HK1026708B - A preparation derived from shark cartilage for treatment of diseases related to excessive phf or excessive intracellular calcium - Google Patents

Description

Preparation from shark cartilage for treating diseases related to PHF excess or intracellular calcium excess

Technical Field

The present invention relates to anti-parathyroid hypertensive factor (anti-PHF) derived from shark cartilage. The compounds of the invention are useful in the treatment of hypertension and other diseases associated with increased intracellular calcium (e.g., non-insulin dependent diabetes mellitus, atherosclerosis, congestive heart failure, cancer (including breast, colon, renal, and leukemia), inflammatory bowel disease, and asthma).

Background

Hypertension generally refers to arterial systolic and/or diastolic blood pressure above the normal level of 140/90 mmHg. Diseases associated with hypertension include atherosclerosis, hypertensive renal failure, stroke, congestive heart failure and myocardial infarction. Although many treatments have been found to be effective in reducing arterial blood pressure, the cause of essential hypertension is still not well understood. Genetic induction leading to hypertension is generally accepted, but a number of various drugs that have been found to be effective in treating hypertension and the fact that these drugs appear to act by eliciting different pharmacological responses, suggest that essential hypertension may have different primary causes.

Many studies have suggested that one or more circulating factors may play a role in the generation or maintenance of hypertension. [ see, Wright et al, hypertensive substances found in the blood of spontaneously hypertensive rats, Life sciences (Life Sci.)1984, 34: 1521-1528; dahl et al, humoral transmission of hypertension: evidence from xenogeneic symbiosis, cyclic studies (circ. res.)1969, 24/25 (supplement I): 21-23; greenberg et al, evidence of circulating factors as a cause of venous overgrowth in spontaneously hypertensive rats, am.j. physiol.1981, 241: H421-H430; tobian et al, circulating fluid pressor in Dahl S rats with salt hypertension, clinical science (clin. sci.)1979, 57: 345s-347 s; zidek et al, humoral factor in the pathogenesis of primary hypertension, klin. wochenschr.1985, 63 (supplement II) D: 94-96; hirta et al, Hypertension-producing factor in the serum of rats with Dahl salt-sensitive Hypertension (Hypertension)1984, 6: 709-716]. For example, in xenobiotic and cross-cycling tests, an increase in blood pressure in normotensive animals can be induced by exposing the blood of a normotensive animal to the blood of a hypertensive animal. Subcutaneous injection of erythrocyte-associated factor from Spontaneously Hypertensive Rats (SHR) into normotensive Wistar-Kyoto (WKY) rats induced hypertension in the WKY rats, and elevation of blood pressure in normotensive salt-sensitive Dahl rats was also induced by injection of serum from Dahl rats with salt-sensitive hypertension.

There have been some reports of circulating factors in hypertensive rats and hypertensive patients, which increase intracellular calcium. [ see, Banos et al, two factors involved in the increased calcium uptake by platelets in essential hypertension patients, Clin. exp. hypertens.1987, 9: 1515-; zidek et al, the effect of plasma from hypertensive individuals on calcium transport in human permeable neutrophils, J.J., 1988, 74: 53-56; linder et al, effect of circulating factors on intracellular free calcium in normal platelets in patients with essential hypertension, n.eng.j.med.1987, 316: 509-; the increase in plasma free calcium in platelets in Bruschi et al, spontaneously hypertensive rats and essential hypertensive patients, in 1985, 68: 179-184; wright et al, spontaneous hypertensive rat erythrocyte extract with hypertensive properties stimulation of calcium uptake by aortic tissue, Can, j.physiol.pharmacol.1986, 64: 1515-1520]. Since vascular tone is affected by intracellular calcium levels, there may be a link between factors that elevate blood pressure and factors that increase intracellular calcium. Evidence has accumulated in this regard suggesting the involvement of calmodulin in certain forms of hypertension [ see: l.m.resnick, am.j.med.82 (supplement IB), 16 (1987). Parathyroid hormone (PTH) is a calcineurin. 30% or more of essential hypertensive patients are a group of hypertensive patients characterized by elevated levels of immunoreactive parathyroid hormone (ir-PTH). [ see: laragh et al, Kidney int.34 (supplement 35), S162(1988) ]. For SHR rats, elevated PTH levels have been reported [ see: McCarron et al, "hypertension", 3 (suppl 1), 1162(1981), "it has also been observed that patients with hyperparathyroidism often exhibit hypertension, the severity of which is in most cases alleviated by parathyroidectomy [ see: hellstrom et al, Brit.J.Urol.30, 13 (1958). Similar results have been reported for parathyroidectomy in SHR rats. [ see: schleiffer et al, Jap. circle. J.45, 1272 (1981). Various investigators have suggested that PTH is associated with the development of essential hypertension, although exogenous administration of PTH causes a decrease in blood pressure in mammals and other vertebrates [ see: pang et al, Gen. Comp. Endocrinol.41, 135(1980) ]. The vasodilatory effect of PTH is also associated with a specific region of the molecule that is different from the region where hypercalcemic effects are produced [ see: pang et al, Endocrinology, 112, 284(1983) ]. PTH has also been shown to inhibit calcium entry into vascular smooth muscle [ see: pang et al, Life sciences 42, 1395(1988), PTH inhibits calcium passage into vascular smooth muscle through L-type calcium [ Wang et al, FEBS, Vol.282, No.2, p.331-334 (1991) ]. This argument is further supported by the fact that hypertensive patients with elevated PTH levels also exhibit reduced serum ionized calcium levels [ see: resnick et al, New engl.j.med., 309, 888 (1983); hvarfner et al, actaMed. Scand.219, 461 (1986). It is expected that serum ionized calcium levels will be elevated if PTH levels are elevated primarily.

The presence of circulating factors in the blood of SHR rats was confirmed by a study report published in am.j. hypertens, 2, 26-31 (1989). These studies showed that when plasma from SHR rats was injected into normotensive ratsIn rats (whether by infusion or bolus injection), WKY and SD rats had elevated blood pressure. In addition, the rat tail artery sections also showed that the rat tail artery pairs increased with increasing SHR plasma concentration in the buffered medium⁴⁵Ca absorption in vitro is also increased in a dose-dependent manner. The results of these experiments clearly show that the increase in blood pressure and the increase in calcium uptake by the cells is a dose-dependent manner, the magnitude of which depends on the amount of SHR plasma present in and available to the system. Unexpectedly, the onset of these two events is delayed and progressive, but known endogenous pressor agents such as norepinephrine, angiotensin II, and vasopressin have been observed to increase blood pressure very quickly following administration. Known endogenous pressor agents begin to increase blood pressure and increase cellular calcium uptake about 1-2 minutes after administration, whereas parathyroid hypertensive factor has a 20-30 minute delay before exerting this effect. Another result observed in these studies was that when the infusion of SHR plasma was stopped and replaced with plasma from normotensive rats, the blood pressure quickly dropped to basal levels. The observed drop excludes simple volume effects. In a related experiment, dialyzed plasma from hypertensive patients was infused into normotensive SD rats, and the results showed that SD rats developed hypertension. Plasma from those patients also increased calcium uptake in vitro in the tail artery of rats. Dialyzed plasma from normotensive patients did not produce a significant increase in blood pressure.

The source of circulating factors is unknown, but the unmet report of elevated PTH levels in hypertensive rats suggests that parathyroid glands may be the target of the study. It was found that parathyroidectomies in SHR rats reduced blood pressure, and that plasma from SHR rats whose parathyroidectomies had been excised did not cause an increase in blood pressure in normotensive rats. In contrast, transplantation of parathyroid glands from SHR rats into normotensive Sprague-Dawley (SD) rats resulted in an increase in blood pressure and the appearance of circulating factors in the plasma, as shown by the infusion of isolated plasma into other normotensive rats. [ Pang and Lewanczuk, Amer.J. Hypertens.2, 898(1989) ].

On the basis of these studies, it was established that the parathyroid gland is the source of the circulating factor, and the term "parathyroid hypertensive factor" or PHF was suggested to be used to describe substances that cause elevated blood pressure.

The isolation and purification of circulating factors derived from parathyroid glands has been carried out in SHR rats and in many patients with essential hypertension and is also the subject of related patent application No.603745 filed on day 11, 21 of 1990 which is in turn part of the subsequent application of the now-abandoned patent application No.327, 450 filed on day 3, 22 of 1989. The disclosures of these related patent applications, including the teachings regarding the purification of parathyroid hypertensive factor, are incorporated herein by reference.

As described in the above-mentioned related patent applications, PHF has been shown to regulate extracellular calcium absorption and to be inhibited by increased dietary calcium levels. PHF has been isolated and methods of screening for PHF using antibodies against PHF have also been described. The molecular weight of PHF is about 2700 daltons, and PHF has the property of delaying the increase in blood pressure, which is temporally associated with the increase in the absorption of extracellular calcium by vascular smooth muscle, after administration to normotensive rats. Biopsy data have shown that the factor is substantially similar from human and rat.

The overgrowth of blood vessels is associated with the pathophysiology of many cardiovascular diseases, including essential hypertension. Vascular smooth muscle proliferation may be the cause of excessive blood vessel growth and increased vascular tone. It has been reported that PHF promotes vascular smooth muscle cell proliferation by a mechanism independent of intracellular calcium regulation (Shan et al, Abstract of 17 th national institute of science, Amsterdam, 1998, 6 months 7-11 days).

The inventors of the present invention have discovered antagonists of PHF. The inventors have surprisingly found that shark cartilage can act as a PHF antagonist, causing a drop in blood pressure and affecting the regulation of intracellular calcium. Shark cartilage is known in the art to contain substances that inhibit tumorigenesis (Lee et al, science, Vol.221, 1185-1187 (1983)) and an anti-inflammatory component (Schinitsky, U.S. Pat. No. 4,473,551). The present inventors also found that shark cartilage extract inhibited VSMC proliferation in SHR rats or WKY rats induced by PHF. In view of this, the shark cartilage extract of the present invention is expected to be useful for the treatment of hypertension and other diseases associated with intracellular calcium elevation.

Detailed description of the invention

The present inventors have found that an extract prepared from shark cartilage reduces blood pressure. It is believed that the shark cartilage extract contains a parathyroid hypertensive factor antagonist which binds to the parathyroid hypertensive factor site but does not activate parathyroid hypertensive factor activity.

The shark cartilage extract of the present invention can be obtained from commercially available shark cartilage by further purification, first washing, drying and grinding the commercially available shark cartilage to a fine powder. The milled, dried shark cartilage powder is first extracted with water at 4-120 deg.C (preferably 95 deg.C) for 2-4 hours (preferably 2 hours). The ratio of solute to solvent is 1: 8-1: 12. The resulting suspension was then cooled to 40-60 deg.C (preferably 50 deg.C) and centrifuged at about 5200-. The supernatant (#1) contained about 8% solids and was stored in a refrigerator at 4-8 ℃ while the pellet fraction was subjected to a second extraction. In the second extraction, the precipitate is extracted with water at 4-120 deg.C (preferably 95 deg.C) for 2-4 hours (preferably 2 hours). The ratio of solute to solvent is 1: 4-1: 6 (based on the raw materials). The resulting suspension was then cooled to 40-60 deg.C (preferably 50 deg.C) and centrifuged at about 5200 and 5700rpm to separate it into a supernatant and a pellet fraction. The supernatant was combined with the supernatant from the first extraction and spray dried to obtain the purified shark cartilage extract of the present invention. Said extraction step is carried out at 95 ℃ for 2 hours, wherein a decanter centrifuge is used in said centrifugation step. The process further comprises concentrating the combined supernatants until a solids content of 8-10% is achieved.

The extract of the present invention contains 5-30% of protein, 15-80% of mucopolysaccharide and 1-20% of chondroitin sulfate C. In a pharmaceutical composition for treating hypertension comprising shark cartilage extract, the composition is produced in an amount of 0.1-20mg shark cartilage extract/kg body weight.

The extract of the invention can be administered to a warm-blooded animal in need of such treatment by parenteral, topical, oral or rectal route or by inhalation. The extract can be formulated into parenteral or oral dosage forms by compounding the extract with conventional carriers, excipients, binders, preservatives, stabilizers, colorants, and the like, which are accepted in pharmaceutical practice.

For parenteral administration, 1-10ml of intravenous, intramuscular or subcutaneous injection may be given 1-4 times per day. The injection solution used may contain a shark cartilage extract according to the invention dissolved in an isotonic sterile aqueous solution, or a suspension with or without a preservative such as phenol or a solubilizing agent such as ethylenediaminetetraacetic acid (EDTA). Those pharmaceutically acceptable carriers and solvents which can be used are water, Ringer's solution and isotonic NaCl solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Synthetic mono-and diglycerides, fatty acids (e.g., oleic acid) have been found to be useful as fixed oils in the preparation of injectable formulations.

For rectal administration, the extracts may be formulated in the form of suppositories by mixing with suitable non-irritating excipients such as cocoa butter or polyethylene glycols.

For topical application, the extract may be formulated as an ointment, gel, solution, suspension, or skin patch.

The extract in the powder aerosol can be administered by Spinhaler turbo-inhaler from Fisons corporation (Bedford, Massachusetts) at a rate of about 0.1-50 mg/capsule, for average humans, 1-8 capsules per day. The extract in the liquid aerosol can be administered at a ratio of about 100-1000mg/puff, or the release can be activated with a standard volume of propellant. The liquid aerosol may be administered in an amount of 1-8 puffs per day and the dosage may vary depending on the severity of the condition being treated, the weight of the patient, the distribution of particle sizes in the aerosol, and the like. Fluorinated hydrocarbons or isobutane can be used as propellants for liquid aerosols.

The daily dose administered is about 0.01-200mg/kg body weight (preferably 1-10mg/kg body weight), depending on the activity of the particular compound, the age, body weight, sex and condition of the individual to be treated, the type and severity of the condition, the frequency and route of administration. As is well known, the amount of active ingredient that may be combined with a carrier material to produce a single dose will vary depending upon the host treated and the particular mode of administration.

The shark cartilage extract of the present invention may also be combined with known drugs effective in treating the condition. For example, for the treatment of hypertension, shark cartilage extract can be combined with known antihypertensive agents such as calcium channel blockers (e.g., verapamil, nifedipine, and azone ).

In addition to the treatment of essential hypertension, the extracts of the invention may also be used to treat other diseases (which include, but are not necessarily essential to hypertension). For example, non-insulin dependent diabetes mellitus is often accompanied by hypertension. Conversely, hypertensive patients often also exhibit impaired glucose tolerance. Accordingly, shark cartilage extract is expected to be useful in the treatment of hypertension and other diseases associated with elevated intracellular calcium levels.

The invention includes the isolation, characterization and synthetic preparation of the active ingredient of shark cartilage extract.

The following examples illustrate the invention but are not intended to limit the invention thereto. Various modifications may occur to those skilled in the art without departing from the scope of the invention.

Example 1

Extraction of shark cartilage

Clean, dry, powdered shark cartilage was purchased. The dry powder form of shark cartilage was first extracted with water at 85-90 deg.C for 2 hours at a solute to solvent ratio of 1: 8. The resulting suspension was then cooled to 50 ℃ and centrifuged at 5200rpm (3245g) to separate the suspension into a supernatant and a pellet. The supernatant, containing about 8% solids, was placed in a refrigerator at 4 ℃ while the resulting pellet was subjected to a second extraction. In the second extraction, the precipitate was extracted with water at 95 ℃ for 3 hours. The ratio of solute to solvent was 1: 4.8 based on the raw materials. Subsequently, the resulting suspension was cooled to 50 ℃ and centrifuged at 5200rpm (3245g), and the suspension was separated into a supernatant and a precipitate. This supernatant, containing about 3% solids, was combined with the supernatant from the first extraction and spray dried to obtain the purified shark cartilage extract of the present invention.

Example 2

Effect of injecting shark cartilage extract concentrated bolus (1mg/kg) on SHR and SD rats

6 Spontaneously Hypertensive Rats (SHR) and 3 Sprague-Dawley (SD) rats were injected intravenously with a concentrated bolus of shark cartilage extract designated DF I-40. 5 Spontaneously Hypertensive Rats (SHR) and 3 Sprague-Dawley rats were intravenously injected with a concentrated bolus of shark cartilage extract designated DF II-40. The shark cartilage extract is administered at a dose of 40mg/kg body weight. Blood pressure was measured 90 minutes after injection, and as shown in FIG. 1, shark cartilage extract had no effect on SD rats, but decreased blood pressure in SHR rats.

Example 3

Effect of shark cartilage extract on SHR and SD rats by gavage

Three groups of SHR were gavaged with three different doses of shark cartilage extract (10, 20 and 40mg/kg) designated DF II-53. 11 rats were administered 10mg/kg body weight shark cartilage extract, 4 rats were administered 20mg/ml body weight shark cartilage extract, and 4 rats were administered 40mg/kg body weight shark cartilage extract. Blood pressure was measured after 90 minutes. As shown in figures 2, 2a and 2b, all rats showed a decrease in blood pressure and were dose-dependent. In rats fed higher doses (20-40mg/kg body weight), the rate of blood pressure decline was higher, with the maximum decline occurring at about 50-60 minutes (FIG. 2 a). After 50-60 minutes, fluctuations in blood pressure occurred, probably due to the blood pressure regulation mechanism in rats.

Example 4

Effect of PHF on blood pressure in SD rats in the Presence or absence of shark cartilage extract

7 SD rats were administered 1ml equivalent of PHF by intravenous bolus injection. After administering 1ml equivalent of PHF by intravenous bolus injection to 6 SD rats 10 minutes, shark cartilage extract (DF II-53) was administered in an amount of 40mg/kg body weight. Blood pressure was measured 90 minutes after injection and as shown in figure 3, PHF produced a delayed increase in blood pressure and shark cartilage extract counteracted this response.

Example 5

Effect of PHF on Vascular Smooth Muscle Cell (VSMC) proliferation in the presence or absence of shark cartilage extract.

The tail arteries of male West Kyoto (WKY) rats and Spontaneously Hypertensive Rats (SHR) (100-200g body weight) were dissected out and immersed in cold Ca and Mg-free Hanks Balanced Salt Solution (HBSS) (Gibco, Grand Island, NY). The tail artery was digested twice sequentially with HBSS enzyme solutions I and II. Each digestion lasted 1 hour. HBSS enzyme solution I containing CaCl₂(0.2mM), collagenase/dispase (1.5mg/ml) (Boehringer Mannheim GmbH, WestGermany), elastase (type I, 0.5mg/ml) (Sigma Chemical Co., St. Louis, Mo.), trypsin inhibitor (type I, 1mg/ml) (Sigma Chemical Co., etc.), and Bovine Serum Albumin (BSA) (fatty acid-free, 2mg/ml) (Sigma Chemical Co., etc.). HBSS enzyme solution II contained collagenase (type II, 1mg/ml) (Sigma Chemical Co.), trypsin inhibitor (0.3mg/ml) and BSA (2 mg/ml). The cell suspension was then seeded onto 96-well flat-bottom tissue culture plates (the DM) in DMEM mediumFCS 10% supplemented in EM) and 5% CO at 37 deg.C₂For 36 hours, so that the cells were attached to the bottom of the plate. The medium was changed to DMEM containing 0.4% FCS to allow the cells to rest for 2-4 days. This step places the cells all at G₀-G₁And (5) a boundary period. PHF and shark cartilage extracts were dissolved in DMEM containing 10% FCS. PHF was added to the resting cells alone or together with shark cartilage extract. After 36 hours of incubation, the cells were washed with³H-Thymidine (0.2/hole) pulse mark, and continued incubation for 24 hours. The medium was then discarded and the cells were washed twice with HBSS followed by incubation with 0.1% trypsin for 15-30 minutes at room temperature. The cells were then collected on filter paper using a cell harvester. The amount of radioactivity incorporated into the cells was determined using a liquid scintillation counter. As shown in fig. 4, PHF stimulated cell proliferation of VSMC in WKY rats. FIG. 5 shows that the VSMC stimulating effect of PHF in WKY rats was inhibited by shark cartilage extract. FIG. 6 shows that shark cartilage extract inhibited VSMC proliferation in SHR rats.

Example 6 chemical composition of shark cartilage extract

(1) Determination of protein content

The total protein content was determined using the BCA method. BCA protein assay reagents were purchased from PIRRCE. A standard curve of standard protein of known concentration was prepared using BSA (bovine serum albumin) standard solution provided by the BCA protein assay kit. The 24 glass tubes were arranged in three rows and seven columns for loading of standard samples, and four additional tubes were used for calibration of the spectrophotometer. To each tube in the first row were added 95, 90, 80, 70, 60, 50, 40 and 30. mu.l of 0.9% NaCl solution, respectively. The same steps are repeated for each tube of the second and third rows. To each tube of the first row containing 0.9% NaCl, 5, 10, 20, 30, 40, 50, 60 and 70. mu.l of standard protein (provided by the kit, at a concentration of 2mg/ml) were added, respectively. The same steps are repeated for the second and third rows. Then 2ml of working reagent, a mixture of 50 parts of reagent A and 1 part of reagent B, was added to each tube. All samples were mixed well and incubated at 37 ℃ for 30 minutes. The amount of protein was determined by measuring the absorbance at 562nm with a spectrophotometer (model PU 8620UV/VIS/NIR, Philips). The mean value of each standard concentration was calculated and a standard curve was drawn using the Analysis of the Regression Line No.5 (pharmacy computing System-version 4.2A). The standard curve was used to determine the protein concentration of each unknown sample.

A1% solution of shark cartilage extract was prepared using Double Distilled (DD) water. The protein concentration (mg/ml) of the sample was calculated using a standard curve, and the percentage of shark cartilage protein was calculated according to the following calculation:

protein% (w/w) ═ sample protein concentration (mg/ml) × dilution factor (2.5) ×

Sample concentration (10 mg/ml). times.100

In order to obtain accurate data of the standard curve and the shark cartilage sample, the step of constructing the standard curve and the step of measuring the protein content of the shark cartilage extract are carried out simultaneously, and the working reagent is finally added into each tube of the standard protein sample and the shark cartilage sample.

The protein content was 15.11 (2.79%) in a total of 16 batches of shark cartilage extract.

(2) Measurement of mucopolysaccharide

The methods adopted were adapted to the methods of P.Whiteman (biochemistry.J.131: 351-. Standard sample chondroitin sulfate C, code No. C-4384, Lot No.21H0103, was purchased from Sigma chemical.Co. A standard or sample was prepared by dissolving 10mg of chondroitin sulfate C or shark cartilage extract in 50ml of DD water. 20mg of Aleian Blue 8GX was dissolved in 20ml of buffer (5.07 g of MgCl in 500ml of water)₂And 3.4g of sodium acetate) and 0.2ml of acetic acid. A series of samples containing 40-200. mu.g of shark cartilage extract in 1ml were added to 50ml plastic tubes, respectively. To these tubes 1ml of the reaction reagent was added. The mixture was equilibrated at room temperature for 2 hours with stirring. Adding 20ml of 95 ℃ ethanol, and then separatingAnd (4) a heart. After decanting the supernatant, 3ml of 0.2M CaCl were added to the pellet₂. By measuring precipitated CaCl₂The light absorption of the solution at 620nm determined the mucopolysaccharide content.

The mucopolysaccharide content was 50.33 (2.25%) in a total of 6 batches of shark cartilage extract.

(3) Isolation and determination of chondroitin C

Methods adapted to the method of l.roden et al (Methods in Enzymology) (1972), stage 28, carbohydrate complex, part B, edited by v.ginsburg) were used. Amberlite IR-120 was purchased from Sigma Chemical Co⁺Numbered IR-120⁺. A calcium acetate buffer was prepared by adding 1.2L of DD water to 62.5 g of calcium acetate, and adjusting the pH to 4.5 with 35.5ml of glacial acetic acid. 2 g of shark cartilage extract was added to 400ml of calcium acetate buffer contained in a 2-liter glass flask, and the sample solution was heated in a water bath at 37 ℃ for 20 minutes and then cooled to room temperature. Ethanol (100%, 116.25ml) was added very slowly to the sample solution at room temperature, and the solution was stirred vigorously while adding ethanol. The flask was placed in a 4 ℃ bath for 3 hours, then centrifuged at 11000rpm (19000g) at 4 ℃ for 15 minutes, and the precipitate was dissolved in DD water and lyophilized. The supernatant was allowed to warm to room temperature and 80ml (100%) ethanol was slowly added thereto with vigorous stirring. The flask was again placed in the 4 ℃ bath overnight with slow stirring. The solution was centrifuged (11000rpm) for 15 minutes at 4 ℃. The second precipitate was dissolved in DD water and lyophilized. The supernatant was warmed to room temperature and 100ml ethanol was added slowly with vigorous stirring. The flask was again placed in the 4 ℃ bath overnight with gentle stirring. Then centrifuged at 11000rpm for 15 minutes. To the third precipitation 125ml DD water was added and loaded onto Amberlite IR-120⁺(Na⁺Type) column (2.5X 16cm, about 60g of Amberlite IR-120⁺) The above. The column was washed with 75ml of DD water. After adding 1.168g NaCl to make the solution 0.1M, it was eluted with 3 volumes (600ml) of absolute ethanol under vigorous stirring. Again, the flask was placed in a 4 ℃ bath overnight and then centrifuged at 11000rpm at 4 ℃The last precipitate was dissolved in DD water for 15 min and lyophilized. The weight of the last precipitate represents the amount of chondroitin sulfate C.

The chondroitin sulfate C content in 2 batches of shark cartilage extract was 5.9 (1.98%).

Brief Description of Drawings

FIG. 1 shows the results of intravenous injections of concentrated pellets of shark cartilage extract into SHR and SD rats. As shown in FIG. 1, shark cartilage extract had no effect on SD rats, but decreased blood pressure in SHR rats.

FIG. 2 shows the results of feeding shark cartilage extract to SHR rats by gavage. As shown in FIG. 2, shark cartilage extract decreased blood pressure in all rats.

Figures 2a and 2b show that blood pressure drops in a dose-dependent manner and reaches a minimum at about 50-60 minutes.

FIG. 3 shows that PHF delays the increase in blood pressure and that shark cartilage extract counteracts this response.

FIG. 4 shows that PHF stimulates VSMC proliferation in WKY rats in a dose-dependent manner. At 0.625X 10^-3、1.25×10^-3And 2.5X 10^-3At doses of the absorbed units, PHF increased cell proliferation by 120 (8.5%, P < 0.05, n-16), 137.91 (12%, P < 0.01, n-16) and 181.9 (14.3%, P < 0.05, n-16), respectively.

FIG. 5 shows the effect of PHF on VSMC proliferation in WKY rats in the presence of shark cartilage extract. At a dose of 50g/ml, shark cartilage extract significantly inhibited PHF-induced VSMC proliferation.

FIG. 6 shows the effect of shark cartilage extract on VSMC in SHR rats. At doses of 5, 50 and 500g/ml, shark cartilage extract inhibited VSMC proliferation in a dose-dependent manner.

Claims (14)

1. A shark cartilage extract having activity against parathyroid hypertensive factor, wherein the shark cartilage extract is prepared by the steps of:

extracting clean, dried and powdered shark cartilage with water at 85-120 deg.C for 2-4 hr,

the resulting suspension was cooled to 40-60 c,

the cooled suspension was centrifuged at 5200-,

placing the supernatant fluid 1 in a refrigerator at 4-8 ℃,

extracting the precipitate with water at 95-120 deg.C for 2-4 hr,

the resulting suspension was cooled to 40-60 c,

the cooled suspension was centrifuged at 5200-,

combining supernatant 1 and supernatant 2, and

the combined supernatants were spray dried to obtain the shark cartilage extract described above.

2. The extract according to claim 1, wherein said extract comprises 5-30% protein, 15-80% mucopolysaccharide and 1-20% chondroitin sulfate C.

3. Use of the shark cartilage extract according to claim 1 for the preparation of a pharmaceutical composition for the treatment of hypertension.

4. The use according to claim 3, wherein the composition is produced in an amount to be administered of 0.1-20mg shark cartilage extract per kg body weight.

5. Use of a shark cartilage extract according to claim 1 for the preparation of a pharmaceutical composition for the treatment of a disease associated with an excess of parathyroid hypertensive factor.

6. Use of the shark cartilage extract according to claim 1 for the preparation of a pharmaceutical composition for the treatment of a disease associated with intracellular calcium elevation.

7. A pharmaceutical composition comprising a shark cartilage extract having anti-parathyroid hypertensive factor activity according to claim 1 and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition comprising a shark cartilage extract having anti-parathyroid hypertensive factor activity according to claim 1, an antihypertensive substance, and a pharmaceutically effective carrier.

9. Use of a shark cartilage extract according to claim 1 for the preparation of a pharmaceutical composition for counteracting the activity of parathyroid hypertensive factor.

10. A process for preparing a shark cartilage extract having anti-parathyroid hypertensive factor activity according to claim 1, comprising the steps of:

extracting clean, dried and powdered shark cartilage with water at 85-120 deg.C for 2-4 hr,

the resulting suspension was cooled to 40-60 c,

the cooled suspension was centrifuged at 5200-,

placing the supernatant fluid 1 in a refrigerator at 4-8 ℃,

extracting the precipitate with water at 95-120 deg.C for 2-4 hr,

the resulting suspension was cooled to 40-60 c,

the cooled suspension was centrifuged at 5200-,

combining supernatant 1 and supernatant 2, and

the combined supernatants were spray dried to obtain the shark cartilage extract described above.

11. The method according to claim 10, wherein said extracting step is carried out at 95 ℃ for 2 hours.

12. The method of claim 10 wherein a decanter centrifuge is used in said centrifuging step.

13. The method according to claim 10, further comprising concentrating the combined supernatants until a solids content of 8-10% is achieved.

14. Use of the shark cartilage extract according to claim 1 for the preparation of a pharmaceutical composition for inhibiting vascular smooth muscle cell proliferation.