1965_ohta_pyridoxine-3,4-diacylates and their use in cosmetics

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J. Soc. CosmeticChemists, 16, 349-358 (1965)

Pyridoxine-3Their Use in

4-DiacylatesCosmetics

and

HARUYASU OHTA, M.Sc.*

Presentedbefore he Third Congress f the I.F.S.C.C.,June 21-2, 19, New York City

Synopsis--Pyridoxine-3,4-diacylates can be prepared by a new process n good yield andhigh purity. Data are presented to demonstrate the oil solubility and the heat and lightstability of these esters. Acute and chronic toxicities of these esters indicate that they aresuitable for inclusion in cosmetics. It is further shown that the esters are hydrolyzed bytissue homogenates at rates which are influenced by the acid moiety used for esterification.Finally, clinical data are presented to demonstrate the utility of pyridoxine-3,4-dipalmitatein dermal therapy.

INTRODUCTIONVitamin B0 is one of the most important factors for nutrition andfor health and beauty of the human skin. Hence, many attempts havebeen made to use vitamin B0 as a cosmetic additive. Pyridoxinehydrochloride,which is a commercially available form of vitamin B6, isnot suitable for topically applied cosmeticsbecauseof its insolubility inoils and fats and its instability to heat and light. It is therefore de-sirable o preparea heat- and light-stable, at-solubleand percutaneouslyabsorbable yridoxinederivative for cosmetic se.In 1956, Sakuragiand Kummerow (1-4) first synthesizedong chainfatty acid triesters of pyridoxine and confirmed that they are sourcesof fat-soluble, heat- and light-stable and biologically active vitamin B0when applied to the rat.In 1961, Rocheggiani (5) reported on the cutaneous actions of

* Nihon Surfactants Industries Co. Ltd., 29, 3-chome, Hasune-cho, Itabashi-ku, Tokyo,Japan.

349


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35O JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTSTABLE I

Physical Properties of Pyridoxine-3,4-DiacylatesPyridoxineDiacylate M.P., C Formula M.W.

Dibutyrate 57-58 C16H230,N 309.37Dioctanoate 69-71 Cs4H39OsN 421.58Di-iso-octanoate Liquid C24H90N 421.58Dilaurate 79-80 CHaO.N 533.80Dipalmitate 88-89 C40H7Or, 646.01

TABLE IISolubilities of Pyridoxine-3,4-dioctanoate

Solvent g./100 g. at 25CIsopropyl myristate 2Olive oil 1Oleyl alcohol 3.5Oleic acid 5Liquid paraffin 0.0770% ethanol 0.3 at -- 5 - -- 10C60%ethanol 0.1 at - 5 - - 10;C

TABLE II I

Solubilities of Pyridoxine-3,4-dipalmitateSolvent g./100 g. at 25C g./100 g. at 75C

Ethanol 0.5Isopropyl Inyristate 0.1 3Olive oil 0.1 5Oleyl alcohol 0.2 5.5Oleic acid 0.1 5.5Liquid paraffin 0.01 0.5

TABLE IVSunlight Stability of Pyridoxine Derivatives

DerivativesIrradiation Time, Transmittanee,

hr. %Pyridoxinc hydrochloride

Pyridoxine,B,4-dibutyrate

Pyridoxine-3,4-dioctanoate

0 9915 9230 87

0 10015 9930 98

0 10015 99.530 98


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PYRIDOXINE-3,4-DIACYLATES IN COSMETICS 351

pyridoxine tripalmitate. He concluded from a series of experimentsthat pyridoxine tripalmitate is very effective in keeping the human skinhealthy and beautiful. The large-scale preparation of pyridoxinetripalmitate is not easy, nor is this compound satisfactory for cosmeticuse becauseof its insufficient at-solubility.A new commercialmethod for the synthesisof heat- and light-stableand fat-soluble derivatives of pyridoxine, pyridoxine-3,4-diacylates,hasbeen developed n this laboratory. Patents for this method of prepara-tion are now pending in the United States of America, Great Britain,France, Switzerland, West Germany, and Japan.The chemical and physical properties, biological activity, toxicityand effectson the skin of these diacylateshave been examined. It ap-pears that the higher fatty acid diesters are heat- and light-stable, fat-solubleand hydrolyzed nto free pyridoxine n vivoand show he biologi-cal activity of vitamin B6.The present report deals mainly with the dibutyrate, dioctanoate,dilaurate, and dipalmitate of pyridoxine.

EXPERIMENTAL AND DISCUSSIONMaterials

All the diesters used were white crystalline powders, except thedi-iso-octanoate. Some of the properties of these diesters are tabulatedin Tables I, II, and III.Heat Stability

Two grams of pyridoxine-3,4-dipalmitate was dissolved n 10 g.of olive oil and heated for six hours at 150-160C. At the end of thisheating period, the oil was brown. The oil was then diluted with 100ml. of petroleum ether and cooled n the refrigerator for two days. Theprecipitate was removedby filtration and washedwith petroleum ether.The crystalline precipitate was pure white and weighed 1.6 g. Thecrystals showed no depression n melting point when mixed with au-thentic pyridoxine-3,4-dipalmitate. The dilaurate and dioctanoatewere tested in a similar manner, and the recoveredunchangedestersweighed 1.4 and 1.1 g. respectively. The dibutyrate, however, couldnot be recovered rom the heated brown oil, presumablybecausemost ofthe diester was destroyedby heat.

Light StabilityOne gram of each of the pyridoxine derivatives was dissolved n100 g. of 50% aqueousethyl alcohol. Samplesof these solutionswere


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352 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTSTABLE V

LDs0 of Pyridoxine-3,4-dipalmitate and Related Compounds Mice)LDs0, g./kg.

Compound Administration 24 Hr. 7 DaysPyridoxin e-3,4-dipahnitate

Pyridoxine hydrochloride

Oral 7.1 5.1Subcutaneous 4.4 1.6Oral 3.8 3.8Subcutaneous 1.7 1.4

Palmitic acid Oral 5.0 4.2Subcutaneous . . . 4.2

sealed nto test tubes and placed nto sunlight. The transmittanceofthe irradiated solutionswas read at 435 mu with a spectrophotometer.The data shown n Table IV indicate hat the estersare not severelydiscoloredduring long exposure o sunlight and are more stable thanpyridoxinehydrochloride.Toxicity*

Acute ToxicityThe acute toxicity of pyridoxine-3,4-dipalmitate was studied inmice and is recordedas LD0 in Table V. l?yridoxine ydrochlorideandpalmitic acid were also tested as control materials. The ester andpalmitic acid were dissolvedn ethanoland then mixed with propyleneglycol. Pyridoxine hydrochloridewas studied n an aqueoussolution.The mixture and the solutionof sampleswere administeredorally andsubcutaneously o mice.From the resultsshown n Table V, it can be concludedhat pyr-idoxine-3,4-dipalmitates less oxic than pyridoxinehydrochloride.

Chronic ToxicitySix groupseach of five male and five female rats weighing rom 85to 135 g. wereused n this test and fed for six monthsasfollows'

Group A'Group B'Group C'Standard DietStandardDiet + 40 mg./kg./day of pyridoxine-HC1Standard Diet + ,50mg./kg./day of palmitic acid

* This work was conducted t the OsakaCity Institute of Hygiene (6).


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Group D: Standard Dietpalmitate

Group E: Standard DietdipalmitateGroup F: Standard Diet

dipalmitate

q- 7 mg./kg./day of pyridoxine-di-70 mg./kg./day of pyridoxine-700 mg./kg./day of pyridoxine-

Groups A, B, and C were control groups. The weights of the ratswere recorded daily for six months, and the growth rate is shown inFig. 1. When pyridoxine-3,4-dipalmitate was orally administereddaily to rats (in an amount of 000 the oral LD:0 for mice) for six340' Croup

4D300,

.- ._ .. 250- / /' / -'- ..... ' 9-./' .... 200 //'. .....

20 60 80 100 120 l 160Figure 1.Change in body weight of rats fed with various amount of pyridoxine-3,4-di-

palmitate

months, the increaseof the animal's weight was greater than when theequivalent amount of pyridoxine hydrochloride was administered. Allrats used n this test survived during the test and were killed at the endof the test for observation of organs. No harmful effect of pyridoxine-3,4-dipalmitate was observed n organsat autopsy.PercutaneousAbsorption (8)

Rabbits weighing from 2 to 3 kg. were used for this test. An area of5 X 15 cm. 2 on the abdomensof the rabbits was clipped, and the hairwas completely removed with a depilatory. After 24 hours, 6 g. ofhydrophilic ointment containing one of the pyridoxine derivatives was


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354 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

applied after ascertaining hat there was no inflammation on the partto be tested. Blood sampleswere collected from the ear lobe by in-travenous puncture and assayed or vitamin B0 content.The compositionof the hydrophilic ointment is as follows:Isopropyl myristate 1 g.Cetanol 0.25 g.Stearic acid 0.4 g.Paraffin wax 1 g.NIKKOL BL-9* 0.2 g.NIKKOL BC-5t 0.15 g.Mineral oil 1.35 g.10% Triethanolamine solution 1ml.Pyridoxine derivative 1Water to make 1 g.

In the preparation of the ointment containing pyridoxine hydro-chloride, the triethanolamine solution was omitted from the aboveformulation.

The vitamin B6 content in blood was determined as follows:Standard curve: In the case of pyridoxine hydrochloride and

of pyridoxine-3,4-dibutyrate,1 ml. of an aqueoussamplesolutionofknown concentration (about 0.5 m/mi.) was acidified with 6 mi. of15% H2SO4, and the solution was heated at 100C for one hour.After cooling, the pH of the solution was adjusted to 5.4, and thevolume made up to 50 ml. with water. Samples of this solution(0.25, 0.5, 1 and 2 mi.) were pipetted into test tubes and diluted withwater to 2.5 mi. After adding 2.5 mi. of culture medium and steriliz-ing at 100C for 15 minutes, 1 drop of preincubatedsuspension fSaccaromyces arlsbergensisulture was added, and the solution wasincubated at 30 C for 20 hours. Absorbancy was then measuredat610 mu, and a standard curve was constructed.For the construction of the standard curve for pyridoxine-3,4-dioctanoate, 1 mi. samplesof ethanolic solutionswere hydrolyzedas mentioned above.Pyridoxine assay n blood: A mixture of 1 g. of blood and 6 mi.of 15% H2SO4was heated at 100C for one hour. After cooling, hepH of the solutionwas adjusted o 5.4, and the liquid was centrifugedto remove blood pigments. The supernatant solution was separated,and its volume was adjusted to 50 mi. with water. The microbio-* Polyoxyethylene lauryl alcohol ether. Nikko Chemicals Co., 1, 1 chome, NihonbashiBakurocho, Chiyoda-ku, Tokyo.t Polyoxyethylene cetyl alcohol ether. Nikko Chemicals Co.


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logical assaywas carried out similarly as mentionedabove, and theamount of pyridoxine was determined by reference o the standardcurves.

The resultsare shown n Fig. 2. Pyridoxineand pyridoxine-hydro-chloride were absorbed mmediately, but their content in blood de-creased apidly. In contrast, the dibutyrate was also absorbed m-mediately, but the blood concentration emained at a constant level.The absorptionof the dioctanoate and di-iso-octanoatewas delayed,but the blood concentration increased gradually. The absorption ofdilaurate and dipalmitate was not studied in this experiment. These1000'

- 8oo.o rldorlne 600-

o Pyrldo lbutMrat 400-

/ Pyridoxlnel-Ionicanoe 200- Pyrldoxlnioota?lme ( hrs. )

Figure 2. Percutaneousbsorption f pyridoxineand its derivative (1% in 0/W creambase)factsmight suggesthat cosmetics r ointments ontainingntermediatechainfatty acid diesterof pyridoxinemaintain higher evelsof vitaminB in the living body or a longerperiodof time than products ontainingpyridoxie.

Hydrolysisof Diacylates n Organs )Liver, kidney, intestine,and blood of the mousewere used n thisexperiment. Liver, kidney, and intestine, rom which as much bloodas possiblewas removed,were washedwith water. One gram of liver,0.4 g. of kidney,0.7 g. of intestine nd0.4 ml. of bloodwerehomogenized

with Tyrode's solution. These homogenates ere poured nto 50 ml.Edenmeyer lasks,and the contentswereadjusted o 7 g. with Tyrode's


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356 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTSTABLE VI

Hydrolysis of Pyridoxine-3,4-dibutyrateSample

No.Time for

HydrolysisAmount Hydrolyzed, wt. %

Liver Kidney Intestine Blood

Average

Average

Average

1 hr.

0.5 hr.

105.8 98.9 98.9 80.194.6 90.9 ... 74.295.4 106.8 104.5 81.898.6 98.8 101.7 78.7

106.6 108.0 92.8 70.4... 97.5 107.6 69.6

98.4 98.9 104.4 66.2102.5 101.4 101.6 68.7105.6 106.2 99.2 64.689.2 104.8 106.8 71.1

107.9 108.0 88.4 56.7100.9 106.3 98.1 64.1

TABLE VIIHydrolysis of Pyridoxine-3,4-dioctanoate

SampleNo.

Amount Hydrolyzed, wt. %Tine forHydrolysis Liver Kidney Intestine Blood

Average

Average

Average

91.5 82.3 82.2 02 hr. 87.6 69.2 82.3 0

101.7 79.0 87.2 093.6 76.8 83.6 074.5 66.8 84.2 0

1 hr. 85.2 85.8 84.2 084.7 75.6 82.7 083.9 76.1 80.2 063.1 72.4 64.1 0

0.5 hr. 62.9 61.6 59.8 084.0 76.4 79.9 070.0 70.1 67.9 0

TABLE VIIIHydrolysis of Pyridoxine-3,4-dilaurate

SampleNo.

Time forHydrolysis Liver

Amount Hydrolyzed, wt. %Kidney Intestine

Average2 hr.

11.1 0 0.97.6 0 1.0

10.1 0 0.89.6 0 0.9


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PYRiDOXINE-3,4-DIACYLATES IN COSMETICS 357

solution. One mi. of one of the pyridoxine-3,4-diacylates solutions wasadded, and the flasks were shaken for 0.5, 1 or 2 hours at 37C tohydrolyze the ester. After shaking, 3 mi. of 10g aqueous richloro-acetic acid solution was added, and the mixture was centrifugcd. Fivemi. of the supernatant was neutralized with 1.5% NaOH solution.For 1 mi. of the solution thus obtained, concentration of pyridoxinewas determinedby using 2,6-dibromoquinone hloroimideat 650 mt.The results are shown in Tables VI, VII, VIII, and IX. It can beseen that the dibutyrate was rapidly hydrolyzed in the blood and

TABLE IXHydrolysis of Pyridoxine-3,4-dipalmitate

Amount Hydrolyzed, wt. %Sample Time forNo. Hydrolysis Liver Kidney Intestine

1 0 0 02 2.3 0 03 2 hr. 1.3 0 04 0.4 0 0

Average 1.0 0 0

organ homogenates. The dioctanoate was not hydrolyzed in the bloodbut slightly in the organ homogenates. On the other hand, dilaurateand dipalmitate were not hydrolyzed even in the liver homogenate.

Derreal TherapyDuring the clinical test, which was designed o demonstrateefficacyagainst several kinds of dermatitis, such as eczema and seborrhoea,hydrophilic ointments and lotions containing 0.2% and 0.02% pyr-idoxine-3,4-dipalmitate respectively were applied topically. The re-sults showed hat pyridoxine-3,4-dipalmitate was an effective treatment

for skin diseaseswithout any harmful side effects.It is also noted that this substance showed marked antidandruff andanti-itching properties, as judged from the following studies. Fourteendermal outpatients (7) suffering from eczemas and seborrhoea weretreated with a hydrophilic ointment containing 0.2% pyridoxine-3,4-dipalmitate for seven to twenty days. This treatment was very effec-tive in six cases,satisfactory n six casesand ineffective in two cases.In three cases of seborrhoea and especially dermatitis of the face,patients felt a cooling sensation mmediately after application; sebumvanishedday by day. In one caseof rosacea of the first degree), ed-dening was reduced,oily luster vanishedafter ten days, and the diseasewas cured completely on the fourteenth day. In two casesof seborrhoea


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358 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

capiris neonatorum, the sebum leaking scab was no longer observedafter four days, and it was nearly cured n eight to ten days.In two casesof facial pimples,no new onesdevelopedafter ointmentapplicationhad begun, and the number of existing pimples decreaseduntil none were observed t all. In two casesof desquamating czema,scale decreased emarkably after four days, and the scaleon the head andface almost vanished. Further, in two casesof eczemaerythematosumand contactdermatitis,reddeningdecreased fter three days.In the two ineffective cases, the symptoms were those of seriouscases f dermatitis; even oral or topical steroidhormone herapy (evenin massive doses)was not fully effective, especially f the side effectinduced by this hormone s considered.Further, 162 cases of pityriasis simplex capiris and seborrhoeacapiris were treated with a lotion containing ethyl alcohol, Tween80* and 0.02% pyridoxine-3,4-dipalmitate. This lotion was effectivein 75% of casesof scaling, n 77% of casesof itching and in 85% of casesof dry dandruff(10).SUMMARY

Pyridoxine-3,4-diacylates ave been shown to be useful cosmeticingredients.The diestersof pyridoxine are considered o be more effective thanthe correspondingriesters sincethe former's content of the pyridoxinemoiety is considerably arger.When heat- and light-stability, fat-solubility, percutaneous ab-sorbability and the effective content of pyridoxine are taken into ac-count, pyridoxine-3,4-dioctanoatemerges s the most suitable deriva-tive for cosmetic purpose.

(ReceivedSeptember14, 1964)REFERENCES

(1) Sakuragi,T., and Kummerow, F. A., J. Am. Chem.Soc.,78,839 (1956).(2) Sakuragi,T., and Kummerow, F. A., J. Am. Oil Chemists' oc.,33, 116 (1956).(3) Sakuragi,T., and Kummerow, F. A., J. Nutrition, 58, 557 (1956).(4) Sakuragi, T., and Kummerow, F. A., Arch. Blochem.Biophys.,63, 32 (1956).(5) Rocheggiani,G., Soap, Perfumery Cosmetics,4, 547 (1961).(6) Osaka City Institute of Hygiene, reports in files of Nihon Surfactants Industries Co.Ltd.

(7) Dept. Dermatology,OsakaMedical College, eport in file of Nihon Surfactants ndus-tries Co. Ltd.Kamada, A., Personal communication.Kamada, A., Personal communication.Yasuda, T., Japan. J. Dermatol., 73,487 (1963).

* Atlas Chemical Industries, Wilmington, Del.

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1965_ohta_pyridoxine-3,4-diacylates and their use in cosmetics

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