate 0 Emulsion solutlon 83,480* Chloroform phase - 0 *) Activated with cysteine (i)A large amount of pepsin was dissolved in water and the pH was adjusted to 5.5.

Then the pH was further adjusted to 3.0, and the 2 time volume of acetone was added.

The precipitate was dissolved in water again, and precipitated again with the 3 time volume of acetone.

NO RSH* +RSH*

Acetone precipitate (insoluble) 0 0 Re-dissolved (soluble) Re-precipitate fraction high protease activity Supernatant solution 0 140 Acetone supernatant solution Insoluble concentrate (modified protein) 0 0 pH 9.5 ethyl acetate-extracted 32 52 concentrate solution Whole activity 3 19 [CAU/g (preparation)] *) Presence and absence of cysteine activation

Claims

WHAT WE CLAIM IS:

1. A biologically active substance which is not a protease but which has casein aggregating activity, having the following properties: (a) it is readily soluble in water and hydrophilic and lipophilic solvents, (h) it is capable of being steam distilled, (c) it can be extracted from an alkaline, aqueous solution using an organic solvent, (d) its ultra-violet spectrum (determined on an aqueous solution of concentration 2 CAU/cc and pH 9.5) shows a single sharp peak at about 253 nm, which is replaced by a strong peak at about 280 nm when said solution is treated with acid to adjust its pH to 2.0, (e) it prevents the aggregation of ADP-induced blood platelets, and (f) it promotes casein decomposition such that when an aqueous solution of refined active substance having a CAU activity of 25 is added to 100 cc's of an aqueous solution of 1% Hammersten casein at a temperature of 37"C, the optical density of the solution is substantially as follows: time after addition of solution of active substance to casein solution (in minutes) optical density 0 0 4 5 90 20 150 60 170 40 210 40

2. A process for preparing a biologically active substance as defined in claim 1, which process comprises treating pineapple roots with water at a temperature of at least 500C to extract the biologically active substance therefrom, and recovering said substance from the resulting aqueous solution.

3. A process according to claim 2, wherein boiling water is employed.

4. A process according to claim 2, substantially as hereinbefore described in Example 1 or Run 1 of Example 4.

5. A biologically active substance as defined in claim 1, whenever prepared by a process as claimed in claim 2, 3 or 4.

6. A process for preparing a biologically active substance as defined in claim 1, which process comprises contacting pineapple stem or fruit juice with an organic solvent to precipitate the biologically active substance together with peptides and carbohydrate therein, and recovering said substance from the resulting precipitate.

7. A biologically active substance as defined in claim 1, whenever prepared by a process as claimed in claim 6.

8. A process for preparing a biologically active substance as defined in claim 1, which process comprises treating a crude protease preparation so as to remove the protease component therefrom, and recovering the biologically active substance from the resulting solution.

9. A process according to claim 8, substantially as hereinbefore described in Example 2 or any one of Runs 1 to 6 of Example 3 or Runs 2 to 4 of Example 4.

10. A biologically active substance as defined in claim 1, whenever prepared by a process as claimed in claim 8 or 9.

11. A pharmaceutical composition comprising a biologically active substance as claimed in any one of claims 1, 5, 7 or 10, together with a pharmaceutically acceptable carrier or diluent.

12. A cosmetic composition comprising a biologically active substance as claimed in any one of claims 1, 5, 7 or 10, together with a cosmetically acceptable carrier or diluent.

13. A composition for activating, promoting or adjusting enzymatic processes in organisms, comprising a biologically active substance as claimed in any one of claims 1, 5, 7 or 10, together with a biologically acceptable carrier or diluent.