US3893995A - Method for obtaining polyfructosans - Google Patents

Abstract

A kidney function diagnostic agent comprising high water-soluble polyfructosans derived from the storage organs of monocotyledon plants.

Description

United States Patent Nitsch et a1.

July 8, 1975 METHOD FOR OBTAINING POLYFRUCTOSANS Inventors: Ernst Nitsch; Siegried Muhlbock, both of Linz, Austria Assignee: Laevosan-Gesellschaft mlbH & Co.

KG, Linz, Austria Filed: Dec. 6, 1971 App]. No.: 205,293

Foreign Application Priority Data References Cited UNITED STATES PATENTS 5/1958 I-Iill 260/209 R OTHER PUBLICATIONS Schlubach et al., Chem. Abst, Vol. 51, 1957, pp. 16310g16311b.

Schlubach et al., Chem. Abst., Vol. 52, 1958, p. 3698 d.

Schlubach et al., Chem. Abst, Vol. 52, 1958, pp. 19,963 i l9,965 0.

Primary Examiner-Johnnie R. Brown Attorney, Agent, or Firm-Sughrue, Rothwell, Mion, Zinn & Macpeak [57] ABSTRACT A kidney function diagnostic agent comprising high water-soluble polyfructosans derived from the storage organs of monocotyledon plants.

7 Claims, N0 Drawings METHOD FOR OBTAINING POLYFRUCTOSANS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a new kidney diagnostic agent which, when administered parenterally, is neither degradated nor altered in the body and which is able to be eliminated through the kidneys. On the basis of the elimination speed and the quantity eliminated, one can draw conclusions as to the function of the kidney.

2. Description of the Prior Art About 35 years ago, inulin, a polyfructosan from the storage organs of certain types of composites (for example, Inula helenium, Helianthus tuberosus, Cichorium intybus), were introduced into kidney diagnosis as a clearance substance for the determination of the size of the glomerulum filtrate; see Richards A. N., Westfall B. B. and Pott A. A. Proc. Exper. Biol. (N.Y.), 32, 73 (1934); Shannon .1. A. and Smith H. W., Clin. Invest. 20, 169 (1935). For this purpose, a known volume of inulin solution is administered parenterally and the elimination of the inulin is followed up with a periodic concentration determination in the blood and the urine. The prerequisite for this use is the property of inulin to be neither stored, nor chemically altered in the body. In spite of certain practical disadvantages, inulin has so far proved to be excellent for this task as a standard substance. The chief disadvantage in the use of inulin resides in the fact that it is difficult to dissolve in cold water. The normally employed percent solution crystallizes at cold temperatures, and is accompanied by the formation of a crusty precipitate. Before it can be given to a patient, this precipitate must once again be dissolved through heat. At this point there is a danger that the inulin could be hydrolyzed into low molecular fractions and fructose, which in contrast to inulin, are broken down by the body; on the other hand, after injection of insufficiently dissolved preparations blockage of the thinner blood vessels of the body may occur which could precipitate serious physiological complications.

SUMMARY OF THE INVENTION In accordance with this invention, there is now proposed, new kidney diagnostic agents, themselves longknown, comprising the watersoluble polyfructosans from the storage organs of certain monocotyledon plants, and especially, from the families of the Liliaceae and the Gramineae.

Thus, the object of this invention is to provide a kidney function diagnostic agent with a polyfructosan content, which is characterized by the fact that it contains water-soluble polyfructosans easily soluble in water from the storage organs of monocotyledon plants, and especially from the families of the Lilicaeae and Gramineae.

DETAILED DESCRIPTION OF THE INVENTION For reasons of economy, one takes into prime consideration the use of Sinistrin, a polyfructosan from the sea onion (Urginea maritima) see Schmiedeberg O., l-loppe Seylers Z. Physio]. Ehem (Journal of Physiological Chemistry), 3, 1 12 (1879) and Schluback H. H. and Florshein W., Ber. dtsch. (Reports of the German Chemical Society) 62, (1929), and the polyfructosan from garlic (Allium sativum), as well as Graminin and Triticin, see Klein 6., Handbuch der Pflanzenanalyse (Handbook of Plant Analysis), Spring 1932, 1st part, pages 866873, and Paech K. and Tracey M. V., Moderne Methoden der Pflanzenanalyse (Modern Methods of Plant Analysis) Springer 1955, 2nd Volume, pages 184-196.

All of these polyfructosans are characterized in that l they are easily water-soluble, (2) they are easily hydrolyzed by acids, (3) their average molecular weight is about 3,000, corresponding to about 18 monosaccharide units, and (4) their fructose residues are primarily B-1,2-bound, see Schlubach H. H., Sing. 0. K., Liebigs Ann. (Liebigs Yearbook) 544, 1 11 (1940). Furthermore, these compounds prove to be completely non-toxic when tested clinically and pharmacologically.

For the production of the above-mentioned watersoluble polyfructosans, the methods described in the literature to date leave much to be desired (Schmiedeberg 0., I-Ioppe Seylers Z. Physio]. Chemistry 3, 112 (1879) and Klein G. Handbuch der Pflanzenanalyse, Springer 1932, 1st part, pages 866-873.) Basically, they reside in the preparation of a watery extract from which a part of the ballast substance is removed through precipitation with vinegar of lead. This is followed by subsequent removal of the lead and the polyfructosans are then precipitated as lime or barite compounds by the addition of milk of lime or baryta water. After filtering by suction and washing, they are broken down into carbonic or oxalic acid, after which the polyfructosan solution thus purified is concentrated into a syrup and precipitated with alcohol. Since the precipitation very stubbornly produces monosaccharides and other impurities, numerous re-precipitations are normally required in order to attain a sufficient degree of purity.

A further object of this invention is to provide a method for the production of polyfructosans for use as a kidney function diagnostic agent, which comprises extracting the particular drugs with low alcohols or with acetone having a water content of -40 percent. The extract is preferably brought to a pH of 9 with calcium hydroxide, filtered, and concentrated under reduced pressure, into a thick syrup, whereupon the thickened extract, after addition of about half its weight in a low alcohol (preferably methanol) is aciditied with a strong acid (preferably hydrochloric acid) to a pH of between 1 and 3. The polyfructosan is then precipitated with a 2 to 8 fold weight of a low alcohol (preferably methanol) or with acetone, and the precipitate thus obtained is converted into a powdery crystalline form through repeated kneading with a water-free precipitation agent. If desired, the solution of the polyfructosan thus obtained can be completely desalted via ion-exchange, treated with active carbon (active charcoal), filtered, and then either filled, after removal of the organic solvent residues by means of distillation, directly into ampoules, or optionally it can be dried, after concentration into a syrup consistency, in a vacuum, to a thin layer.

The method just described is best carried out as follows:

The coarsely comminuted plant material is repeatedly extracted with aqueous alcohols or acetone (preferably methanol, ethanol, or isopropanol) or with acetone having 80-40 percent water by means of percolation, if necessary, at increased temperatures. The function of the organic solvent is (1) to prevent degradation by microorganisms, (2) to suppress the effect of the polyfructosan-decomposing ferments, and (3) to prevent the dissolution of interfering pectin and shiny substances. The extracts thus obtained are alkalized to a pH value of 9 with milk of lime (calcium hydroxide), filtered and the resultant filtrate is evaporated in a vacuum to produce a syrup of about 70-80 percent dry substance content. After cooling, it is, portion by portion, mixed with so much of a low alcohol illustrated above (preferably methanol) or with acetone until no remaining precipitate develops; concentrated hydrochloric acid is then added to obtain a pH of l to, 3 'and preferably 2.5. Next, we add a sufficient amount of solvent (same as above) to complete precipitation. 1n adding the acid, we assure that a large part of the existing salts and dyes, which otherwise would also be precipitated, remain in solution. The precipitate, which consists of a white, viscous mass, is separated from the supernatant liquid and is thoroughly kneaded several times with small portions of solvent, thereby forming a fine-grained crystalline product which finally is liberated from the adhering liquid by centrifuge or suction filter. The substance thus obtained is already very pure but, for parenteral application, it must be dissolve again in water and further purified by'means of ion exchange and filtration of the desalted solution with active carbon. Finally, the solution thus obtained can either be filled directly into ampoules after removal of the solvent residues through distillation and adjustment' of the EXAMPLE 1 Productionof Polyfructosan (Sinistrin) From the Onion Bulbs of the Composite Urginea Maritima 100 Kg of commercially available dried red sea onion were coarsely ground in a knife-mill with a screen insert having a 4-mm perforation diameter, they are premoistened with 200 Kg of a percent watery metha'v nol solution, and percolated, after standing, for 24 hours with the same solvent at room ter'nperaturehav-v ing a runoff speed of about 4 Kg per hour. A total of 300 Kg of extract were obtained. The extract was brought to a pH of 9 with a suspension of 250 g of calcium hydroxide in a little water,the precipitate was filtered off, and the liquid was evaporated, in a vacuum, to a thick syrup. About 75 Kg of syrup containing 80 percent of solid substance was obtained. Accompanied by forceful stirring, 30 Kg of methanol wereintroduced into a thin jet and the almost clear solution, was cooled down to 15C. Accompanied by furtherforceful stirring, a cooled off mixture consisting of 3 liters of concentrated hydrochloric acid and 8 Kg of methanol was added. As a result, Sinistrin was precipitated as a viscous, nacreously-brilliant, yellowish-white mass. The solution, which was clear yellow after standing briefly, was poured off as completely as possible. It contained .salts, fructose and low-molecular oligosaccharides.

Methanol can then be recovered from it by distillation and rectification. I

' The Sinistrin precipitate, about 40 Kg, was forcefully kneaded three times with 40 Kg of methanol, each 4 time, in a kneading machine. The supernatant methanol was poured off in each case and was combined with the one obtained during the firstprecipitation, whereby the entire mass was converted into a fine-grained cyrstallin'e suspension. This was contrifuged on a centrifuge and the substance isolated (about 35 Kg).was dissolved in 70 Kg of water. The solution was then stirred with 3 Kg of active carbon, filtered, and it was then moved at about 15 liters per hour over a mixing bed consisting of 5 liters of a slightly acid cation exchanger in the PH form and 10 liters of a strongly basic anion exchanger in the 01-1 form. The Rein-Sinistrin solution thus obtained was concentrated into'a thick syrup (in tary power: i I

[a] =39.2 (c 10, H O). Yield: 31.5 Kg.

' EXAMPLE 2 Production of Polyfructosan From Garlic (Allium I Sativum) Stalks, roots, and external dry skins were stripped coarsely from 100 Kg of garlic and the rest was run through a meat grinder. The pap was immediatelyforcefully mixed with 30 Kg of isopropanol and was percolated, after standing about 12 hours, with a 30% watery isopropanol solution, at a runoff speed of about 2.5 Kg per hour. A total of 200 Kg of extract was obtained. Further processing was accomplished exactly in the manner of Example 1. Yield: 10.5 Kg. Optical rotary power:

[a] 40.5 (c= 10, B 0). The other properties exhibited were as described in Example 1.

Although the present invention has been adequately described in the foregoing specification and'examples included therein, it is readily apparent that various changes and modifications can be made without departing from the spirit and scope of the invention.

c. filtering and concentrating said extract into a thick syrup under reduced pressure,

d. adding to said extract, in an amount equal to about half of said extracts weight, a low alcohol,

e. acidifying said extract with a strong acid, to obtain .a pH of between 1 and 3,

f. subsequently precipitatingthe polyfructosan with a member selected from the goup consisting of a low alcohol and acetone, in an amount of 2 to 8 times the weight of said extract, and

g. converting the precipitate thus formed into a powdery crystalline state by repeatedly kneading the same with a water-free precipitation agent.

2. The process of claim ll, further comprising the step of desalting the polyfructosan thus obtained by means of ion exchange.

3. The process of claim 2, further comprising the step of filtering the thus desalted polyfructosan with active carbon.

4. The process of claim 3, further comprising the step of removing the organic solvent residues remaining through distillation, or drying said polyfructosan after concentration, into a syrup consistency in a vacuum at a maximum temperature of 80C.

5. The process of claim 1, wherein said low alcohol vacuum at a maximum temperature of C.

Claims (7)

1. A METHOD FOR OBTAINING THE HIGLY WATER-SOLUBLE POLYFRUCTOSANS FROM THE STORAGE ORGANS OF MONOCOTYLEDON PLANTS, WHICH COMPRISES, A. EXTRACTING SAID POLYFRUCTOSAN WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF A LOW ALCOHOL AND ACETONE HAVING A WATER CONTENT OF FROM 80-40 PERCENT, B. BRINGING SAID EXTRACT TO A PH OF ABOUT 9 WITH CALCIUM HYDROXIDE, C. FILTERING AND CONCENTRATING SAID EXTRACT INTO A THICK SYRUP UNDER REDUCED PRESSURE, D. ADDING TO SAID EXTRACT, IN AN AMOUNT EQUAL TO ABOUT HALF OF SAID EXTRACT''S WEIGHT, A LOW ALCOHOL, E. ACIDIFYING SAID EXTRACT WITH A STRONG ACID, TO OBTAIN A PH OF BETWEEN 1 TO 3, F. SUBSEQUENTLY PRECIPITATING THE POLYFRUCTOSAN WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF A LOW ALCOHOL AND ACETONE, IN AN AMOUNT OF 2 TO 8 TIMES THE WEIGHT OF SAID EXTRACT, AND G. CONVERTING THE PRECIPITATE THUS FORMED INTO A POWDERY CRYSTALLINE STATE BY REPEATEDLY KNEADING THE SAME WITH A WATER-FREE PRECIPITATION AGENT.

2. The process of claim 1, further comprising the step of desalting the polyfructosan thus obtained by means of ion exchange.

3. The process of claim 2, further comprising the step of filtering the thus desalted polyfructosan with active carbon.

4. The process of claim 3, further comprising the step of removing the organic solvent residues remaining through distillation, or drying said polyfructosan after concentration, into a syrup consistency in a vacuum at a maximum temperature of 80*C.

5. The process of claim 1, wherein said low alcohol is a member selected from the group consisting of methanol, ethanol and isopropanol.

6. The process of claim 1, wherein said strong acid is HCl.

7. A method for purifying the polyfructosan obtained in claim 1, which comprises: a. desalting the polyfructosan thus obtained by means of ion exchange, b. filtering the thus obtained desalted polyfructosan with active carbon, and c. removing the organic solvent resiDues remaining through distillation, or drying said polyfructosan after concentration, into a syrup consistency in a vacuum at a maximum temperature of 80*C.