Physicochemical properties of [3-glycyrrhizin ([3-G, a triterpenoid saponin) and ot-glycyrrhizin (or-G, a stereoisomer of [3-G, newly developed as part of this ...
j. Soc.Cosmet. Chem.,37, 177-189 (May/June1986)
Physicochemical propertiesand applicationsof and I-glycyrrhizins,naturalsurfaceactiveagentsin licorice root extract MITSUO KONDO, HIROMI MINAMINO, GENICHIRO OKUYAMA, KEIICHI HONDA, HISANAO NAGASAWA, and YASUHISA OTANI,
Cosmetics
Laboratory, Kanebo,Ltd., 5-3-28 Kotobuki-cho, Odawara250, Japan.
Received August17, 1983. Presented at the12th IFSCC Congress, Paris,September 1982. Synopsis
Physicochemical propertiesof [3-glycyrrhizin([3-G, a triterpenoidsaponin)and ot-glycyrrhizin (or-G, a stereoisomer of [3-G, newlydeveloped aspart of this study)wereexamined for cosmetic applications. Both or-and [3-G exhibitedsimilarconsiderable interfacialactivity. However,the aqueous solutionof [3-G formedan extremelyrigid gel in acidicmedia,whereas ot-Gshowedno signof gelation.[3-Gcanemulsify variousoily materialsover a wide rangeof requiredHLB values,while ot-G hassolubilizingability for severalperfumematerials.
A seriesof experiments wascarriedout to obtainsomeinformationon the solubilizing,emulsifying,and gellingmechanisms of or-and [3-Gusing•3C-NMRandscanning electronmicroscopy with variousderivatives of Gs.
The resultssuggested that the [3-G moleculewhichwasfoundto be cyclicallyconstructed constitutes the micelieswhichin turn orientanisotropically to form a rigid gel and to stabilizethe emulsion.
INTRODUCTION
•3-glycyrrhizin (•3-G), a triterpenoidsaponinpresentin licoriceroot extract,hasbeen widely utilized in cosmeticsowing to its excellentanti-inflammatoryaction (1). In addition to suchcharacteristics, this compoundwas found to displayunique physicochemicalbehavior,particularlysurfaceactivity (2-4) and gel formation(5), evenin dilute aqueousmedia. An isomer,(x-glycyrrhizin((x-G, a new compound),waspreparedto comparephysical propertiesof the two isomers.The presentpaperdealswith the surfaceactivity, solubilizing andemulsifyingmechanisms, andapplicationto cosmetics of bothGs. Also, the mechanism of gelationby •3-G is discussed. EXPERIMENTAL MATERIALS
(x- and [3-glycyrrhizins. (x-G waspreparedby base-catalyzed isomerization of [3-G ac177
178
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS QH
,co¸
CH30 c'•_1•8•••
O,'•••-•¾' •',•/•• ' 17 •'H3
COOH • CH3CH3 Osj_ O TANS H
H
/
OM
i
OM
OOH
O
CMa
.'a•• HO•'Sb cH3 COOH V¸• HOOL•co0 H o dh,
U
H
I
I
OH OH Figure 1. Chemicalstructures of o•-and[•-Gs.
cIs
cordingto the methodreportedby Miyashitaeta/. (6). o•-Ghada purity determinedby HPLC analysis(7) of not lessthan 93.7%.
[3-G was commerciallyavailable(MaruzenKaseiCo., Ltd.; purity 98.5%) and used without further purification.Sinceboth Gs were only slightly solublein water, their monoammoniumsaltswere usedin our experimentsunlessotherwisenoted. a- and[•-glycyrrhetins. Both o•-and [3-glycyrrhetins, whichareaglycones corresponding to respectiveGs (glycosides),werecommercialproducts(MaruzenKaseiCo., Ltd.) and subjectedto the experimentswith no refinement.The minimal purity of both compoundsexceeded97%.
&lethy/•-g/ycyrrhizinate. Reactionof dipotassiumsalt of •-G with a large excessof etherealdiazomethaneat room temperatureyieldedthe dipotassiumsalt of methyl •glycyrrhizinate ascolorless crystals;IR: 1740 cm-z O/C= 0); z3C-NMR(D20):8 52.3 (-OCH3), 177.9 (-CO2CH3). Anal. Calc. for C43H62Oz6K2: C, 56.56; H, 6.84. Found:C, 56.46; H, 6.98. Methyl •-glycyrrhizinatewasobtainedby dissolvingthe abovesalt in water, subsequent acidificationwith aceticacid, and additionof acetone. A white precipitatewascollected,washedwith acetone,and dried in vacuo,mp 268.0268.5øC (dec).
[•-g/ycyrrhizin pentaacetate. •-G wassubjected to the esterification with eightequivalents of aceticanhydride at 40øC.Methanolwasaddedto the reactionmixtureto decompose an excessof aceticanhydride.After evacuatingthe volatilesand washingthe residue
with ether,colorless crystals wereobtained;mp 225-227øC;IR: 1740cm-• O/C= O);
PROPERTIESOF o•-AND [B-GLYCYRRHIZINS
179
o • 40 .•_ 30
lO
I
I
-5
-4
I
-3
I
-2
log C ( mol/I ) Figure2. Interfacial tensionof Gsat water-liquid paraffininterface (25øC).
13C-N. MR(CD3OD + CDC13): 8 20.7,20.9(•H3COO-), 170.8,171.0,171.1 (CH3COO-). Anal. Calc. for C52H72017: C, 60.45; H, 7.02. Found:C, 60.59; H, 7.12.
Other chemicalsusedin the presentstudywere all of reagentgrade. INTERFACIAL
TENSION
Measurements were carriedout with a RigoshaAuto Tensionmeter,Model 6801 ESD,
at 25øCby the ring method.The innerdiameterof the ring and the diameterof the platinumwire were 1.909 and 0.060 cm, respectively. NUCLEAR
MAGNETIC
RESONANCE
(NMR)
•C-NMR spectrawere recordedon a JEOL JNM-FX 100 Spectrometer with tetramethylsilaneas the externalstandard. Pulsewidth: 10 •sec (45ø). Pulse repetition: 1.0 sec. Spectralwidth: 6024 Hz. SCANNING
ELECTRON
MICROSCOPY
(SEM)
A Hitachi ScanningElectronMiscroscope, Model S-450, equippedwith a cryosystem
180
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
ß
x
m
O ß
2
3
4
5
6
7
pH Figure 3. pH dependence of viscosity of Gs aqueous solution(1.0 wt%, 20øC).
Table I
Gelling behaviors of G derivatives
H3Ci,•C OO R3 CH3 (• '•
H3 3
R1
R2
R3
H
H
H
/3
gel-formation ++
K(H}
H(K}
H
/3
++
K K
K K
H K
/3 /3
K(H}
H (K} CH3
-
/•
K salt of G
Pentaacetate
/3
K salt of
H
)-o o i
OH
OH
COOR2
Glycyrrhetin
o•
J-
PROPERTIESOF or-AND [3-GLYCYRRHIZINS
•
6
• -g lycyrrhet in
d, -g lycyrrhet in
!
,+
3
181
ß
/
\
,
I I
•,•ø 0
o
1
2
Shearstress (dyne/cm 2)X10-4 Figure4. Flowcurves of gels(10 wt%), Cone-plate viscometer, 30øC,0-30 rpm.
wasused.The samplewasfrozenin a sample-holder usingliquid N2 andcracked.Its
surface wasetchedin vacuo at about- 80 - - 90øC.Thestructure ofthefrozengelwas directlyobserved with the microscope. RHEOLOGICAL
MEASUREMENTS
Flowcurvesof the gelswereobtainedwith a Cone-plate Viscometer (B.E.E. Co., Ltd.)
at 20øC.Maximalshear rateandsweep timewere500sec-•andtwomin,respectively. Theviscosities of or-and[3-Gsolutions weremeasured at 20øC,24 hr aftertheirpreparation.A Brookfield Viscometer (SeikiKogyoLaboratory, rotorNo. 1 and3, 12 rpm) wasusedfor [3-G. A Ubbelohde's Viscometer (ShibataKagakuKiki Kogyo)wasutilized for ot-G. SOLUBILIZATION
TEST
Ten gramsof ethanolsolutions of solubilizates (0.5 wt%) wereaddedto 90 g of ot-G aqueous solutions of variousconcentrations (all of whichwereadjustedto pH 4.5 with V3oM phosphate buffer).The mixtureswereshakententimes,left standing for 24 hr at
30øC,andsubjected to themeasurement oftransmittance ona HitachiRecording Spectrophotometer,Model EPS-3T. Appraisalof the solubilizingability of ot-G wasbased on the transmittanceof the resultingquaternarysystemsat 450 nm, at which wavelengththe solubilizates havelittle opticalabsorption. EMULSIFICATION
TEST
Emulsionswerepreparedby the followingprocedure.To 70 g of the aqueous solution
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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
of I3-G and/orsodiumcetylsulfate(SCS),30 g of a prescribed oily materialwasaddedat 80øC, and the mixturewasagitatedwith a TK Auto HomomixerType M (Tokushu
Kika KogyoCo., Ltd.). The resultingemulsionwascooledto 30øCwhile stirring continuously.The stabili.tyof emulsionswas determinedby the volume ratio of the
residualemulsifiedlayer after centrifugingat 3000 rpm for 15 min at 30øC.The emulsifyingability was examinedby the appearance of emulsions.Oils with various HLB valueswerepreparedby changingthe weight ratio of liquid paraffinand 2-hexyldecanol. RESULTS
and DISCUSSION
PHYSICOCHEMICAL NATURES of or- and [3-GLYCYRRHIZINS
Chemicalstructuresof Gs are shownin Figure 1. They are tribasiccarboxylicacids.
3
2
1
1
2
45
5
80 øC
25øC
ß
•
I
I
I
I,,
200
180
ppm
200
*SO
ppm
Figure 5. Temperature dependence of t3C-NMR spectraof individualGs (10 wt% D20 solution).1: C=O of triterpenemoiety. 2: COOH of triterpenemoiety. 3,4: COOH of glucuronicacid moiety. 5:
- • = C- C= O oftriterpene moiety.
PROPERTIES OF or- AND IB-GLYCYRRHIZINS
183
ot-G hasa transconfiguration, while IB-Gis cisin which D and E ringsof the triterpene moietyare bent at the C•7-C•8 bond. Theseisomershaveno structuraldifferenceexcept at this point and the configurationof the hydrogenat C•8. One of the most importantphysicochemical propertiesof or- and IB-G is that both haveinterfacialactivity as shownin Figure 2. The interfacialtensionbetweenwater and liquid paraffin wasdiminishedby 25-30 dyn/cmby additionof eachG. This activityis basedon the peculiarstructurehaving a triterpenemoiety as a lipophilic group and glucuronicacid moietyasa hydrophilicgroup. AlthoughC.M.C. valuesof both compounds arealmost
equal(ca. 2-3 x 10-4 mol/1),the physicochemical naturesof the two isomers were remarkablydissimilar.The aqueoussolutionof IB-Gformedan extremelyrigid gel in acidicmedia, whereasot-G showedno signof gelation(Figure3). It is alsonotablethat, evenin dilute solutionbelow0.1 wt%, in spiteof beingsucha smallmolecule(MW =
840), I•-G formeda rigid gel in acidicmediabelowpH 4.5. SOLUBILIZING AND GELLING MECHANISMS OF GLYCYRRHIZINS PROPERTIES OF VARIOUS DERIVATIVES OF GLYCYRRHIZINS
Variousderivativesof Gs were prepared,and their gelling propertieswere examinedin orderto investigatethe structuralfactorsresponsible for the gelling mechanisms. The resultsare shownin Table 1. It wasconfirmedby preliminaryIR and NMR investigationsthat salt formationoccurredfirst at carboxylgroupsof the glucuronicacidmoiety and subsequently at that of the triterpenemoiety.
Table 1, representing the gelling behaviorof severalderivativesof Gs alongwith their chemicalstructures,demonstrates that the freecarboxylgroupof the triterpenemoiety is indispensable for gelationby IB-Gand that the gelationneveroccurswhen all of the
I
35 4
5
I
200
I
180
I
•
ppm
200
I
180
I
ppm
Figure 6. Effectof Eu(fod) 3 on •3C-NMR spectraof Gs (10 wt% D20 solution),Eu/G = 0.05 (molar ratio). c•-G wasmeasured at 25.0øCand •-G at 80.0øC.
184
JOURNALOF THE SOCIETYOF COSMETICCHEMISTS
carboxyl groupsand/orsomeof the hydroxylgroupsof the glucuronic acidmoietyare blockedby salt formationor esterification. Therefore,the factorsessential for gelation are as follows:
1. The carboxylgroupof the triterpenemoietyshouldbe free. 2. At leastoneof the carboxylgroupsof the glucuronicacid moietyshouldbe free. 3. Someof the hydroxygroupsof the glucuronicacidmoietyshouldbe free. The potassiumsalt of tx-glycyrrhetin,which takesa more planar conformation,was readyto producea coagel(soap-type gel), in contrastto the caseof Gs, therebysuggestingthat a rigid gel producedby I3-G is not of a coagelstructure(Figure4). BEHAVIOR
OF GLYCYRRHIZIN
MOLECULES
IN MICELLAR
STATE
Dynamic behavior of G moleculesin a miceliar state was examined by •3CNMR(CMR). Temperaturedependenceof CMR spectraof individual Gs is shownin Figure5. The spectraof tx-G wasslightlyaffectedby temperature,while that of I3-G
variedmarkedlywith the elevationof temperature. At 25.0øC,all signalsof I3-G can hardlybe discerned.This undoubtedlyreflectsthe strongrestrictionof molecularmotion of I3-G due to gel formation.Furtherexperimentswereconductedon the effectof
Figure 7. Scanningelectronmicrographimageof lyophilizedgel of I3-G aqueoussolution(!.0 wt%).
PROPERTIES OF tx- AND [3-GLYCYRRHIZINS
185
shift reagentson the resonances of Gs. Lanthanideshift reagentshave beenwidely utilized in NMR spectroscopy (8) to elucidatechemicalstructureor conformationof variouscompounds. This is dueto their ability to causea noticeable paramagnetic shift of resonance inducedby the interactionbetweenthe shift reagentsand a Lewisbase moietyof a givenmolecule.Figure6 showsCMR spectraof miceIlarsolutionsof Gs in whicha shiftreagent,Eu(fod) 3, is solubilized. As Eu(fod) 3 is oil-soluble,thisreagentis adequate to probethe internalnatureof micellesandthusobtainconformational information on the triterpenemoiety servingas a lipophilic group. The reagentcaused strikingbroadening of signalsof the triterpenecarbonylandcarboxylcarbons of or-G, whichmaybe ascribable largelyto the spin-nucleus relaxationbroughtaboutby strong interactionbetweenEu and carboxyland/orcarbonylcarbons.In contrast,no appreciablebroadening of signalswasobserved in [3-G, thougha slightparamagnetic shiftof resonances of triterpenecarboxylcarbonswasinducedby the additionof the reagent. Thesefindingsstronglysuggest that the carboxylgroupof the triterpenemoietyof is blockedby somemolecularinteraction,whereas that of ot-Gis muchmorefree. SEM OBSERVATIONS/AQUEOUS SOLUTIONS OF GLYCYRRHIZINS
An SEM image of a lyophilizedgel of [3-G is shownin Figure 7, wherea regular
/.
:
. f-
Figure 8. Scanningelectronmicrographimageof lyophilizedsampleof o•-Oaqueous solution(l. 0 wt%).
186
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
O
CH3
(;:a CH• ø•c•
HO I
CH:]
HO ]'!%., / O, /O•,,/! o.... 'C; CHa
.o
HO' •
'OH
....
OH Figure 9. Cyclicmodelof •-G in aqueoussolution.
lOO
•
80
E
•
60 I
/
c
O: Geranyl
40
acetate
•:
•
20
El .' Ionone
•: 0 0
Geraniol
0.5
Concentration
Dipentene
I
•
ß
1.0
1.5
2.0
of c•-G (wt%)
Figure 10. Solubilizingability of(x-G for severalperfumematerials(30øc , pH 4.5). Ethanol:9.95 wt%. Solubilizate: 0.05 wt%. Water + (x-G: 90.00 wt%.
PROPERTIESOF o•-AND [3-GLYCYRRHIZINS
HLB
10.2
187
17
SCS
Figure 11. Emulsifyingability of •-G for oils with variousHLB values(after l0 days).Liquid paraffin, 2-Hexyldecanol: 30 wt%. I•-G or sodiumcetylsulfate (SCS):3.0 wt%. Water: 67 wt%. The numbergiven to eachbottle indicatesthe weight ratio of liquid paraffin/2-hexyldecanol.
networkstructureis clearlydiscernable.In contrast,suchstructurecannotbe observed in o•-G, asdemonstratedin Figure8. The c•-G moleculetaking an almostlinear conformationis likely to form lamellastructuresin the miceliarstateas do conventional soaps.
Theseresultssuggestthat intermicellarassociation and orientationof the miceliesof [3-G seemto resulteventuallyin the formationof a rigid gel. All theseresults,combinedwith our previousESR study (9), suggestthat the moleculein a miceIlar state is cyclicallyconstructedby intramolecularinteractionbetweena carboxylgroupof the aglyconmoietyand oneof the glucuronicacidmoiety. A proposedcyclicmodelof the [3-G moleculeis illustratedin Figure9. APPLICATION
TO
COSMETICS
Basedon the alreadydescribedproperties,o•-G shouldbe utilized asa solubilizer,and [3-G as an emulsifierand a stabilizerin cosmetics.Figure 10 showsthe solubilizing ability of o•-G for severalperfumematerials.In this experiment,geranylacetate,geraniol, ionone,anddipentenewereselectedfor typicalperfumematerialsandsolubilized in the aqueoussolutionof o•-G. It is obviousfrom Figure 10 that the transmittanceof the solubilizingsystemriseswith the increaseof o•-G concentration.This systemappearsclear when the transmittancebecomesover 80%. Therefore,the solubilizing ability of o•-G is satisfactoryas evidencedfor the perfumematerials.This seemsto ensureextensivepracticalapplicabilityasa solubilizer. On the other hand, the emulsifyingability of [3-G for oils with variousHLB valuesin comparison to SCS, which is widely used,is shownin Figure ! 1. The HLB valuesof
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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
1.0
0.6 E
wt%
ß Liquid paraffin
28
0.4. ßi•-G+SCS
2
ßGlyceryl monostearate
0.2
0
2
ß Water
68
I
I
0.2
0.4
I•-G/( (wt.
I
0.6
I
0.8
1.0
jS-G + SCS ) rat io)
Figure 12. Effectof [•-G on emulsionstability. Centrifugation:3000 rpm, 30oC , 15 min.
oils in this experimentrangedfrom 10.2 to 17.0. Stabilityof the emulsions observed afterten daysstorageat roomtemperaturedemonstrated that [3-G hasenoughemulsifying ability for variousoils lying overa wide rangeof HLB values.Furthermore,the effectof [3-G on emulsionstabilitywasinvestigatedby partiallyreplacing•3-G with SCSas shownin Figure 12. Stability of the emulsionwas judged by volumeratio of residualemulsifiedlayer after centrifugation.It is obviousthat the additionof givesriseto prominentstabilizationof emulsionsprobablydue to gel formation.
ACKNOWLEDGEMENTS
We wish to thank Dr. S. Yamazakiand Dr. A. Okubo, Departmentof Agricultural Chemistry,Facultyof Agriculture,Universityof Tokyo,for the measurement of NMR spectraandcriticaldiscussion. We areindebtedto Dr. H. Yoshioka,Shizuoka College of Pharmacy,for valuableadvice.Thanksarealsodueto Mr. H. Futagoishi,emeritus
PROPERTIESOF or-AND [3-GLYCYRRHIZINS
189
generalmanagerof our laboratory,to Mr. M. Yamagishi,generalmanagerof our laboratory,and to Mr. T. Yoneyawho encouraged us throughoutthis work.
REFERENCES
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(1980).
(2) A. Otsuka,Y. Yonezawa,K. Iba, T. Tatsumi, and H. Sunada,Physico-chemical propertiesof glycyrrhizicacid in aqueousmedia.I. Surface-active propertiesand formationof molecularaggregates, Yakugaku Zasshi,96, 203-208 (1976). (3) A. Otsuka, Y. Yonezawa,and Y. Nakamura, Physicochemical propertiesof glycyrrhizicacid in aqueousmedia. II. Effect of flocculation-deflocculation behaviorof suspensions of sulfathiazoleand graphite,J. Pharm.Sci., 67, 151-154 (1978). (4) Y. YonezawaandA. Otsuka,Physico-chemical propertiesof glycyrrhizicacidin aqueous media.III. Solubilizingpropertiesfor dyesand medicinalsubstance, Yakugaku Zasshi,101, 829-835 (1981). (5) B. E. Azazand R. Segal,Glycyrrhinzinasgelling agent,Pharm.ActaHelv., 55, 183-186 (1980). (6) A. Miyashita,K. Imoto, T. Kuramoto,K. Kakegawa,andY. Otani, Reactionofglycyrrhizicacid. I. Base-catalyzed isomerization.Abstractsof papers,The 100th Annual Meetingof the Pharmaceutical Society ofJapan,Tokyo, April 1980, p 202. (7) K. Tsubone,S. Ohnishi, and T. Yoneya, Separationof glycyrrhizinicacid isomersby high-performanceliquid chromatography, J. Chromatogr., 248, 469-471 (1982). (8) A. F. Cockerill, G. L. O. Davies,R. C. Hardon, and D. M. Rockham,Lanthanideshift reagentsfor NMR spectroscopy, Chem.Rev., 73, 553-588 (1973). (9) H. Yoshioka,K. Honda, and M. Kondo, Spin probestudy of the aqueoussolutionsof or- and [3-glycyrrhizins,J.ColloidInterface Sci., 93, 540-544 (1983).
