HU190569B - Process for producing inflammation inhibiting active substance - Google Patents

Abstract

The present invention also relates to pharmaceutical or cosmetic compositions comprising an anti-inflammatory activity comprising the above active ingredients. -1-

Description

The present invention relates to antiinflammatory agent and to pharmaceutical compositions containing it. The active ingredient can be prepared from vegetable raw materials and is polysaccharide-like.

Polysaccharides as used herein are monosaccharides having furan or pyranose structure and are optically active heteropoysaccharides with glycoside or ether linkages, the basic or acidic functional groups of which may optionally be linked to protein and / or peptide-type compounds (peptidoglycans or glycoproteins). Antiinflammatory activity is understood to mean any effect that rat foot carrageenin edema test allows or does not exclude. The anti-inflammatory inhibitors found active in rat foot edema tests may be antirheumatic agents, anti-ulcer agents, possibly external ointments, gels or solutions, or therapeutic cosmetics.

The non-natural anti-inflammatory drugs currently used in medicine, on the one hand, contain steroids, for which many tolerance problems occur, for example, they are often hormonal, increase the body's sodium retention, and can cause atrophy. Non-steroidal anti-inflammatory drugs, on the other hand, are also of limited use due to their side effects. These side effects are usually related to indomethacin and salicylates, reduce the body's resistance, cause gastrointestinal disorders, internal bleeding, and ulcerogenicity. Due to the toxic side effects, there has been an increasing tendency to seek out natural anti-inflammatory agents. The intensification of research is also justified by the fact that the use of anti-inflammatory agents has been extended to treat tissue oxygen supply disorders such as myocardial infarction, other tissue circulatory disorders and shock conditions.

Many herbs and preparations derived therefrom are used in medicine and cosmetics in areas which are presumed to contain anti-inflammatory agents. The components of the various herbal drugs and the products derived from them, which have the effect, are largely undetected. There are many plant species used as therapeutic drugs or not as therapeutic drugs from which anti-inflammatory ingredients could not be produced. These include Tilia, Malva, Plantago, Linum usitatissimum, Trigonella foenum-graecura, Cydonia oblongata.

The object of the present invention is to isolate a group of biologically active, mainly anti-inflammatory compounds from a plant not used as a therapeutic drug, which can be made by relatively simple processing processes for the preparation of pharmacological and cosmetic preparations having an anti-inflammatory backbone comparable to the presently used formulations.

Surprisingly, it has been found that Cucurbita maxima (pumpkin) can be isolated from the raw material in a manner different from the usual extraction conditions, at ambient temperature and with a small amount of water, with a significant anti-inflammatory effect. This polysaccharide fraction can be converted, if desired, to a concentrate which can be incorporated into pharmaceutical or cosmetic formulations by precipitation and further purification.

Characteristics of the active ingredient from Cucurbita maxima include:

Not more than 2,7% total nitrogens, not more than 5% expressed as Phosphorus, not more than 25% calculated as gluten, not less than 30% as reducing sugars and not less than 40% as total sugars, based on chromatographic analysis of glucose and mannose, contains at least 40% of the anti-inflammatory effect of 10 mg / kg rat carrageenin edema test.

The process of preparing the herbal active ingredient according to the invention consists in treating the freshly used or previously picked and stored drug at a temperature of 0-40 ° C, preferably after pre-comminution or simultaneously with comminution, with water for 1-24 hours. and precipitating the active ingredient from the aqueous portion by addition of C 1-3 alcohol, acetone, or 1-3 divalent cations, which is separated from the aqueous portion and, if desired, further purified by repeated precipitation from an aqueous medium. The amount of water used for the aqueous treatment is i-20 times the weight of the drug. The plant is ground or minced, then the mill is pressed, the press residue is treated again with water and then pressed again. Preferably, the active ingredient is precipitated from the aqueous solution by the addition of methanol or ethanol, and the aqueous active ingredient is purified by repeated dissolution and precipitation and / or slurrying with a C 1-3 alcohol. The separation of the protein fraction in the aqueous extract is carried out by heating to 40-80 ° C. A preferred method of treatment is to disintegrate the drug in an aqueous medium. The aqueous extraction may be carried out at room temperature or at temperatures above + 30 ° C.

Preference is given to continuous processing, during which the starting material is wet-comminuted and then pressed again at low or high pressure, and possibly pressed again with water. Extracted and extracted and

The liquid separated by pressing -2190566 contains the polysaccharide fraction to be recovered as the final product. The concentrate is precipitated from the aqueous medium with C 1 -C 3 alcohol or acetone, optionally with a 1-3 valence cation. The individual precipitation methods can also be combined. The weight of the active ingredient recovered ranges from 0.1 to 1.0% by weight based on the dry weight of the starting material. Further processing is to extract the chopped plant material with 1-20 times the weight of water, heat the extract at 60 ° C, purify the heat treated extract by centrifugation and filtration, and precipitate the polysaccharide from the solution with 2-3 volumes of alcohol and After standing for 24 hours, the precipitate was separated from the mother liquor by centrifugation and filtration and washed with alcohol. The precipitated crude drug was purified by dissolving the wet material in 10 times water, warming to 75 ° C, removing the precipitated material by centrifugation, and mixing the supernatant with 3x volume of alcohol. After 24 hours, the precipitate was collected by centrifugation and filtration, washed with alcohol and dried under vacuum at <RTI ID = 0.0> 40 C </RTI>.

Further purification can be achieved by dissolving the dried product in 30-40-fold amounts of water at 50-75 ° C, purifying by centrifugation, and filtering at a pressure of 3-10 att through a membrane that is permeable to materials with a molecular weight of less than 75,000, then precipitating the polysaccharide from the solution on the high molecular weight side of the membrane with alcohol. The polysaccharide can also be precipitated from the aqueous solution with acetone.

The anti-inflammatory composition of the invention comprises the active ingredient isolated by any of the above methods and optionally a non-steroidal anti-inflammatory drug, in particular indomethacin or acetylsalicylic acid or derivatives thereof, in association with conventional excipients and carriers. The pharmaceutical composition is suitable for oral or parenteral administration in the form of tablets, dragees, capsules, infusions, emulsions, injections, solutions, suppositories, implants or topically applied solutions, creams, ointments or gels. Anti-inflammatory compositions may be used for therapeutic or cosmetic purposes and may be prepared by mixing the active ingredient with excipients and / or carriers customary in the preparation of pharmaceutical or cosmetic compositions.

The anti-inflammatory activity of the active ingredient of the present invention is determined by C.A. Winter et al., Proc. Soc Exp Bioi. Med., 111. 544 (1962), rat foot was examined by carrageenin edema test. The active ingredient has significant specific activity and associated beneficial anti-inflammatory activity with extremely low toxicity.

The isolation and pharmacological activity of the active compound according to the invention is illustrated by the following embodiments:

EXAMPLE 1 Cucurbita maximae fructus, purified from its peel and kernel, is mixed with 1.5 liters of tap water at 20 ° C in a disintegrator and pressed at a hydraulic pressure of 130 att. The press juice was filtered through a Seitz filter plate K-5 to give 1500 ml of filtrate, which was concentrated at 40 ° C under reduced pressure to 40% solids. To the 140 ml concentrate was added 300 ml of 96% ethyl alcohol with stirring. The mixture containing the precipitate was allowed to stand at room temperature for 12 hours. The pellet was separated by centrifugation and then triturated with 100 ml of 96% ethyl alcohol, filtered and dried under reduced pressure to give 3.0 g of the active ingredient.

Example 2 kg of Cucurbita raaxima fructus containing 0.15% of active ingredient were cut in half and the seeds removed. The starting material is comminuted with a disintegrator to a coarse pulp and then taken up in a 10 L tap. The mixture was heated to 35 ° C with stirring, kept under vigorous stirring for 1 hour and then squeezed out after 6 hours. 12 liters of pressed juice are obtained. The press residue is suspended in 10 L of tap water, stirred for 1 hour and then pressed to give 12 L of press juice again.

The two press broths were combined, heated to 60 ° C with stirring, then centrifuged and the protein precipitate discarded. The centrifuged solution was filtered through Seitz K-5 filter paper and the precipitate was discarded. The pellet-free solution was concentrated under reduced pressure at 60 ° C to a dry matter content of 10-20%. The concentrate, cooled to room temperature, is centrifuged and the precipitate is discarded. Three times the volume of methyl alcohol was added to the supernatant with vigorous stirring and the precipitated solution was allowed to stand at room temperature for 24 hours. The supernatant is decanted and discarded. The precipitated solution was centrifuged and the precipitate was disintegrated in 500 ml of methanol and centrifuged. The precipitate obtained by centrifugation was washed twice more with methyl alcohol. After the third disintegration of the precipitate, the precipitated solution is poured onto a vacuum suction filter and the mother liquor is sucked off.

The wet white precipitate was disintegrated with 10 times distilled water, heated to 75 ° C with stirring and centrifuged hot. To the purified solution was added three volumes of methyl alcohol with stirring and allowed to stand at room temperature for 24 hours. After standing, centrifuge, the resulting precipitate was disintegrated with 300 mL of methanol and centrifuged again. The methanolic wash and disintegration was repeated two more times, and after the third purification step, the precipitate was filtered through a vacuum suction filter and the filtered precipitate was dried under vacuum at 40 ° C. 21.1 g of a white powder are obtained.

For further purification, the crude drug was dissolved in 600 ml of distilled water at 50 ° C, allowed to stand for 1 hour, and then centrifuged. The supernatant was filtered at 5 att using a membrane filter (Diaflo XM 300, manufactured by Amicon Co, USA), which retains compounds of molecular weight greater than 75,000. The solution remaining on the high molecular weight side of the membrane filter was diluted three times with distilled water and the solution was again forced through the membrane filter. Finally, the solution remaining on the high molecular weight side of the filter was centrifuged and three times the volume of methyl alcohol was added to the supernatant with stirring. Allow to stand for 24 hours at room temperature. The precipitate was filtered off with suction and the filtered product was dried at 40 ° C. The purified active compound weighed 13.2 g.

Example 3

1050 kg of Cucurbita maxima fructus are coarsely ground, the seeds are removed and the 1000 kg seed-free material is finely ground with a hammer mill. The resulting pulp is suspended in 1000 liters of water and separated on a decanter into 500 kg of fibrous slurry and 1500 kg of aqueous supernatant. The fibrous slurry was suspended in 470 L of tap water at 20 ° C, stirred for 2 hours and then decanted again. 570 kg of slurry and 400 liters of aqueous solution were obtained. The fibrous slurry obtained after the second decantation is pressed at 150 att, with an initial layer thickness of 50 mm, to finally yield 240 kg of press residue and 370 kg of press juice. The pressed residues were discarded and the aqueous solutions were combined to give a total of 2270 kg of an aqueous solution which was adjusted to pH 4.3-4.4 with stirring with 20% aqueous citric acid. The mixture was then allowed to stand at 20 ° C for 12 hours. A portion of the precipitate precipitated during this time, the supernatant was partially removed by decantation and partly by separation to give 12080 kg of an aqueous solution and 120 kg of separator sludge. The aqueous solution is concentrated in vacuo so that the temperature of the concentrate is 40-42 ° C, the temperature of the steam chamber is 70-75 ° C, the weight of the concentrate is 157 kg, the dry matter content of the concentrate is 28.5% and the pH of the concentrate is 4.50 and the compression time should be 7 hours. The concentrate was purified by separation of the precipitated precipitate during suction to give a purified concentrate (153 kg), which was stirred with vigorous stirring at 38 ° C for 385 liters of methanol, stirred for 30 minutes and allowed to stand for 20 hours. The precipitated solution was first decanted and then filtered through a filter centrifuge, and the resulting precipitate was washed with methanol (10 L) to obtain a wet precipitate (20 kg) which was resuspended in a shake mixer in the presence of 80 L of methanol. filtered through a filter centrifuge and washed again with 10 L of methanol. The above procedure is repeated 3 times in total. The final cer tri-wet wet precipitate was dried at 80 ° C in flowing air until a dry matter content of 50% was achieved. It is then granulated, dried to a particle size of 2 mm, and then dried in air at 50 ° C to constant weight. The product weighed 7.0 kg. Analytical characteristics of the product:

White amorphous powder, soluble in alcohol, acetone, chloroform, ether, ethyl acetate.

After boiling for 2 hours in boiled distilled water, 80% of the 5% weighing solution is dissolved.

Aqueous solution: pH 5.0 (5.0-7.0) at 5% loading.

Total sugar content: 42.2% (min: 40%)

Reducing sugar: 33.0% (min: 30%)

Total N: 1.94% (min: 1.5%)

N content with Biuret reagent: 2.66% (greater than 1.5%)

P content: 1.15% (min: 1.0%)

Residual ignition: 22.74% (max: 25%)

Sugar composition for gas chromatography:

glucose 76.9% mannose 4.9% arabinose 2.1% rhamnose 1.4% unidentified 14.7%

Anti-inflammatory: min. 40% 10 mg / kg i.p.

dose at the rat foot edema test.

To determine the molar distribution, 38 mg of product is chromatographed on a Sepharose CL-6B column. Column diameter: 27 mm, length: 260 mm, empty volume: 150 cm ³ , bracket: Blue dextran 2000 standard, thermostated at 65 ± 3 ° C with flowing hot water. Filling: 150 cm ³ , Sepharos: CL-6B suspension and 6 g glass fiber. The fractions of 5 cm ³ each, of the determination: spectrophotometry phenol reaction occurs acid, glucose and reference substance. The eluent is distilled water containing 0.2% NaNOa. Proceeding as above, the following molecular weight distribution was obtained

Fraction number

Molecular Weight Fraction Polysaccharide;

content of the eluted total; % of polysaccharide; expressed;

10 17 greater, equal 10 ⁶ 37 17 - 25 greater, equal 10 ⁵ 16 25 - 30 greater, equal 10 ⁴ 6 30 - 40 smaller, equal 10 ³ 4

Example 4

The anti-inflammatory activity of the resulting active compound was determined by a rat foot carrageenin edema test. p. dosing. The results are shown in Table 1. 20

Table 1

Anti-inflammatory activity of Cucurbita maxima PS concentrate in a 5 hour rat foot carrageenin edema test i.p. after dosing

agent Dose in mg / kg % Anti-inflammatory; Example 3 0.5 21 of product 1 24 2 44 5 46 • 10 54 20 56 50 75 100 80 indomethacin po 10 46

The active ingredient LD50 values are shown in Tables 2 and 3.

Table 2

24 hour LD50 values of Cucurbita maxima PS concentrate in male CFLP mice, mean 30 g, after intravenous, intraperitoneal and oral administration

Method of administration LD50 mg / kg arc* 188 ip 214 po greater than 3010

Note: the first 8 hours have no toxic symptoms, death is between 16-24 hours.

Table 3

Mortality rate after eight days of administration of Cucurbita maxima PS concentrate in groups of 30 g male CFLP mice

Method of administration Dose mortality ip LDso / 3 0 ip LD / 10 0 po 3010 mg / kg 0

The antiinflammatory activity was tested in the rat leg kaolin and cellulose sulfate edema test.

Data are shown in Tables 4 and 5.

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Table 4

Examination of the anti-inflammatory limit of Cucurbita maxima PS concentrate in rat foot kaolin edema test

agent Dose mg / kg i.p. % Anti-inflammatory Example 3 2 9 5 36 10 44 20 55 50 69 100 79

Table 5 Cucurbita maxima on PS test anti-inflammatory effect of concentrate rat foot cellulose sulfate edema agent Dose mg / kg i.v. inflammation %: Example 3 0.5 37 RINTEL 1 51 5 51 10 57

The effect of the combination of the active ingredient and indomethacin is shown in Table 6.

Table 6

Cucurbita maxiraa PS concentrate and indomethacin i.p. and p.o. combinations of anti-inflammatory drugs in the rat foot carrageenin edema test

agent Dose input mode Edema% Inhibition% control (Fiziol.só) 1 ml ip 21.4 ± 2.91 - Example 3 2 mg / kg Ip 13.3 + 2:28 37.9 by indomethacin 5 mg / kg po 13:04 & 1:17 39.3 Example 3 and 1 mg / kg ip 11.812.93 44.9 indomethacin 2.5 mg / kg

-612

The gastric ulcer inhibitory activity of Cucurbita maxima PS concentrates of the present invention was further investigated in the following ulcer models: Indomethacin ulcus: K.D. Rainsford, Agents and Actions 7, 573, 1977. 5.Shay ulcus: Shay H. et al., Gastroenterology 5, 43, 1945. Stress and salicylate ulcus:

Nagy 1. et al .: Experimental Ovostud. 34, 201, 1982. The results were evaluated according to the method of A. Szilágyi et al., Experimental Medicine 30, 1. 1978. We use the following terms in the evaluation system:

Erosion number: (Ei) The number of lesions on the inner surface of the stomach incised along a small curve using a ten-fold stereo microscope.

Severity Index: (Si) The number expressing the amount of gastric mucosal erosion. Its numeric value is between zero and four, as follows:

Si = 0 when intestinal mucosa is intact, i.e. no lesion;

1 if the diameter of the erosion is less than 1 mm

2, if the erosion diameter 1 and 2 mm between there is 3 if the erosion diameter 2 and 4 mm between there is 4, if the erosion diameter 4 mm •than

bigger.

Actual Severity Index: (TSi) Same Severity! index number of erosions in an animal. This number actually expresses the ulceration of the stomach of a given animal.

Actual Severity Index Mean: (TSi) Equal to the sum of the actual severity indices of the animals in each experimental group divided by the number of individuals in the group.

The ulcer inhibitory effect is illustrated in FIGS. The following table illustrates:

Table 7

Inhibitory effect of Cucurbita maxima PS machine concentrate on indomethacin ulceration based on actual severity index mean (dose-effect relationship). Input method: i.p.

Dose in mg / kg Actual severity index average Inhibition% control 86.61 0.1 71.26 17.8 0.5 59.9 30.8 2.0 51.56 40.5 10 40.98 52.7 50 15.92 81.2

Table 8

Inhibitory effect of Cucurbita maxima PS machine concentrate on indomethacin ulceration in different severity index groups (dose-effect relationship). Input method: p.o.

Severity index

Dose ———————————————————————————————————————

2 3 4

mg / kg erosion song Avg. inhibition X erosion song Avg. inhibition X erosion song Avg. inhibition X erosion song Avg. inhibition X contact roll 27.17 7:50 3:11 3:05 0.5 21:06 22.5 3.89 48.2 2:38 23:34 2.78 9.2 2 21:11 22.3 6.78 9.7 2:00 35.70 1:56 49.1 10 16.61 38.9 3:56 52.7 0.83 73.2 0:22 92.8 50 18:22 33.0 3:50 53.34 1:00 67.8 0:50 83.6

-Ί14

Table 9

Inhibitory effect of aspirin ulcer on Cucurbita maxima PS concentrate based on actual severity index mean (dose-effect relationship), in non-pilorus fed animals, route of administration: i.p.

Dose in mg / kg Actual severity index average Inhibition 5454; control 47.15 "» 0.5 14.80 70.0 2 20.99 55.5 10 7:22 84.7 50 2.67 95.3

Table 10

Stress-ulcer inhibitory effect of Cucurbita maxima PS concentrate on the basis of actual severity index mean (dose-effect relationship), steeping for 5 hours, administration method i.p.

Dose in mg / kg

Actual severity index average

Inhibition%;

control 211.0 - 0.1 171.6 18.7 0.5 165.8 21.4 2 140.9 33.2 10 131.1 37.8 50 125.5 40.5

Example 5;

From the product of Example 3, the following antacid ulcer inhibitor formulation was prepared:

2.00 g of the compound of Example 3

1.40 g MgO

4.00 g of Al (OH) 3

24.00 g Hydrogelum methylcellulosum Ph.

Hg. VI.

10.00 g of sorbitol

0.1 g of sorbic acid

If necessary, flavor one tablet with a commercial sweetener and one drop of flavoring agent (e.g. Oleum aurantiae).

The above additives were diluted to 100 cm ³ with distilled water and vigorously homogenized. The ulcer inhibitory activity of the preparation was tested on indomethacin-ulcus and the results are shown in Table 11:

Table 11

The inhibitory effect of the preparation of Example 5 on indomethacin ulceration was based on the mean of the actual severity index (dose-effect relationship). Input method: p.o.

Dose in mg / kg

Actual severity index average

Inhibition at 54b

control 22.1 - 1.0 15.4 30.3 2.0 4.8 78.3 3.0 8.8 60.2

-816

Table 12

The preparation of Example 5 had indomethacin ulcer inhibitory activity of varying severity! index (dose-effect relationship). Input method: p.o.

Dose ----------------------- Seriousness! index mg / kg 1 2 3 4 erosion inhibition erosion inhibition erosion inhibition erosion inhibition song % song % eye % song % Avg. Avg. Avg. Avg.

Kon ι-

roll 7:40 - 5.70 - 1:10 - 0.0 0.0; 1.0 2.70 36.7 1.70 70.2 0.70 36.4 0.0 0.0; 2.0 2.70 36.7 0:30 94.7 0:50 54.4 0.0 0.0; 3.0 3.70 50.0 1.80 68.4 0:50 54.4 0.0 0.0;

έ. example

For the preparation of an externally applied gel, a 3% aqueous solution of Carbopol 934 is adjusted to pH 7.0 with 1N sodium hydroxide, previously in the aqueous phase at 3%.

The active ingredient of Example 3 was dissolved. The gel is preserved with 0.5% sorbic acid or 0.1% Nipagin.

Example 7

For the preparation of an ointment, the preservatives of Example 6 and 5% of the active ingredient of Example 3 are dissolved in water and mixed at a 1: 1 volume ratio with 1000 g Unguentum emulzificans / composition 100 g carboxethene stearate, 100 g paraffin, 300 g cetyl alcohol stearate 500 g of Vaseline Album) and homogenize.

Ultraviolet light Inhibition of erythema test in rabbit

Cucurbita maxim PS (5% hydrophilic cream) 42%

Phenylbutazone (5% ointment) 50%;

Dexamethason (Dexatopic Ointment) 69%

Claims (9)

Claims 1. A process comprising an anti-inflammatory polysaccharide having a total sugar content of 40-60%, a reducing sugar content of 30-50%, a total nitrogen content of 1.5-3% (according to Kjeldahl), and a total nitrogen content (by biuret reaction) 2-4%, 1-2% phosphorus, 15-25% glow residue, at least 70% glucose, at least 10% glucuronic acid, total anti-inflammatory effect at least 40% 10 mg / kg i.p. a rat foot pad for edema test, characterized in that the Cucurbita maxima fructus (pumpkin) plant is treated with water for 1-24 hours after pre-comminution or simultaneously with comminution, if desired, at a temperature of 0 ° C to 40 ° C. and precipitating a stock concentrate from the aqueous portion, optionally after separation of the proteinaceous materials, by addition of C 1-3 alcohols, acetone or 1-3 divalent cations, which is separated from the aqueous phases and optionally purified by repeated precipitation from an aqueous medium; 35 ² ·. The process of claim 1, wherein the plant is ground or comminuted, then the mill is pressed, the press residue is repeatedly treated with water and then pressed again. 3. The process of claim 1, wherein the plant is ground or comminuted, then the mill is pressed, the press residue is repeatedly treated with water and then pressed again. 45 The process of claim 1, wherein the extract is heated to a temperature of 40-80 ° C to separate the proteinaceous material in the aqueous extract and the resulting 50 precipitates were collected. 5. A process according to claim 1, wherein the starting material is wet-comminuted and optionally pressurized to low or high pressure. It is pressed several times, and the pressed material is repeatedly treated with water. 6. The method of claim 1, wherein the chopped plant material is Extract with 60 to 20 times water, heat the extract at 60 ° C, purify the heat treated extract by centrifugation and filtration, and precipitate the polysaccharide from the solution with 2 to 3 volumes of alcohol and stand for 24 hours. 65 precipitates by centrifugation and filtration -918 1!) From mother liquor and washed with alcohol. 7. The process of claim 1, wherein the dried product is dissolved in 30-40-fold water at 50-75 ° C, purified by centrifugation, and filtered through a membrane under pressure of 3-10 att. Substances with molecular weights of less than 000 are permeable and the polysaccharide is precipitated from the solution on the high molecular weight side of the membrane with alcohol or acetone. 8. A process for the preparation of an anti-inflammatory pharmaceutical composition comprising the process of any one of claims 1-7. The active ingredient produced by the process of claim 1 in combination with customary excipients and excipients in the form of tablets, dragees, capsules, infusion solutions for oral or parenteral administration. in the form of an emulsion, injection, solution, suppository, implant, or topically applied solution, mucus, cream, ointment or gel. Cosmetic composition, characterized in that it comprises a composition according to any one of claims 1-7. The active ingredient of the method of claim 1 is formulated as a solution, emulsion, lotion, cream, ointment, gel, foam, soap, toothpaste, milk, chewing powder, in admixture with excipients customarily used in cosmetic formulations for external use.