FR2617710A1 - TOOTHPASTE COMPOSITION - Google Patents

Abstract

Toothpaste composition for preventing dental caries, containing hydroxyapatite in which the glucanase is immobilized.

Description

The present invention relates generally

  a toothpaste composition to prevent tooth decay

  keep silent, and more particularly a toothpaste composition containing hydroxyapatite in which glucanase etc ... are immobilized. "Glucanase" is a general term for the enzymes breaking down glucan. However, it should be noted that the term "Glucanase" here designates a levanase

  which breaks down levan, a dextranase which breaks down dex-

  trane and a mutanase which breaks down the mutan.

  It is well known that the onset of tooth decay

  shut up is due to the dental plaque that forms on the surface

  teeth under the effect of polysaccharides produced by various

  his mouth bacteria. It is believed that prevention of tooth decay is achieved by the elimination of this dental plaque. Levane, mutane, and dextran are all polysaccharides produced by bacteria in the mouth, and have been shown to be a factor in the formation of

  dental plaque. Among the enzymes that break down these po-

  lysaccharides, it is well known that dextranase provides

261771'0

  a toothpaste composition having an improved effect for the

  prevention of tooth decay because it is capable of

  weld plaque due to its ability to decompose

  position of dextran, as disclosed in the Japanese patent

  nais N 782 154. When the enzyme is incorporated into toothpaste products, however, it is easily broken down and deactivated due to its relative instability. To keep it in a stable state, it is necessary to use

  an appropriate stabilizer. Dextranase is also used

  seed with various stabilizers. For example, it has been proposed

  to use dextranase with a hydrocarbon-based scent

  re terpene or aliphatic alcohol (Japanese patent N

  9 242 229), with carvone or e-menthol in a report

  completed (Japanese patent application N 56-110 609), or with

  aluminum oxide used as a polishing agent (from-

  Japanese patent application N 56-63,915). We can suppose that

  the use of an enzyme, not to mention dextra-

  nase, in a toothpaste would require the incorporation of a sta-

  biliary, as is the case with dextranase. Like a den-

  tifrice is used in the mouth, it should be on and common

  having a refreshing feeling. Obviously, we can think

  ser that considerable limitations are imposed on the choice of stabilizers. For this reason, no

  toothpaste compositions containing mutanase or le-

  vanase, despite the fact that they are considered effective

  caces in the prevention of dental caries.

  One of the aims of the present invention is to provide

  a toothpaste composition containing as an enzyme, in particular

  to bind an enzyme breaking down the polysaccharides usable for the prevention of dental caries, more particularly levanase, dextranase or mutanase, which eliminates the problems related to the safety and the feeling of the user resulting from the use of stabilizers, and which exhibits stable enzymatic activity over a prolonged period without having recourse to the use of any stabilizer. Since enzymes are relatively unstable and soluble, it is good

  known that it is better to immobilize them for a use

  more efficient. In general, the immobilized enzymes are found to be stable even in the form of an aqueous solution. Although there are various methods for immobilizing

  enzymes, the classic process is based on physical adsorption

  if that. As is the case with active carbon, kaolinite, kaolin etc ..., hydroxyapatite is used as a support to immobilize enzymes by physical adsorption, and it is known that it is suitable for use as an agent polishing. It is therefore assumed that if levanase,

  dextranase and mutanase are immobilized with hydroxy-

  apatite, it should then be possible to prepare a composition

  Toothpaste containing an enzyme which does not require any

  biliary. Following extensive studies carried out on this presumption, the applicant has succeeded in developing

  a process for immobilizing these enzymes with hydroxyapa-

  tite. It consists in adding the hydroxyapatite used as a polishing agent to the solution containing glucanase and a protein such as lysozyme, albumin, casein and cytochrome C which can be strongly absorbed on hydroxyapatite and which are harmless for l 'organization

  human and then add a glue solution dropwise

  taraldehyde to this solution, shaking vigorously

  below room temperature, and stir the solution for several hours to complete the reaction at the same

  temperature after addition of the glutaraldehyde solution.

  The immobilized hydroxyapatite is obtained by centrifugation.

  As immobilized hydroxyapatites are easily prepared

  by this process, toothpaste compositions which do not

  do not hold stabilizers but glutanase and

  which have an improved dental caries prevention effect

  re are prepared using this hydroxyapatite as an agent

  polishing according to the classic toothpaste formula

than.

2 6 1 7 7 1 0

  For a better understanding of the invention and to show how it can be put into practice, we

  will now refer, by way of example, to the examples of work

following and reference.

  EXAMPLE 1 Toothpaste (% by weight) Levanase immobilized on hydroxyapatite 13.2 Calcium phosphate 25.0 Sodium salt of CMC 0.3 Carrageenan 1.2 Glycerol 10.0 Sorbitol 15.0 Sodium lauryl sulfate 2.0 Fragrance 1.2 Saccharin sodium salt 0.1 Silica 2.0 Water 30.0 EXAMPLE 2: Mutanase toothpaste immobilized on hydroxyapatite 20.0 Calcium pyrophosphate 10.0 Sodium salt of CMC 0.5 Carrageenan 0 , 6 Glycerol. 20.0 Sorbitol 10.0 Sodium lauryl sulfate 2.0 Fragrance 1.0 Sodium salt of saccharin 0.1 Silica 2.0 Potassium chloride 2.0 Magnesium chloride 0.3 Sodium phosphate 0 , 5 Water 30.0 EXAMPLE 3: Dextranase toothpaste immobilized on hydroxyapatite 35.5 Sodium salt of CMC 1.0 Carrageenan 0.3 Glycerol 35.5 Sodium lauryl sulfate 2.0 Fragrance 1.0 Sodium salt of saccharin 0.1 Silica 2.5 Sodium chloride 2.5 Magnesium chloride 0.1 Potassium chloride 1.0 Water 20.0 EXAMPLE 4: Dextranase toothpaste immobilized on hydroxyapatite 90.8 L sodium auryl sulfate 2.0 Fragrance 1.5 Sodium salt of saccharin 0.2 Sodium chloride 5.0 Magnesium chloride 0.5 EXAMPLE 5: lubricating toothpaste Dextranase immobilized on hydroxyapatite 63.0 Calcium phosphate 10.0 Sorbitol 10.0 Sodium lauryl sulfate 2.0 Fragrance 1.5 Sodium chloride 3.3 Magnesium chloride 0.08 Saccharin 0.12 Water 10.0

REFERENCE EXAMPLE 1

  (1) Preparation of a dextranase immobilized on hydroxyapatite

  To a mixture of 50 mg of dextranase, 50 mg of ly-

  sozyme and 5 g of hydroxyapatite used as a polishing agent

  wise, add 500 ml of potassium phosphate buffer solution

  sium having a concentration of 0.05 mole and a pH of 6.8. To the solution obtained, 500 l of a 0.2% aqueous solution of glutaraldehyde are added dropwise, with stirring and at 40 ° C.

  and the stirring is continued at 4 ° C. for 5 hours. We collect-

  the reaction product and wash 3 times with 100 ml of the above buffer solution to remove material

  not reacted if present. A subsequent freeze-drying

  higher gives 5.06 g of a powder. We weigh exactly 1 g of the above powder, add to the weighed powder 20ml

  of a potassium phosphate buffer solution having a concentration

  tration of 1 mole and a pH of 6.8, and stirring is carried out for three hours, after which it is centrifuged. The precipitate is washed with a buffer solution, the filtrates are combined to measure the quantity of protein contained therein by the Lowry method, and the precipitate is washed with water

  pure to dry and weigh it. We confirm that 10.9 ml of pro-

  teins are absorbed per gram of dried hydroxyapatite.

  Measuring the dextranase activity of this protein by a method which will be described below indicates that 0.513 g of dextran per gram of the protein absorbed are broken down

in 2 hours.

  (2) Preparation of levanase immobilized on hydroxyapatite 50 ml of pure water are added to a mixture of 100 mg of lysozyme, 100 mg of levanase and 2 g of hydroxyapatite, after which the mixture is cooled to -4 C. slowly drip,

  shaking vigorously, to the solution obtained 2 ml of a so-

  aqueous solution containing 28 mg of glutaraldehyde in 100 ml of water, while maintaining the temperature at 4 C. Agitation is then continued at this temperature for 2 hours. Centrifugation gives a solid which is washed 3 times with 50 ml of pure water, then subjected to lyophilization to obtain 2.05 g of powder. Analysis carried out in the same way as in (1) indicates that 11.7 mg of protein are bound per gram of hydroxyapatite. The measurement of the levanase activity of the absorbed protein, carried out by the following method, indicates

  0.47 g of levan are broken down per gram of protein

  not adsorbed on hydroxyapatite. (3) Preparation of immobilized mutanase on hydroxyapatite

  We operate under the same conditions as in (2), except

  We use mutanase instead of levanase to obtain immobilized hydroxyapatite. Analysis performed in the same way as in (2) indicates that 15.0 mg of protein

  are bound per gram of hydroxyapatite. The measurement of the activity

  t of the mutanase, performed as will be described below, indicates that 0.48 g of mutan is broken down.

gram of the bound protein.

REFERENCE EXAMPLE II

  Measurement of the immobilized hydroxyapatite titer A precisely weighed quantity of each sample is added (1 ml of the non-dried sample before lyophilization for the immobilized hydroxyapatite and 2 g of the sample for the toothpaste 2 to 10 ml of a potassium phosphate buffer solution containing 1% of each substrate and having a pH of 7.0 and a concentration of 0.05

  mole, and the solution obtained is stirred at 35 for two hours.

  res, then centrifuged. The residue is washed with the same solution.

  buffer and combined with a centrifuge to determine the amount of each decomposition product. In the case of dextranase, the substrate is dextran and the amount of glucose formed by decomposition is measured by the glucose oxidase method. In the case of mutanase, the substrate

  is mutane, and the amount of glucose formed by decomposing

  tion is measured by the same method. In the case of the leva-

  nase, the substrate is levan, and the amount of fluctuates

  produced by decomposition is determined by chromatography

high performance liquid phie.

REFERENCE EXAMPLE III

  The results of the

  measurement of the variation over time of the enzymatic activity

  as the sample of toothpaste prepared in the examples

1, 2 and 3.

  Variation of the specific activity over time at 37 and at pH 7.0 (the activity immediately after preparation is posed as equal to 100) days 40 days 60 days 80 days 100 day Example 1 100 154.0 84.7 70.5 60.3 52.2 Example 2 100 155.0 85.3 68.4 55.4 46.4 Example 3 100 327.0 185.5 125.4 96.4 84.3

  It is obvious that the toothpaste containing the hydro-

  immobilized xyapatite obtained by the present process has

  an increase in activity which increases immediately after the pre-

  preparation and goes through a maximum activity value after a certain time, after which there is a gradual decrease in activity. However, the toothpaste retains a

  enzymatic activity higher than its initial activity during

for an extended period of time.

  The dentifrice composition in accordance with the present

  vention includes hydroxyapatite in which enzymes

  such as levanase, dextranase and mutanase, used

  for the prevention of dental caries, are immobili-

  sées. It does without any stabilizer and has excellent stability, unlike toothpaste containing

  a conventional enzyme.The toothpaste composition of this

  The invention is also advantageous in that it can be prepared using an immobilized hydroxyapatite obtained in the present invention in place of the polishing agent

  classic in the usual way, without resorting to any

do special technique.

2617 7 1 0

  9. The present invention is not limited to the examples

  which have just been described, it is on the contrary

  re susceptible of modifications and variations which will appear

to the skilled person.

Claims (1)

CLAIM   1. Toothpaste composition containing hydroxyapa-   tite, in which glucanase is immobilized.