[1]United States Patent Office 3@522@229 3,522,229 GLYCOPROTEIN FROM SALIVA OR SALIVARY GLANDS HAVING GASTRIC ACID SE, CRETION INHIBITORY AS WELL AS ANTI-ULCERATIVE ACTIVITY AND METHOD OF COLLECTING THESAME Masaaki Yamamoto and Masayoshi Kobayashi, Kawasaki-shi, and Tokutaro Miki, Tokyo, Japan, assignors to Teikoku Hormone Mfg. Co., Ltd., Tokyo, Japan, a corporation of Japan No Drawing. Filed Mar. 25, 1968, Ser. No. 715,579 Claims priority, application Japan, Mar. 29, 1967, 42/19,209; Mar. 15, 1968, 43/16,517 Int. Cl. A61k 17100; C07g 7/00 U.S. Cl. 260-112 16 Claims ABSTRACT OF THE DISCLOSURE A glycoprotein having gastric acid secretion inhibitory and antiulcerative activity, obtained from saliva or salivary glands. A method of collecting the glycoprotein which comprises in combination steps of acidifying saliva or an aqueous extraction of salivary glands to pH 4.0 stepwise, adding zinc salt to mother liquor to precipitate complex, redissolving the precipitate by chelating agent, subjecting to dialyzation, and adding watermisci'ble organic solvent to precipitate the glycoprotein. This invention relates to a new glycoprotein having gastric acid secretion inhibitory as well as anti-ulcerative activity and a method of collecting said glycoprotein from saliva or salivary glands. It is well known that the gastric juice of animals contains gastrone, a substance which demonstrates gastric acid secretion inhibitory activity. It is also pointed out that a similar activity is also present in saliva, the external secretion of the same alimentary canal. Namely, Code et al. have pointed out the presence of an antigastric acid secretory activity in the saliva of human and canine (Fed. proc. 8, 26, 1949). While it was thus known to date that saliva contained a gastric acid secretion inhibitory substance, the essence of the effective substance as well as its isolation was not known as yet and, furthermore, it was also unknown tO date that this substance had an anti-ulcerative activity. We succeeded in isolating a new glycoprotein from the saliva of man and mammals and also found that this new glycoprotein not only had a pronounced gastric acid secretion inhibitory activity but also had a therapeutic activity on gastric ulcers, as well as an inhibitory activity on their development. It was also found that the aforesaid new glycoprotein could also be isolated readily from the salivary glands of mammals. An object of this invention is to provide a new glycoprotein which is of value in the therapy of gastric hyperacidity and ulcerous disorders. Another object is to provide a new glycoprotein which is in a ptirified form suitable for administration to tjle human body and moreover possesses pronounced gastric acid secretion inhibitory as well as antiulcerative activity. A ftirther object is to provide a commercially feasible method of isolating the aforesaid glycoprotein in a purified form from saliva and salivary glands. This invention is to provide a method of collecting a glycoprotein having gastric acid secretion inhibiting Patented July 28, 1970 2 activity and antiulcerative activity, the method being characterized by acidifying an aqueous extraction of saliva or salivary glands to eliminate impure proteins having isoelectric points of about from pH 5.4 to pH 5.6 and about from pH 4.0 to pH 4.6, adding a water-soluble zinc salt to the resulting mother liquor to precipitate a complex of the zinc salt and proteins, adding a chelating agent for zinc to the so formed precipitates, redissolving the precipitates into an aqueous medium, subjecting the 10 so obtained solution to a dialyzing treatment to eliminate the chelate of zinc, and thereafter carrying out the following procedures (a) and (b) singly or in combination: (4) Adding a water-miscible organic solvent to the dialysate while adjusting its concentration up to 30 volume 15 percent to eliminate precipitated inactive proteins and then adding the water-miscible organic solvent to the filtrate while adjusting its concentration up to 60 volume perc ent to precipitate effective substances, and/or (b) Adding a sulfate of ammonia or an alkali metal 20 to the dialysate while adjusting its concentration up to O' 5 - saturation, collecting the so formed precipitates and subjecting the precipitates to a dialyzing treatment to eliminate the sulfate. As the saliva, useable, besides the as-obtained saliva 25 of man and mammals, are such as saliva concentrated to on6- half to one-fifth of its original volume, powdered saliva obtained by adding such as acetone or alcohol to saliva or concentrated saliva followed by making precipitates therefrom and drying the precipitates, or pow30 dered saliva obtained by drying the as-obtained saliva or concentrated saliva by a drying technique by which the activity thereof is not impaired, such as direct spray drying or freezing and drying. The term "saliva," as used herein and in the appended claims, will include the fore35 going concentrated saliva as well as the aqueous solution of the aforesaid powdered saliva. While as the salivary glands, either the sublingual, submaxillary or parotid glands of mammals can be used, the preferred ones are those which abound in mucus, such 40 as the sublingual and submaxillary glands, the parotid gland being somewhat inferior with respect to the yield of the glycoprotein. These glands are used ground or as a dried powder thereof. In the present invention, mixtures of sublingual glands, submaxillary glands and parotid 45 glands can be also used. As the source of saliva or salivary glands of mammals, useable are those of the ungulate animals such as bovine, equine, swine and ovine, and mammals other than the ungulate animals, such as canine, cats, rabbits, mice and 50 whales. The aforesaid salivary glands are made into a liquid extract by extraction in an aqueous medium showing a pH 6-8 such, for example, as water, a weakly alkaline water or saline, after removing, if desired, the fat from 55 the glands. The resuliing liquid extract of saliva or salivary glands, in its as-obtained state or, if necessary, after being submitted to a solid-liquid sepatation operation such as filtration or centrifuging to eliminate the idsoluble portion 6o therefrom, is acidified to thereby precipitate the impure proteiins having isoelectric points of about from pH 5.4 to pH 5.6 and about from pH 4.0 to pH 4.6. As the acid to be used, conveniently useable are the inorganic acids such as hydrochloric, sulfuric and hydrobromic acids. The pre65 ferred procedure is to first adjust its pH to 5.4-5.6 and remove the precipitates separating out and thereafter adjust-
[2]3 ing the pIl of the supernatant to 4.5-4.6 and again removing the resulting precipitates. In this manner, most of the ineffective protein substance, including the parotins, are rernoved and a clear mother liquor is obtained. For example, it is disclosed in the Ann. New York Acad. Sci., 85 Art. 1, pp. 228-310 (1960) that when the pH is adjusted to either 5.4 or 4.6 in the step of extracting parotin and salivaparotin from saliva or salivary glands, they are separated out as isoelectric point precipitates at respective foregoing pH value, but in the case of the invention glycoptotein it does not precipitate out but remains behind in the upper layer mother liquor. Again, it is known that, as other substances similar to parotin, alpha-parotin Endocrinol. Japan, 12, 69,249 (1965) and beta-parotin Endocrinol. Japan, 12, 69,249 (1965) are obtained from the parotid gland, while S-parotin Ann. N.Y. Acad. Sci., 85, Art. 1, pp. 228-310 (1960) is obtained from the submaxillary gland. Further, as a substance similar to salivaparotin, salivaparotin-A has been extracted from saliva (Ann. N.Y. Acad. Sci., 85, Art. 1, pp. 228-310, 1960). The alpha-parotin and Sparotin are, however, separated out as isoelectric point precipitates at either pH 5.4 or pH 4.5-4.6 as in the case of parotin. Hence, they can be separated froin the invention glycoprotein in the hereinbefore described treatment step. Salivaparotin-A is a low molecular weight protein which can only be obtained by hot extraction with - Iacial acetic acid and does not contain any sugar at all. Again, parotin and S-parotin are also simple proteins which do not contain any sugar ancl are clearly distinguishable from the invention glycoprotein. On the other hand, beta-parotin is a substance which can only be obtained by autolysing the parotid gland at 37' C. and it only contains about 3% of sugar. It thus is clearly different from the invention glycoprotein. The precipitated ineffective protein substances are separated from the mother liquor containing the glycoprotein of the invention by an optional solid-liquid separation operation such, for example, as filtration, centrifugation or decantation. A water-soluble zinc, salt is added to the resulting mother liquor and the precipitates of a complex of the zinc salt and protein are formed. The mucoid present in the saliva or salivary glands remains in the liquor. As the water-soluble zinc salt, any salt may be used so long as it is one in which a soluble chelate salt can be formed by chelating when the zinc in the zinc-protein complex is acted upon by the chelating agent, zinc salts conveniently used in the invention include zinc chloride, zinc bromide, zinc acetate and the like. The zinc salt is added to the mother liquor to make its zinc concentration in the latter becomes 0.01-0-05 mole. Normally, a 0.1-0.5 mole aqtieous solution of the zinc salt is added at a neutral pH in an amount one-tenth that of the mother liquor. The resulting zinc-protein complex precipitates are separated by an optional solid-liquid separation operation such, for example, as filtration or centrifugation. In this case, a water-miscible organic solvent such as acetone or methanol can be mixed for accelerating the sedimentation of the complex. Next, a chelating agent of zinc is added to act on the resulting precipitates and then the precipitates are redissolved in either water or other aqueous media. As the chelating agent of zinc, any of those known sueb, for example, as hydroxy acids, keto acids, keto-acids esters, beta-diketone, amino acids, nitrolotriacetic acid and ethylenediamine tetraacetic acid can be used. Chelating agents which are particularly suitable for use in this invention are sodium citrate, glycine and EDTA. It is, of course, to be desired that the chelating agent is added in an amount equivalent to or greater than the zinc. This is followed by submitting the resulting solution to dialysis to eliminate the chelate of zinc and thereby obtaining a dialysate containing the glycoprotein of the invention. The diaiysis, which is performed in accordance 3,522,229 4 with the method which is known per se, is carried out readily by dialyzing the redissolved solution obtained in the hereinbefore described step against running water throu.-h the intermediary of a dialytic membrane such as cellophane. The so obtained dialysate is a liquor containing mainly effective substances. For the purpose of collecting the effective substances in more purified forms, preferably after addition of a small amount of sodium chloride, a 10 water-miscible organic solvent such as acetone and ethanol is added to the dialysate to make the concentration thereof is adjusted up to 30%. Next, unnecessary precipitates formed by the addition of said solvent are eliminated by means of centrifugal separation and to the supernant 15 fluid is added the same organic solvent while the concentration thereof is adjusted between 30 to 60%, and in the consequence, the intended active substances of the present invention are obtained. In the case of using only submaxillary glands or mixed 20 saliva and/or mixed glands, it is preferred to add a sulfate of ammonium or an alkali metal while adjustin.- its concentration up to 0.5 saturation and collect the resulting precipitates. As the sulfate of alkali metals, useable are sodium sulfate and potassium sulfate, but the use 25 of ammonium sulfate is to be most preferred. The effective glycoprotein of the invention can be separated out as precipitates at this time while, on the other hand the other protein substances remain bebind in the soluti@n. Namely, in the case of the submaxillary gland, the liquid 30 extract contains the nervous growth promoting factor (NGF) and, as in the case of the invention -lycoprotein, the NGF is also separated as zinc complex precipitates. However, since NGF can be precipitates by fractional precipitation procediure with sulfate in the concentration 35 0.5 and 0.71 saturation (24-33 w./w. percent), but can not be precipitated in the concentration below 0.5 saturation and remained in the supernatant, it can be distinguished from this invention glycoprotein. Furthermore, NGF is distinguished from this invention substance in 40 respect of a simple protein not containing any sugar. When these precipitates obtained with the 0.5 saturation of a sulfate is dissolved in water, removed of its sulfate by dialyzing by the same technique as hereinbefore described, and thereafter the dialysate is frozen and 45 dried, the purified effective glycoprotein of the invention is obtained. However, the more purified glycoprotein can be obtained by adopting both said treatments with an water-missible organic solvent and said sulfate fractional procedure iri combination. 50 The yields of the invention purified glycoprotein from the various starting materials are, generally speaking, as follows: 100 mg. per liter from saliva, 1000 mg. -per kg. froul sublingual gland, 700 mg. per kg. from submaxillary gland and 100 mg. per kg. from parotid gland, there being 55 a tendency that the purified glycoprotein from the glands being some)what inferior to that obtained from saliva in purity and also slightly lower in activity. The foregoing glycoprotein obtained by the invention r-riethod is fully purified and moreover has a pronounced 60 gastric acid secretion inhibitory as well as anti--ulcerative activity and hence r-an be used for the various gastric disorders in a form suitable for oral administration or iiijection. Again, since this glycoprotein is readily water soluble, it can be further homogenized by the oustomary 65 techniques of purifying proteins. The most preferred techniques are the ionexchange chromatography, the gel filtration technique and the starch block electrophoresis, which may be used singly or in combination. When the purified glycoproteins obtained from various 70 starting materials are further more homogenized, homogenized glycoproteins similar activity and composition. The homogenized glycoprotein is a non-dialyzable high molecular weight glycoprotein having a molecular weight of above 150,000. Its elemental analysis values are: 75 H=7.68%, C=42.31%, N@9.15%, and it is coniposed
[3]3)522)229 5 6 of 34.9% sugar and 62.6% peptide. It has the maximum view point that the foregoing substances do not denionabsorption in the ultraviolet region at 280 m/.& and the exstrate the anti-gastric acid secretory and anti-ulcerative tinction coefficient at said wavelength is activities at all, it is clear that the invention glycoproE,'% .=4.67 tein is a novel substance. Further, no anti-gastric acid secretory or antiulcerative activity whatsoever is obThe isoelectric point measured by electrophoresis with 5 served in any of the parotins and their analogous subacetylcellulose mem@brane as carrier shows a pH of 2.6. stances or in NGF, too. Further, the analytical results of the sugars and amino When the gastric acid secretion and gastric ulceration acids composition are as shown in Table I. inhibitory effects of the glycoprotein obtained by the . ention method were investigaed by using pylorusTABLE I.-AMINO ACIDS AND SUGARS COMPOSITION OF 10 InV THE HOMOGENIZED NOVEL GLYCOPROTEIN ligated rats, it was found in the case of gastric secretion Amino Acid that by the administration of 500 Ag.lkg. of rat weight Sugar, of saliva-derived purified glycoprotein, there was a deM.mol./g. Mg./g. mg./g. crease, after 6 hours of ligature, of 67% iii the amount Lysine ------------------------------- 0.39 5o -- -------- 1,5 of gastric secretion, 76% in the free acidity and 35% in Histidine ----------------------------- 0.11 15 ------ ---- the total acidity, as compared with those of the control Arginine ----------------------------- 0.19 30 ------ ---- Aspal-atic acid plus Asparagine ------- 0.52 60 ------ ---- group. On the other hand, as regards the ulceration of Glutamic acid plus Glutamine ------- 0.37 41 ------ ---- the anterior region of the stomach, no ulcerations ocThreonine ---------------------------- 0.30 26 ---------- Serine -------------------------------- 0.67 89 ------ ---- curred at all even after 20 hours of ligature, as compared Prolin --------------------------------- 0.28 27 ------ ---- 20 viith the occurrence of -ulcerations in the control group alycine ------------------------------ 0.26 15 ------ ---- Alanine ------------------------------ 0.47 33 ------ ---- even after only 12 hours of ligature. Valine -------------------------------- 0.39 9 ---------- On the other hand, when 5 mg./kg. of salivary glandIsoleucine ---------------------------- 0.13 15 ---------- Leucine ------------------------------ 0.40 45 ------ ---- derived glycoprotein were similarly administered to pyTyrosine ----------------------------- 0. 15 24 ------ ---- lorus-ligated rats, tllere was a decrease, after 6 hours of Phenylalanine ------------------------ 0. 20 29 ---------- Cystine ------------------------------ 0. 08 is ---------- 25 ligature, of 76% in the amount of gastric juice, 88% in Tryptophan -------------------------- 0.12 22 ------- the ftee acidity and 44% in the total acidity, as comGalactose ---------------------------------------------------- d with the control group. Further, the occurrence of Fucose ---------------------------------------- --------------- 41 pare Glucosamine ----------------------------- -------------------- 55 ulcerations in the anterior region of the stomach was comSialic acid ------------------------------------- -------------- 8 - pletely inhibited even after 20 hours of ligature. Total -------------------------------- -------- 578 349 30 As a result of these experiments in which pylorusligated rats were used, it is clear that the purified glycoThe lipide and phosphorus contents of this glycoprotein is not more than 0.1% and it does not contain fats or nucleic acids. It tends to loose its activity in an alkaline solution, and its activity demonstrates a pronounced de- 35 cline when treated for three hours at a pH above 10. It is stable in an acid solution, no change at all being observed even though it is treated for three hours at a pH 1.5. It is also relatively stable against digestive enzymes. The invention glycoprotein is a novel substance differing 40 from the other known protein substances which are present in saliva and the salivary glands. As previously noted, parotin and the parotin-like substances such as S-parotin and salivaparotin, and NGF protein of saliva and salivary glands possesses a pronounced gastric acid secretion as well as ulceration inltibitory effects. Next, when the effect on the inhibition of restraint ulcer was examined by an experiment with rats, it is as shown in Table 11, that when saliva-derived purified glycoprotein was adrninistered in a dose of 2 mg./kg., the incidence of ulceration was 40% and most cases were of slight condition, as compared to the control group in which the incidence was 100%, all of which were of serious condition. Even with an administration of 0.5 mg./ kg., the incidence was 65%, most of which were of slight condition. TABLE II.-THE INHIBITORY EFFECTS OF PURIFIED GLYCOPROTEIN ON RESTRAINT ULCERS OF RAT Lesion I LeSion 2 Morphological Orders Incidence (Numbers of animals) Animal Number, Sample Dosag@s, mg./kg. B.W. Numbers percpnt....................................... Saline (Control) -------------------------------- 20 20 (100) 0 0 4 9 7 Purified Glycoprotein obtained from Saliva: 2.0 ------------------------- ----------------- 20 8 (40) 12 3 5 0 0 0.5 ------------------------- ----------------- 20 13 (65) 7 2 7 0 4 Purified Glycoprotein obtained from Glands: 20.0 ---------------------- ---------------- 20 6 (30) 14 2 2 2 0 5.0 ---------------------- -------------------- 20 12 (60) 8 5 5 2 0 1 Number and percentage of animals shonving at least one focalized lesion, ulcer or focus, accompanied with ificaut circulatory disturbances. Slgn 2 These signs are defined as follows: --No significant focalized lesions (ulcer, foci, hyperemia, edema, erosion and hemorrhagic coating). +-Slight fooalized lesion, significant hyperemia but not reacti hemorrhagic coating. ++-Significant focalized lesion (lio ulcer) hemorrhagic coating, below 1 mm. size. +++-Significant focalized lesion (ulcer) not expanded over V5 area of stomaeli. ++++-Significant focalized lesion (ulcer) expanded over Y5 area of stomach. are simple proteins, which differ also from the invention glycoprotein in respect of their isoelectric point. On the other hand, alpha-parotin and beta-parotin differ from the invention glycoprotein in respect of their isoelectric 65 point as well as sugar content. Aside from the foregoing parotins and NGF, it is known that saliva and salivary glands contain such enzymes as amylase, lyzozyme and protease and such biologically active substances as callicrein. However, all 7o of these are simple proteins not containing sugars and differ from the invention glycoprotein in their @,hemical properties. From the fact that the foregoing enzymatic and biological activities are not observed in the invention glycoprotein itself and, in contrast thereto, from the 75 On the other hand, in the case of the purified glycoprotein obtained from salivary glands, the incidence of ulcers was 30% by an administration of 20 mg./kg. and 60% by an administration of 5 mg./kg., both of which were slight cases. It is thus clear that the purified glycoprotein obtained from saliva as well as salivary glands demonstrates a pronounced effect in inhibiting the incidence of restraint ulcers. The novel glycoprotein of the present invention can be used in relief of various gastric diseases by incorporating a therapeutically effective amount thereof with a pharmaceutically acceptable carrier after dry freezing of the glycoprotein. The therapeutically effective amount is considerably varied depend on symptoms, bodyweight, administra-
[4]7 tion times and the like. Generally speaking, an amount ranging from 0.1 to 100 m.-. is optionally selected as one dosage. The novel glycoprotein of the present invention is applied to patients by means of oral administration or intramuscular injection. In the case of oral administration, the novel glycoprotein of the present invention is administered in a form of granules, tablets or pills prepared by employing lactose, starch, talc and the like as a carrier, or in a state charged into a -elatin capsule. In the case of intramuscular injection, frozen dry powder of the glycoprotein is dissolved into a physiological solution of sodium chloride or sterilized distilled water, and the solution is applied to patients. EXAMPLE 1 Ten liters of human saliva are centrifuged, following which its pH is adjusted to 5.4 by adding to its upper layer 5 N-HCI with stirring under cooling conditions and thereafter allowed to stand overnight in a refrigerator. The resulting precipitates are eliminatcd by centrifuging and, after adjusting the pH of the solution to 4.6 by a further addition of 5 N-HCI to its Lipper layer, it is allowed to stand overnight in a refrigerator. The resulting precipitates are eliminated and the solution is adjusted to a pH of 7 by adding to its upper layer 5 N-NAOH with stirring. One liter of a 0.3 mole solution of ZnCI2, is added to 10 liters of the foregoing solution with stirring under coolin.- conditions, after which the solution is allowed to stand overnight in a refrigerator. The resulting precipitates are separated by centrifugation. A fourfold amount of 1-3 % sodium citrate solution is added to these precipitates and extraction thereof is carried out for 2 hours. The liquid extract is centrifuged and to the precipitates, a further addition of a fourfold amount of 1% sodium citrate solution is made followed by extraction for one hour. Centrifugation is similarly carried out to separate liquid extract which, after being commingled with the first liquid extract, is poured into a cellophane tube and dialyzed against runnin,@ water for 48 hours. 1.25 grams of NaCl is added to 250 ml. of the dialysate followed by the addition of 107 ml. of acetone with stirring under c(>oling conditions to make the concentration 30%, after which the solution is allowed'to stand overnight. The resulting precipitates are eliminated by centrifuging and the supernatant is brought to a concentration of 60% by a further addition of acetone. The precipitates which form as a result of standing the supernatant overnight are separated by centrifugation. A saturated ammonium sulfate solution is added to a solution of these precipitates in a fivefold amount of water to bring the saturation of the solution to a 0.5 saturation (24 w./w. percent). The pH of the solution is then adjusted to neutral, after which it is left standing ovemight in a refrigator. The precipitates which have deposited are collected by either centrifugat-ion or filtration and, after being dissolved in a small quantity of water, the solution is placed in a cellophane tube and dialyzed against distilled water. When, after several changes of the outer liquid, the dialysate is lyophilized, a salivaderived glycoprotein powder is obtained in an amount of one gram. For homogenizing this purified glycoprotein powder, any of the various techniques that are used customarily in purifying proteins can be employed. If, by way of illustration, one is described, the followin.- method is useful. After thorougbly washing 30 grams of a DEAE-cellulose with distilled water, the DEAE-cellulose is packed in a 4 x 40 cm. column and buffered by passing a phosp oric acid buffer solution of a pH 7.4 and finally a 0.01 mole phosphoric acid biiffer solution. One gram of the aforesaid piirified glycoprotein powder is dissolved in a small qliantity of a 0.01 mole phosphoric acid buffer solution and charged to the column, following which a 0.01 mole phosphoric acid buffer solution and then a 0.05 mole phosphoric acid buffer solution are 3;522;229 8 passed through the column, and the unexchanged or eluted non-effective substances are removed. Now, when a 0.05 mole phosphoric acid buffer solution to which has been added 0.5 mole of NaCl is passed through the colunin, the effective substance will be eluted. This eluted solution is separately collected with a fraction collector into 5 ml. portions and the optical absorption at 280 ml& of these portions are determined. When only those portions showing a protein signal peak are collected, 10 concentrated under reduced pressure, dialyzed and thereafter the dialysate is lyophilized, first-stage purified powder of the glycoprotein is obtained. Next, after thoroughly swelling Sephadex G-75 (crosslinked dextran) with water, it is packed in a 2.5 x 50 cm. 15 column. After the Sephadex has been fully equilibrated by passing a 0.05 M NH4HCO3 solution through the column, a solution in which 120 mg. of the glycoprotein powder purified by the hereinbefore described ion-exchan.-C chromatographic method is dissolved in a small 20 quantity of a 0.05 M NH4HCO3 solution, is charged and developed and eluted with the same NH4HCO3 solution. When the eluted solution is separately collected every 3 ml. portions and those exhibiting the characteristic protein peak of absorptions at 280 mu are collected, 25 they can be divided into three components. Since the effective substance is only contained in the first component, this portion only is concentrated under reduced pressure and frozen and dried, and thereby obtained purified powder of the glycoprotein in the second-stage. 30 Next, as the third stage, after repeatedly washing about 400 grams of starch first with N-HCI and then with distilled water, the starch is buffered thoroughly in 0.05 M veronal buffer at a pH 8.6 followed by repeated stirring and decantations to make it in a pasty mass. By 35 pouring the so obtained pasty mass into a 1.5 x 7 x 40 cm. cell, a starch block is prepared. One hundred rng. of the purified glycoprotein powder obtained in the hereinbefore described in the second stage are dissolved in a small quantity of veronal buffer 40 followed by adding starch thereto to form a pasty mass. This pasty mass is embedded in the middle of the starch block, and the starch block electrophoresis is carried by either the horizontal or vertical method by passing an electric current using veronal buffer as the electrode 45 solution. After conducting the electrophoresis in a low temperature chamber for 20 hours with 2 ma./CM.2, the starch block is cut into pieces 0.5 cm. in width, and each of the small starch pieces is extracted with 10 ml. of 5o water. After removing the starch by filtration, the absorption at 280 mu of the filtrates is determined. Those portions exhibiting the characteristic protein peak are collected. The active substance is only contained in the liquid 55 extraction obtained froni the starch block 5-7 cm. in the plus direction from the origin. Hence, when this portion is concentrated under reduced pressure dialized and the dialysate is frozen and dried, the purified powder of glycoprotein in the third-stage is obtained. 60 This purified product exhibits a single migratory figure during the electrophoresis with polyacrylamide gel as carrier and thus a homogeneous glycoprotein is obtained. The yield from one gram of the salvia-derived purified 65 gylcoprotein is 50 mg. (5 mg. per liter of salvia) and the rise in the antigastric acid secretory activity is ten times. EXAMPLE 2 70 Fifty liters of saliva from which residue has been removed by centrifugation are concentrated to 10 liters under reduced pressure at 30' C. Ten liters of this concentrated saliva are then treated as in Example I to obtain 15 grams of the saliva-derived purified glyco75 protein.
[5]9 3)5 22)229 10 The homogenization operation is carried out as in Example 1. EY.AMPLE 3 To 100 liters of saliva (or 20 liters of concentrated saliva) are added 200 liters of acetone (40 liters i'in the 5 case of concentrated saliva), and after standing overnight the precipitates are separated by centrifugation Two hundred grams of the so obtained dry powder are extracted with 10 liters of water (or 0.9% NaCl solution) for 3 hours while maintaining the pH in the 10 neighborhood of 8. EXAMPLE 4 Salivary glands of animals (parotid, submaxillary and sublingual glands), after being removed of their fat and 15 other tissues, are ground, following which one kg. of the ground glands is extracted with a fivefold amount of water (or @0.9% NaCl solution) for 3 hours while maintaining the pH in the neighborhood of 8 with NAOH solution followed by centrifugation to ol@tain a liquid 20 extract. The operation described in Example I are carried out on this liquid extract. Similar results are also obtained when an acetonedried powder of the glands is used instead of the ground 25 glands. The yield and the rise in activity of the glands-derived purified glycoprotein powder after fractional precipitation as well as those of a homogenized glycoprotein powder obtained by carrying out the additional three-stages 30 purification operation as in Example 1 are shown in Table Ill. TABLE III.-YIELD AND ACTMTY OF THE NOVEL GLYCOPROTEIN OBTAINED FROM SALIVARY GLANDS Novel Glycoprotein 35 Purified Homogenized Yield t ActiVity 2 Yield I ActiVity 2 Glands (-g-) (Ig.) (-g) (tg.) Parotid ------------ 100 1, 000 0.6 io 40 Subm,,lxillary ------- 700 1,000 3.5 10 Sublingual --------- 1, 000 1,000 5.0 10 I Per kilogram of glands. Per 200 grarns body weight of rat, minlinum effective dose of antisocretory activity in the pylorus-ligation method. We
