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Sanguinarine, a new antiplaque agent: retention and plaque specificity
ARTICLES
From teeth-cleaning sticks to a modern oral rinse, use of this herbal extract spans the centuries.
Sanguinarine, a new antiplaque agent: retention and plaque specificity G. Lee S outhard, P hD R ichard T. Boulw are, M S D uane R. W alborn
T
JL he use of herbs has been beneficial to the o ral h e a lth of n a tiv e c u ltu re s th ro u g h o u t th e w o rld for m any c e n tu ries .1 Sanguinaria, an herbal extract, is one such exam ple, but the use of san guinaria extract has not been reported in the dental literature. S a n g u in a ria ex tract (SaE) has been used in a variety of m edications for dec ades in the U nited States and other coun tries .2 It is used in m odern cough syrups and cold rem edies as an expectorant. Its use as a hom eopathic and folklore rem edy predates its m odern-day use. The extract is principally a m ixture of benzophenathridine alkaloids, the chief constituent alkaloid being sanguinarine (Sa). San g u in arin e is h ig h ly fluorescent u n d er long-w ave light, w h ich m akes it rela tively easy to m onitor and analyze. The chem ical structure of sanguinarine is sim ilar to the benzophenathridine alk a lo id a l c o m p o n e n ts of th e F a g a ra zanthoxyloides species of plant, w hich is used in Africa as tooth-cleaning sticks and are reported to be beneficial to the oral hygiene of these native cultures .3'5 C h lo rh e x id in e an d q u a te rn a ry am 338 ■ JADA, Vol. 108, M arch 1984
W ilm a J. G roznik, RDH Eileen E. Thorne, M S Sam L. Y ankell, PhD , RDH
m onium com pounds are two other sub s ta n c e s th a t h av e b e e n e x te n s iv e ly studied for retention, oral cavity concen tration, and antiplaque effects .6,7 The es sential property for antiplaque agents is long-term retention and slow release into the oral cavity .8,9 This study discusses the antiplaque effects of sanguinaria extract an d th e re te n tio n of san g u in a rin e in plaque and saliva quantitatively, visu ally, and photographically.
M aterials and m ethods ANTIPLAQUE METHODOLOGY. T w en ty -fo u r subjects w ere selected for a study conducted at the U niversity of Pennsylvania Dental School. Twel ve subjects were random ly assigned to the tw o test groups and six to the placebo control group. Two blind-coded oral rinses containing sa n g u in aria e x tract (0.045% ), sa n g u in aria extract (0.03%) and zinc chloride (0.2%), and a placebo control w ithout sanguinaria extract or zinc chloride w ere used daily in five rinse periods for seven days. All test products were identically flavored and test subjects could not distinguish the products based on taste. Four rinse periods w ere supervised (two in the m orning and two in the afternoon) for five days. The fifth rinse period and those on the
w eekends were done at home, unsupervised. Rinses lasted for 15 seconds w ith two 15-ml rinses each period. All subjects were super vised w hile they brushed w ith a comm ercially available soft toothbrush and a nonfluoride toothpaste once daily just before the first rinse in the m orning. No other oral hygiene w as al low ed. Safety ex am inations w ere m ade on days 1 ,3 , and 5 just before and im m ediately after the first scheduled m orning rinse, and one hour after the exam ination that followed the brushing session. An additional examina tion was made on day 8. Plaque was scored be fore the start of the study and, on day 8, before product use or brushing. The plaque area was m onitored on the facial surfaces of the maxil lary right first molar, left central incisor and first prem olar, and the m andibular right cen tral incisor,10 using the m ethod described by Turesky and others11 w ith disclosure by m eans of sodium fluorescein and an ultraviolet wave source (wave-length 4,800 A). ANALYSIS OF SALIVA AND PLAQUE. N ineteen volunteers, ranging in age from 18 to 65 years, were instructed to refrain from oral hygiene, eating, and chewing gum for eight hours before using the assigned test rinses. Nine subjects participated in the saliva analysis and ten sub jects participated in the plaque analysis. Each day the subjects w ere assigned a test product,
A R T IC L E S
Fig 1 ■ Sanguinarine baseline.
Fig 2 ■ Sanguinarine one hour later.
Fig 3 ■ Erythrosine baseline.
Fig 4 ■ Erythrosine one hour later.
Fig 5 ■ Fluorescein baseline.
Fig 6 ■ Fluorescein one hour later.
instructed to rinse w ith tw o consecutive 15-ml rinses for 15 seconds, and w ere not allow ed to rinse w ith w ater or eat or drink during the test. Saliva was collected before rinsing at 15, 45, and 90 m inutes after rinsing; the subjects p u t 6 ml of saliva into separate, capped and marked 16- x 150-mm test tubes contained in ice-filled styrofoam containers. At the tim e of collection, 3 ml of the saliva sam ple was used to determ ine antiglycolytic activity.12 An additional 3 ml w as tak en a n d m ix ed w ith 2 m l 0.01 M triethanolam ine phosphoric acid (TEAPA)
buffer and analyzed for sanguinarine by high perform ance liquid chrom atography. Sim i larly, approxim ately 5 to 10 mg of plaque was collected on different days at 30, 60, 90, and 120 m inutes after rin sin g and m ixed w ith TEAPA buffer. Collection was m ade w ith a curette from th e buccal and lingual surfaces and deposited in alum inum tared w eighing containers, 30 x 15 mm in size. Im mediately after being w eighed, the plaque sam ple was deposited into a 16- x 150-mm disposable cul ture tube and rinsed down into the tube w ith
0.01 M TEA PA b u ffe r. T h e sa m p le w as a n a ly z e d fo r s a n g u i n a r i n e b y h ig h perform ance liquid chrom atography.
Visual and photographic analysis13 Selected subjects w ere instructed to refrain from overnight oral hygiene and to rinse w ith a test rinse containing 0.03% SaE, erythrosine, or sodium fluorescein on separate days. The
S outhard-O thers : SANGUINARINE AS ANTIPLAQUE AGENT ■ 339
A R T IC L E S
The use of sanguinarine in oral rinses is highly specific for dental plaque and is retained in plaque at high levels for several hours after rinsing.
plaque areas in subjects using SaE were visu alized using long-wave ultraviolet light and determ ined w ith the m ethod described by Turesky and others11 one hour after rinsing. The plaque area of subjects using erythrosine was assessed under am bient light. The plaque area of subjects using sodium fluorescein was determ ined under an ultraviolet light source (Plak-Lite). P ho to g rap h y w as done w ith a M inolta system w ith Kodacolor films (details are available from the author).
High perform ance liq u id chrom atography Plaque or saliva sam ples in TEAPA buffer were ultrasonicated on a m icroultrasonicator and added by syringe to a Cis Sep-Pak, w hich was previously wet with m ethanol. After the col lection tube was w ashed w ith 2 m l TEAPA, and the w ashings were added to the Sep-Pak, the Sep-Pak w as placed on a 25-ml pearbottom flask and diluted in two stages w ith 12 and 3 ml of m ethanolic hydrochloric acid. The eluate was evaporated to near dryness in vacuo. The Sep-Pak was w ashed and eluted successively tw o additional tim es and the combined eluates w e re e v a p o r a te d to d r y n e s s in v a c u o . M ethanolic p h o sp h o ric acid (0.5 ml) w as added to the residue, the flask was stoppered, and the m ixture w as vortexed for one m inute. A sam ple size of 100 /¿I was injected into a high-pressure liquid chrom atograph, w hich h ad a M-45 delivery system, UKG variable vol um e injector, radia com pression module, 5 CNIOU Radial-Pak Column, a detector with 280 nm filter, and a reporting integrator. Elu tion was done at a flow rate of 0.05 ml/min using a mobile phase of an 84:16 by volum e m ixture of 5 x 10'3 m olar triethylam ine and phosphoric acid in m ethanol and water. The am ount of sanguinarine present was read d i rectly from the integrator.
had significantly low er (P < .05) posttreatm ent plaque scores than the placebo. No subject experienced oral mucosal irrita tion during the test period. When rechallenged and exam ined on day 21, the subjects did not have any signs of sensitization.
P laque retention The fluorescent properties of sanguinarine were used to compare its retention in plaque w ith the conventional plaque disclosants— erythrosine and fluorescein—by m easuring the plaque area im m ediately after rinsing and one hour later. The SaE plaque area showed that subjects were sim ilar to erythrosine and sodium fluorescein im m ediately after rinsing but significantly greater (P < .05) at one hour after rin sin g (Table 1). These observations w ere recorded photographically (Fig 1-6). Plaque retention and saliva levels of sangui n a rin e w ere q u an titativ ely d eterm in ed by h ig h -p erfo rm an ce liq u id ch rom atography. Plaque levels (Table 2) of Sa in subjects treated w ith SaE w ere higher than saliva levels (Table 3). In general, both plaque and saliva levels varied daily among the same people and be tw een people, indicating a dependence on fac tors beyond the controls established for the test. Variability among collected plaque sam ples was greatest at 15 m inutes after rinsing.
Table 1 ■ Visual assessm ent of plaque reten tion of sanguinarine, erythrosine, and sodium fluorescein.
Disclosing agent Sanguinarine Erythrosine Sodium fluorescein
Initially
One hour later
2.87 ± 0.23 2.86 ± 0.22 2.84 ± 0.23
2.66 ± 0.28 2.18* ± 0.53 1.75* ± 0.45
‘ Significantly different from all other values P < .05.
A ntiplaque activity of SaE oral rinses contain ing SaE (0.045%) alone and SaE (0.030%) com bined w ith 0.2% zinc chloride resulted in re duced plaque scores of 19.4% and 20.4%, re spectively, during the eight-day test period. In contrast, the placebo group show ed a 21.3% plaque grow th during the test period. For both test groups, the posttreatm ent plaque scores w ere significantly low er than baseline scores (P < .05) and the placebo (P < .05). Baseline scores among the three groups w ere not signif icantly different and there was no significant difference in plaque reduction between the two groups containing SaE. Both test groups 340 ■ JADA, Voi. 108, M arch 1984
Inhibition of plaque growth and reduc tion in plaque area scores suggest that sanguinarine rinses have both a preven tive and th erap eu tic effect on den tal plaque (Table 4). The fact that sangui narine rinse had such a dem onstrable ef fect on overnight plaque suggests that an optim um tim e for using sanguinarine rinses may be at bedtime. This procedure would capitalize on the retentive prop erties of sanguinarine in plaque during this critical tim e of building plaque .12 The presence of 0.2% zinc chloride in a rinse containing 0.03% SaE was approx im ately the same antiplaque efficacy as a 0.045% SaE rinse w ithout zinc chloride. The role of zinc in accentuating a n ti plaque effects has been reported. Waler and Rolla 13 noted that chlorhexidine gave a better plaque-inhibiting effect in the presence of 0.3% zinc chloride than w hen used alone. It was concluded that zinc ions and chlorhexidine are competitive for the same receptor sites because pre rinses with zinc caused a distinct reduc tion in the effectiveness of chlorhexidine. It has also been observed 14 that the addi-
Table 2 ■ Plaque retention levels of sanguinarine (/xg/g wet plaque).*__________________ Subject no.
Mean plaque area score ± SD
Results A n tiplaque activity
Discussion
T able 3 ■ Saliva levels of sanguinarine (Atg/ml saliva).* Subject no.
1 2 3 4 5 6 7 8 9
45
90
1.13 1.47 1.96 0.80 0.90 0.47 2.63 0.50 1.65
0.92 0.75 0.79 0.57 0.90 0.17 1.25 0.29 0.65
0.45 0.09 1.00 0.32 0.50 0.22 0.54 0.18 0.60
‘ Assays conducted using HPLC.
1 1A 2 2A 3 3A 4 5 6 7
26 110 29 79 111 160
45
30 . .
.+
96 46 42 46
60
90
120
68 46 46 34
26 30 31 46 36 36 51 46 39 33
27 47 24
22 51 27 56 37 34
‘ Assays conducted using high-perform ance liquid chrom a tography (HPLC). tE llip ses indicate plaque not sampled at this time.
Sample tim es after rinsing (min) 15
Sample tim es after rinsing (min) 15
T ab le4 ■ Plaque indexes and percent plaque reduction of sanguinarine orai rinses. Group Placebo SaE, 0.03% and ZnCh, 0.2% SaCl, 0.045%
Baseline (day 0)
After treatm ent (day 8) Reduction (%)
2.44 (0.73) 2.99 (0.96)
2.96 (0.59) 2.38 (0.57)
+21.3 -2 0 .4
2.68 (0.70)
2.16 (0.37)
-1 9 .4
A R T IC L E S
Antiplaque activity of SaE oral rinses containing SaE (0.045%) alone and SaE (0.030%) combined with 0.2% zinc chloride resulted in reduced plaque sr.orps of 19.4% and 20.4%. respectively.
tio n o f z in c io n s to v a ry in g c o n c e n tra tio n s o f S a a ffected s a liv a g ly c o ly sis to a s ig n if ic a n t e x te n t in rin s e s c o n ta in in g SaE a t 0.015 % . It w o u ld a p p e a r th a t th e ef fe c t o f z in c in S a -c o n ta in in g rin s e s is to e n h a n c e th e a c tiv ity o f S a th r o u g h m e c h a n i s m s s i m i l a r to t h o s e w i t h c h l o r h e x id in e b u t n o t y e t fu lly u n d e rs to o d . T h e m e c h a n is m b y w h ic h S a in h ib its p la q u e m a y be a c o m b in a tio n o f a n tim i c ro b ia l a c tio n a n d re te n tio n . T h e a n ti m ic ro b ia l a c tiv ity a g a in s t a v a rie ty o f o ra l a e ro b ic a n d a n a e ro b ic m ic ro o rg a n is m s of p u r e S a c h lo r id e s h o w e d a m in im u m in h ib ito r y c o n c e n tra tio n (MIC) o f 8 to 32 //.g/ml (J. M. G o o d so n , D D S, P h D , a n d S. S o c ra n s k y , D DS, p e rs o n a l c o m m u n ic a tio n s). T h e r e te n tio n of S a in p la q u e v a rie d a m o n g in d iv id u a ls . S a n g u in a r in e c o n c e n tr a tio n in p la q u e w a s ab o v e th e M IC fo r u p to tw o h o u rs a fte r rin s in g (T a b le 3). R e te n tio n v a ria b ility a m o n g in d iv id u a ls w a s s im ila r to th a t o b se rv e d in th e o ra l r e te n tio n o f c h lo r h e x id in e , c e ty lp y rid in iu m c h lo r id e , a n d h e x a d e c y ltrim e th y la m m o n iu m b ro m id e .9 V a ria b ility o f S a le v e ls in p la q u e a m o n g in d iv id u a ls a n d w ith in in d iv id u a ls at d iffe re n t tim e s m a y b e th e re s u lt o f th e ra n d o m n e s s of p la q u e s e le c tio n site s o r d ie ta ry o r o th e r e n v ir o n m e n ta l in flu e n c e s p re s e n t in i n d iv id u a ls a t th e tim e o f s a m p lin g (or all th re e ). T h e le v e l of Sa in s a liv a (T a b le 4) w a s 10 to 100 tim e s lo w e r th a n p la q u e le v e ls a n d s u b s ta n tia lly b e lo w M IC lev els. T h is d is trib u tio n of Sa d e m o n s tra te s a h ig h s p e c ific ity fo r p la q u e . C o n se q u e n tly , e f fe c ts o n o ra l m ic ro flo ra w o u ld lik e ly b e m o s t p ro n o u n c e d in p la q u e a n d p ro v id e a p la u s ib le e x p la n a tio n fo r a n tip la q u e a c tiv ity . Sa re te n tio n b y v is u a l a n d p h o to g ra p h ic c o m p a ris o n w ith th e p la q u e d isc lo s a n ts , e ry th ro s in e a n d s o d iu m flu o re s c e in (Fig 1-3), fu r th e r illu s tra te s S a s p e c if ic ity fo r p la q u e . Im m e d ia te ly a fte r r in s in g , Sa h a d a ffin ity fo r d e n ta l p la q u e s im i la r to e ry th ro s in e a n d s o d iu m flu o re sc e in , b u t p la q u e a re a s w e re h ig h e r th a n th e s e a fte r o n e h o u r (T a b le 2). U n lik e e r y th r o s in e , Sa a n d s o d iu m flu o re s c e in d id n o t s ta in th e g in g iv a a n d o th e r so ft ti s su e s , in d ic a tin g a s im ila rity a n d h ig h e r p la q u e s p e c i f ic ity o f S a a n d s o d i u m flu o re s c e in . H o w e v e r, s o d iu m f l u o r e s
c e in w a s le s s re ta in e d th a n th e o th e r d isc lo s a n ts. S u c h re s u lts w o u ld s u g g e s t th e a d d itio n a l b e n e fit of u s in g Sa as a p la q u e - d is c lo s in g a g en t. R e te n tio n o f S a c o u ld b e th e re s u lt o f its c a tio n ic n a tu re . G jerm o 8 h a s illu s tra te d th a t th e c a tio n ic n a tu r e of q u a te r n a r y a m m o n iu m c o m p o u n d s w a s re s p o n s ib le fo r p la q u e re te n tio n a n d th a t r e d u c e d a n tip la q u e efficacy c o rre la te d w ith r e d u c e d p la q u e r e te n tio n of a n tip la q u e a g e n ts . G jerm o f u r th e r d e m o n s tra te d th a t m u lti p le rin s e s o f q u a te rn a ry a m m o n iu m c o m p o u n d s le d to in c re a s e d re te n tio n . T h e se r e s u lts p a ra lle l th o s e r e p o r te d h e re . T h e c a tio n ic n a tu r e o f S a d iffe rs c h e m ic a lly fro m a ll o th e r q u a te r n a r y a m m o n iu m c o m p o u n d s te s te d in th e o ra l c a v ity a s th e r e s u lt o f th e p re s e n c e o f th e im in iu m io n . T h e e x a c t c o n tr ib u tio n of th is c h e m is try to t h e c l i n i c a l e ff e c ts o b s e r v e d w ith s a n g u in a rin e is n o t fu lly u n d e rs to o d at th is tim e . T h e u s e of s a n g u in a rin e in o ra l rin s e s is h ig h ly s p e c ific for d e n ta l p la q u e a n d is re ta in e d in p la q u e a t h ig h le v e ls fo r se v e ra l h o u rs a fte r rin s in g . In c o n tra s t, th e le v els o f s a n g u in a rin e in s a liv a a re re la tiv e ly lo w . T h e r e te n tio n o f s a n g u in a r in e in p la q u e m a y b e re s p o n s ib le fo r its c lin ic a l a n tip la q u e efficacy.
Summary S a n g u in a r in e , a c o m p o n e n t o f s a n g u i n a ria e x tra c t, w a s in v e s tig a te d fo r a n ti p la q u e a c tiv ity a n d r e te n tio n in th e o ral c a v ity . O ra l rin s e s c o n ta in in g s a n g u in a r ia e x tra c t sh o w e d a n tip la q u e a c tiv ity in h u m a n s . U p ta k e a n d r e t e n ti o n le v e ls o f s a n g u i n a r in e in p la q u e a n d sa liv a w e re d e te r m in e d b y h ig h - p re s s u re liq u id c h ro m a to g ra p h y a n d s a n g u in a rin e le v e ls in p la q u e w e re h ig h e r th a n in v itro m in im u m in h ib ito r y c o n c e n tra tio n s a g a in s t o ra l a e ro b ic a n d a n a e ro b ic b a c te ria . F u rth e r, s a n g u in a rin e w as a b le to d is c lo s e p la q u e w ith th e a id o f lo n g -w a v e u ltra v io le t lig h t a n d w a s re ta in e d lo n g e r th a n e ry th ro s in e a n d s o d iu m flu o re s c e in as s h o w n b y m e a s u re m e n t of p la q u e area. It w a s c o n c lu d e d th a t s a n g u in a rin e h a s a h ig h sp e c ific ity a n d re te n tio n in d e n ta l p la q u e . T h e p la q u e -re te n tiv e p ro p e rtie s in c o m b in a tio n w ith a n tim ic ro b ia l a c tio n
m a y b e re s p o n s ib le fo r its c lin ic a l a n ti-
T his study was done by Vipont Laboratories, Inc. The inform ed consent of all hum an subjects w ho participated in the experim ental investigation re ported or described in this m anuscript was obtained after the nature of the procedure and possible discom forts and risks had been fully explained. Dr. Southard is vice-president, research and devel opm ent; Mr. Boulware is a senior chem ist; Mr. Walborn is a m icrobiologist; Ms. Groznik is a dental hygienist; and Ms. Thorne is analytical chem ist, V ipont Laboratories, Inc, 220 E Olive, Ft. Collins, Col 80524. Dr. Yankell is research associate professor, U niversity o f P e n n s y lv a n ia , S c h o o l o f D e n ta l M e d ic in e , Philadelphia. A ddress requests for reprints to Dr. Southard. 1. Lew is, W.H., an d E lvin-Lew is, M. M edical botany. New York, Wiley Interscience, 1977, p 515. 2. A m erican P h arm aceu tical A sso ciatio n . N a tional form ulary, rev 7. W ashington, DC, Am erican Pharm aceutical Assoc, 1940, pp 191, 368, 416. 3. O debiyi, O .O ., an d Sofow ara, E.A. A n tim i crobial alkaloids from N igerian chew ing stick (Faga ra zanghoxyloidesj. P lanta M edica 36:204-207, 1979. 4. Messmer, W.M., and others. Fagaronine, a new tum or inhibitor isolated from Fagara zanthozyloides Lam (Rutaceae). J Pharm Sci 61:1858-1859, 1980. 5. Elvin-Lewis, M., and others. The dental health of chew ing stick users of southern Ghana: prelim i nary findings. J Prevent Den 6:151-159, 1980. 6. Gjermo, P.; Baastad, K.L.; and Rolla, G. The plaque inhibiting capacity of 11 antibacterial agents. J P eriodont Res 5:102-109, 1970. 7. Bonesvoll, P., and Gjermo, P. A com parison be tw een chlorhexidine for some quaternary am m onium com pounds w ith regard to retention, salivary concen tration and plaque-inhibiting effect in the oral cavity after m outh rinsing. Arch Oral Biol 23:289-294,1978. 8. Rolla, G.; Loe, H.; and Schiott, C.R. T he affinity of chlorhexidine for hydroxyapatite and salivary m u cins. J Periodont Res 5:90-95, 1970. 9. Rolla, G.; Loe, H.; and Schiott, C.R. Retention of chlorhexidine in the hum an oral cavity. A rch Oral Biol 16:1109-1116,1971. 10.Ramjford, S.P. Indices for prevalence and inci dence of periodontal disease. J Periodontol 30:51-59, 1959. 11. Turesky, S.; Filmore, N.D.; and Glickman, I. R educed p laq u e form ation by th e c h lo ro m eth y l analogue of victam ine C. J P eriodontol 41:41-43, 1970. 12. Yankell, S., and others. Overnight plaque for m ation. J Prev Dent 6:313-315, 1980. 13. Waler, S.M., and Rolla, G. Plaque inhibiting ef fect of com binations of chlorhexidine and the m etal ions zinc and tin. Acta Odontol Scand 38:213-217, 1980. 14. Boulware, R.T., and others. Effects of sangui n arine oral rinses on saliva glycolysis. European Or ganization for Caries Research, XXX Congress, July 1983.
S outhard-O thers : SANGUINARINE AS ANTIPLAQUE AGENT ■ 341
From teeth-cleaning sticks to a modern oral rinse, use of this herbal extract spans the centuries.
Sanguinarine, a new antiplaque agent: retention and plaque specificity G. Lee S outhard, P hD R ichard T. Boulw are, M S D uane R. W alborn
T
JL he use of herbs has been beneficial to the o ral h e a lth of n a tiv e c u ltu re s th ro u g h o u t th e w o rld for m any c e n tu ries .1 Sanguinaria, an herbal extract, is one such exam ple, but the use of san guinaria extract has not been reported in the dental literature. S a n g u in a ria ex tract (SaE) has been used in a variety of m edications for dec ades in the U nited States and other coun tries .2 It is used in m odern cough syrups and cold rem edies as an expectorant. Its use as a hom eopathic and folklore rem edy predates its m odern-day use. The extract is principally a m ixture of benzophenathridine alkaloids, the chief constituent alkaloid being sanguinarine (Sa). San g u in arin e is h ig h ly fluorescent u n d er long-w ave light, w h ich m akes it rela tively easy to m onitor and analyze. The chem ical structure of sanguinarine is sim ilar to the benzophenathridine alk a lo id a l c o m p o n e n ts of th e F a g a ra zanthoxyloides species of plant, w hich is used in Africa as tooth-cleaning sticks and are reported to be beneficial to the oral hygiene of these native cultures .3'5 C h lo rh e x id in e an d q u a te rn a ry am 338 ■ JADA, Vol. 108, M arch 1984
W ilm a J. G roznik, RDH Eileen E. Thorne, M S Sam L. Y ankell, PhD , RDH
m onium com pounds are two other sub s ta n c e s th a t h av e b e e n e x te n s iv e ly studied for retention, oral cavity concen tration, and antiplaque effects .6,7 The es sential property for antiplaque agents is long-term retention and slow release into the oral cavity .8,9 This study discusses the antiplaque effects of sanguinaria extract an d th e re te n tio n of san g u in a rin e in plaque and saliva quantitatively, visu ally, and photographically.
M aterials and m ethods ANTIPLAQUE METHODOLOGY. T w en ty -fo u r subjects w ere selected for a study conducted at the U niversity of Pennsylvania Dental School. Twel ve subjects were random ly assigned to the tw o test groups and six to the placebo control group. Two blind-coded oral rinses containing sa n g u in aria e x tract (0.045% ), sa n g u in aria extract (0.03%) and zinc chloride (0.2%), and a placebo control w ithout sanguinaria extract or zinc chloride w ere used daily in five rinse periods for seven days. All test products were identically flavored and test subjects could not distinguish the products based on taste. Four rinse periods w ere supervised (two in the m orning and two in the afternoon) for five days. The fifth rinse period and those on the
w eekends were done at home, unsupervised. Rinses lasted for 15 seconds w ith two 15-ml rinses each period. All subjects were super vised w hile they brushed w ith a comm ercially available soft toothbrush and a nonfluoride toothpaste once daily just before the first rinse in the m orning. No other oral hygiene w as al low ed. Safety ex am inations w ere m ade on days 1 ,3 , and 5 just before and im m ediately after the first scheduled m orning rinse, and one hour after the exam ination that followed the brushing session. An additional examina tion was made on day 8. Plaque was scored be fore the start of the study and, on day 8, before product use or brushing. The plaque area was m onitored on the facial surfaces of the maxil lary right first molar, left central incisor and first prem olar, and the m andibular right cen tral incisor,10 using the m ethod described by Turesky and others11 w ith disclosure by m eans of sodium fluorescein and an ultraviolet wave source (wave-length 4,800 A). ANALYSIS OF SALIVA AND PLAQUE. N ineteen volunteers, ranging in age from 18 to 65 years, were instructed to refrain from oral hygiene, eating, and chewing gum for eight hours before using the assigned test rinses. Nine subjects participated in the saliva analysis and ten sub jects participated in the plaque analysis. Each day the subjects w ere assigned a test product,
A R T IC L E S
Fig 1 ■ Sanguinarine baseline.
Fig 2 ■ Sanguinarine one hour later.
Fig 3 ■ Erythrosine baseline.
Fig 4 ■ Erythrosine one hour later.
Fig 5 ■ Fluorescein baseline.
Fig 6 ■ Fluorescein one hour later.
instructed to rinse w ith tw o consecutive 15-ml rinses for 15 seconds, and w ere not allow ed to rinse w ith w ater or eat or drink during the test. Saliva was collected before rinsing at 15, 45, and 90 m inutes after rinsing; the subjects p u t 6 ml of saliva into separate, capped and marked 16- x 150-mm test tubes contained in ice-filled styrofoam containers. At the tim e of collection, 3 ml of the saliva sam ple was used to determ ine antiglycolytic activity.12 An additional 3 ml w as tak en a n d m ix ed w ith 2 m l 0.01 M triethanolam ine phosphoric acid (TEAPA)
buffer and analyzed for sanguinarine by high perform ance liquid chrom atography. Sim i larly, approxim ately 5 to 10 mg of plaque was collected on different days at 30, 60, 90, and 120 m inutes after rin sin g and m ixed w ith TEAPA buffer. Collection was m ade w ith a curette from th e buccal and lingual surfaces and deposited in alum inum tared w eighing containers, 30 x 15 mm in size. Im mediately after being w eighed, the plaque sam ple was deposited into a 16- x 150-mm disposable cul ture tube and rinsed down into the tube w ith
0.01 M TEA PA b u ffe r. T h e sa m p le w as a n a ly z e d fo r s a n g u i n a r i n e b y h ig h perform ance liquid chrom atography.
Visual and photographic analysis13 Selected subjects w ere instructed to refrain from overnight oral hygiene and to rinse w ith a test rinse containing 0.03% SaE, erythrosine, or sodium fluorescein on separate days. The
S outhard-O thers : SANGUINARINE AS ANTIPLAQUE AGENT ■ 339
A R T IC L E S
The use of sanguinarine in oral rinses is highly specific for dental plaque and is retained in plaque at high levels for several hours after rinsing.
plaque areas in subjects using SaE were visu alized using long-wave ultraviolet light and determ ined w ith the m ethod described by Turesky and others11 one hour after rinsing. The plaque area of subjects using erythrosine was assessed under am bient light. The plaque area of subjects using sodium fluorescein was determ ined under an ultraviolet light source (Plak-Lite). P ho to g rap h y w as done w ith a M inolta system w ith Kodacolor films (details are available from the author).
High perform ance liq u id chrom atography Plaque or saliva sam ples in TEAPA buffer were ultrasonicated on a m icroultrasonicator and added by syringe to a Cis Sep-Pak, w hich was previously wet with m ethanol. After the col lection tube was w ashed w ith 2 m l TEAPA, and the w ashings were added to the Sep-Pak, the Sep-Pak w as placed on a 25-ml pearbottom flask and diluted in two stages w ith 12 and 3 ml of m ethanolic hydrochloric acid. The eluate was evaporated to near dryness in vacuo. The Sep-Pak was w ashed and eluted successively tw o additional tim es and the combined eluates w e re e v a p o r a te d to d r y n e s s in v a c u o . M ethanolic p h o sp h o ric acid (0.5 ml) w as added to the residue, the flask was stoppered, and the m ixture w as vortexed for one m inute. A sam ple size of 100 /¿I was injected into a high-pressure liquid chrom atograph, w hich h ad a M-45 delivery system, UKG variable vol um e injector, radia com pression module, 5 CNIOU Radial-Pak Column, a detector with 280 nm filter, and a reporting integrator. Elu tion was done at a flow rate of 0.05 ml/min using a mobile phase of an 84:16 by volum e m ixture of 5 x 10'3 m olar triethylam ine and phosphoric acid in m ethanol and water. The am ount of sanguinarine present was read d i rectly from the integrator.
had significantly low er (P < .05) posttreatm ent plaque scores than the placebo. No subject experienced oral mucosal irrita tion during the test period. When rechallenged and exam ined on day 21, the subjects did not have any signs of sensitization.
P laque retention The fluorescent properties of sanguinarine were used to compare its retention in plaque w ith the conventional plaque disclosants— erythrosine and fluorescein—by m easuring the plaque area im m ediately after rinsing and one hour later. The SaE plaque area showed that subjects were sim ilar to erythrosine and sodium fluorescein im m ediately after rinsing but significantly greater (P < .05) at one hour after rin sin g (Table 1). These observations w ere recorded photographically (Fig 1-6). Plaque retention and saliva levels of sangui n a rin e w ere q u an titativ ely d eterm in ed by h ig h -p erfo rm an ce liq u id ch rom atography. Plaque levels (Table 2) of Sa in subjects treated w ith SaE w ere higher than saliva levels (Table 3). In general, both plaque and saliva levels varied daily among the same people and be tw een people, indicating a dependence on fac tors beyond the controls established for the test. Variability among collected plaque sam ples was greatest at 15 m inutes after rinsing.
Table 1 ■ Visual assessm ent of plaque reten tion of sanguinarine, erythrosine, and sodium fluorescein.
Disclosing agent Sanguinarine Erythrosine Sodium fluorescein
Initially
One hour later
2.87 ± 0.23 2.86 ± 0.22 2.84 ± 0.23
2.66 ± 0.28 2.18* ± 0.53 1.75* ± 0.45
‘ Significantly different from all other values P < .05.
A ntiplaque activity of SaE oral rinses contain ing SaE (0.045%) alone and SaE (0.030%) com bined w ith 0.2% zinc chloride resulted in re duced plaque scores of 19.4% and 20.4%, re spectively, during the eight-day test period. In contrast, the placebo group show ed a 21.3% plaque grow th during the test period. For both test groups, the posttreatm ent plaque scores w ere significantly low er than baseline scores (P < .05) and the placebo (P < .05). Baseline scores among the three groups w ere not signif icantly different and there was no significant difference in plaque reduction between the two groups containing SaE. Both test groups 340 ■ JADA, Voi. 108, M arch 1984
Inhibition of plaque growth and reduc tion in plaque area scores suggest that sanguinarine rinses have both a preven tive and th erap eu tic effect on den tal plaque (Table 4). The fact that sangui narine rinse had such a dem onstrable ef fect on overnight plaque suggests that an optim um tim e for using sanguinarine rinses may be at bedtime. This procedure would capitalize on the retentive prop erties of sanguinarine in plaque during this critical tim e of building plaque .12 The presence of 0.2% zinc chloride in a rinse containing 0.03% SaE was approx im ately the same antiplaque efficacy as a 0.045% SaE rinse w ithout zinc chloride. The role of zinc in accentuating a n ti plaque effects has been reported. Waler and Rolla 13 noted that chlorhexidine gave a better plaque-inhibiting effect in the presence of 0.3% zinc chloride than w hen used alone. It was concluded that zinc ions and chlorhexidine are competitive for the same receptor sites because pre rinses with zinc caused a distinct reduc tion in the effectiveness of chlorhexidine. It has also been observed 14 that the addi-
Table 2 ■ Plaque retention levels of sanguinarine (/xg/g wet plaque).*__________________ Subject no.
Mean plaque area score ± SD
Results A n tiplaque activity
Discussion
T able 3 ■ Saliva levels of sanguinarine (Atg/ml saliva).* Subject no.
1 2 3 4 5 6 7 8 9
45
90
1.13 1.47 1.96 0.80 0.90 0.47 2.63 0.50 1.65
0.92 0.75 0.79 0.57 0.90 0.17 1.25 0.29 0.65
0.45 0.09 1.00 0.32 0.50 0.22 0.54 0.18 0.60
‘ Assays conducted using HPLC.
1 1A 2 2A 3 3A 4 5 6 7
26 110 29 79 111 160
45
30 . .
.+
96 46 42 46
60
90
120
68 46 46 34
26 30 31 46 36 36 51 46 39 33
27 47 24
22 51 27 56 37 34
‘ Assays conducted using high-perform ance liquid chrom a tography (HPLC). tE llip ses indicate plaque not sampled at this time.
Sample tim es after rinsing (min) 15
Sample tim es after rinsing (min) 15
T ab le4 ■ Plaque indexes and percent plaque reduction of sanguinarine orai rinses. Group Placebo SaE, 0.03% and ZnCh, 0.2% SaCl, 0.045%
Baseline (day 0)
After treatm ent (day 8) Reduction (%)
2.44 (0.73) 2.99 (0.96)
2.96 (0.59) 2.38 (0.57)
+21.3 -2 0 .4
2.68 (0.70)
2.16 (0.37)
-1 9 .4
A R T IC L E S
Antiplaque activity of SaE oral rinses containing SaE (0.045%) alone and SaE (0.030%) combined with 0.2% zinc chloride resulted in reduced plaque sr.orps of 19.4% and 20.4%. respectively.
tio n o f z in c io n s to v a ry in g c o n c e n tra tio n s o f S a a ffected s a liv a g ly c o ly sis to a s ig n if ic a n t e x te n t in rin s e s c o n ta in in g SaE a t 0.015 % . It w o u ld a p p e a r th a t th e ef fe c t o f z in c in S a -c o n ta in in g rin s e s is to e n h a n c e th e a c tiv ity o f S a th r o u g h m e c h a n i s m s s i m i l a r to t h o s e w i t h c h l o r h e x id in e b u t n o t y e t fu lly u n d e rs to o d . T h e m e c h a n is m b y w h ic h S a in h ib its p la q u e m a y be a c o m b in a tio n o f a n tim i c ro b ia l a c tio n a n d re te n tio n . T h e a n ti m ic ro b ia l a c tiv ity a g a in s t a v a rie ty o f o ra l a e ro b ic a n d a n a e ro b ic m ic ro o rg a n is m s of p u r e S a c h lo r id e s h o w e d a m in im u m in h ib ito r y c o n c e n tra tio n (MIC) o f 8 to 32 //.g/ml (J. M. G o o d so n , D D S, P h D , a n d S. S o c ra n s k y , D DS, p e rs o n a l c o m m u n ic a tio n s). T h e r e te n tio n of S a in p la q u e v a rie d a m o n g in d iv id u a ls . S a n g u in a r in e c o n c e n tr a tio n in p la q u e w a s ab o v e th e M IC fo r u p to tw o h o u rs a fte r rin s in g (T a b le 3). R e te n tio n v a ria b ility a m o n g in d iv id u a ls w a s s im ila r to th a t o b se rv e d in th e o ra l r e te n tio n o f c h lo r h e x id in e , c e ty lp y rid in iu m c h lo r id e , a n d h e x a d e c y ltrim e th y la m m o n iu m b ro m id e .9 V a ria b ility o f S a le v e ls in p la q u e a m o n g in d iv id u a ls a n d w ith in in d iv id u a ls at d iffe re n t tim e s m a y b e th e re s u lt o f th e ra n d o m n e s s of p la q u e s e le c tio n site s o r d ie ta ry o r o th e r e n v ir o n m e n ta l in flu e n c e s p re s e n t in i n d iv id u a ls a t th e tim e o f s a m p lin g (or all th re e ). T h e le v e l of Sa in s a liv a (T a b le 4) w a s 10 to 100 tim e s lo w e r th a n p la q u e le v e ls a n d s u b s ta n tia lly b e lo w M IC lev els. T h is d is trib u tio n of Sa d e m o n s tra te s a h ig h s p e c ific ity fo r p la q u e . C o n se q u e n tly , e f fe c ts o n o ra l m ic ro flo ra w o u ld lik e ly b e m o s t p ro n o u n c e d in p la q u e a n d p ro v id e a p la u s ib le e x p la n a tio n fo r a n tip la q u e a c tiv ity . Sa re te n tio n b y v is u a l a n d p h o to g ra p h ic c o m p a ris o n w ith th e p la q u e d isc lo s a n ts , e ry th ro s in e a n d s o d iu m flu o re s c e in (Fig 1-3), fu r th e r illu s tra te s S a s p e c if ic ity fo r p la q u e . Im m e d ia te ly a fte r r in s in g , Sa h a d a ffin ity fo r d e n ta l p la q u e s im i la r to e ry th ro s in e a n d s o d iu m flu o re sc e in , b u t p la q u e a re a s w e re h ig h e r th a n th e s e a fte r o n e h o u r (T a b le 2). U n lik e e r y th r o s in e , Sa a n d s o d iu m flu o re s c e in d id n o t s ta in th e g in g iv a a n d o th e r so ft ti s su e s , in d ic a tin g a s im ila rity a n d h ig h e r p la q u e s p e c i f ic ity o f S a a n d s o d i u m flu o re s c e in . H o w e v e r, s o d iu m f l u o r e s
c e in w a s le s s re ta in e d th a n th e o th e r d isc lo s a n ts. S u c h re s u lts w o u ld s u g g e s t th e a d d itio n a l b e n e fit of u s in g Sa as a p la q u e - d is c lo s in g a g en t. R e te n tio n o f S a c o u ld b e th e re s u lt o f its c a tio n ic n a tu re . G jerm o 8 h a s illu s tra te d th a t th e c a tio n ic n a tu r e of q u a te r n a r y a m m o n iu m c o m p o u n d s w a s re s p o n s ib le fo r p la q u e re te n tio n a n d th a t r e d u c e d a n tip la q u e efficacy c o rre la te d w ith r e d u c e d p la q u e r e te n tio n of a n tip la q u e a g e n ts . G jerm o f u r th e r d e m o n s tra te d th a t m u lti p le rin s e s o f q u a te rn a ry a m m o n iu m c o m p o u n d s le d to in c re a s e d re te n tio n . T h e se r e s u lts p a ra lle l th o s e r e p o r te d h e re . T h e c a tio n ic n a tu r e o f S a d iffe rs c h e m ic a lly fro m a ll o th e r q u a te r n a r y a m m o n iu m c o m p o u n d s te s te d in th e o ra l c a v ity a s th e r e s u lt o f th e p re s e n c e o f th e im in iu m io n . T h e e x a c t c o n tr ib u tio n of th is c h e m is try to t h e c l i n i c a l e ff e c ts o b s e r v e d w ith s a n g u in a rin e is n o t fu lly u n d e rs to o d at th is tim e . T h e u s e of s a n g u in a rin e in o ra l rin s e s is h ig h ly s p e c ific for d e n ta l p la q u e a n d is re ta in e d in p la q u e a t h ig h le v e ls fo r se v e ra l h o u rs a fte r rin s in g . In c o n tra s t, th e le v els o f s a n g u in a rin e in s a liv a a re re la tiv e ly lo w . T h e r e te n tio n o f s a n g u in a r in e in p la q u e m a y b e re s p o n s ib le fo r its c lin ic a l a n tip la q u e efficacy.
Summary S a n g u in a r in e , a c o m p o n e n t o f s a n g u i n a ria e x tra c t, w a s in v e s tig a te d fo r a n ti p la q u e a c tiv ity a n d r e te n tio n in th e o ral c a v ity . O ra l rin s e s c o n ta in in g s a n g u in a r ia e x tra c t sh o w e d a n tip la q u e a c tiv ity in h u m a n s . U p ta k e a n d r e t e n ti o n le v e ls o f s a n g u i n a r in e in p la q u e a n d sa liv a w e re d e te r m in e d b y h ig h - p re s s u re liq u id c h ro m a to g ra p h y a n d s a n g u in a rin e le v e ls in p la q u e w e re h ig h e r th a n in v itro m in im u m in h ib ito r y c o n c e n tra tio n s a g a in s t o ra l a e ro b ic a n d a n a e ro b ic b a c te ria . F u rth e r, s a n g u in a rin e w as a b le to d is c lo s e p la q u e w ith th e a id o f lo n g -w a v e u ltra v io le t lig h t a n d w a s re ta in e d lo n g e r th a n e ry th ro s in e a n d s o d iu m flu o re s c e in as s h o w n b y m e a s u re m e n t of p la q u e area. It w a s c o n c lu d e d th a t s a n g u in a rin e h a s a h ig h sp e c ific ity a n d re te n tio n in d e n ta l p la q u e . T h e p la q u e -re te n tiv e p ro p e rtie s in c o m b in a tio n w ith a n tim ic ro b ia l a c tio n
m a y b e re s p o n s ib le fo r its c lin ic a l a n ti-
T his study was done by Vipont Laboratories, Inc. The inform ed consent of all hum an subjects w ho participated in the experim ental investigation re ported or described in this m anuscript was obtained after the nature of the procedure and possible discom forts and risks had been fully explained. Dr. Southard is vice-president, research and devel opm ent; Mr. Boulware is a senior chem ist; Mr. Walborn is a m icrobiologist; Ms. Groznik is a dental hygienist; and Ms. Thorne is analytical chem ist, V ipont Laboratories, Inc, 220 E Olive, Ft. Collins, Col 80524. Dr. Yankell is research associate professor, U niversity o f P e n n s y lv a n ia , S c h o o l o f D e n ta l M e d ic in e , Philadelphia. A ddress requests for reprints to Dr. Southard. 1. Lew is, W.H., an d E lvin-Lew is, M. M edical botany. New York, Wiley Interscience, 1977, p 515. 2. A m erican P h arm aceu tical A sso ciatio n . N a tional form ulary, rev 7. W ashington, DC, Am erican Pharm aceutical Assoc, 1940, pp 191, 368, 416. 3. O debiyi, O .O ., an d Sofow ara, E.A. A n tim i crobial alkaloids from N igerian chew ing stick (Faga ra zanghoxyloidesj. P lanta M edica 36:204-207, 1979. 4. Messmer, W.M., and others. Fagaronine, a new tum or inhibitor isolated from Fagara zanthozyloides Lam (Rutaceae). J Pharm Sci 61:1858-1859, 1980. 5. Elvin-Lewis, M., and others. The dental health of chew ing stick users of southern Ghana: prelim i nary findings. J Prevent Den 6:151-159, 1980. 6. Gjermo, P.; Baastad, K.L.; and Rolla, G. The plaque inhibiting capacity of 11 antibacterial agents. J P eriodont Res 5:102-109, 1970. 7. Bonesvoll, P., and Gjermo, P. A com parison be tw een chlorhexidine for some quaternary am m onium com pounds w ith regard to retention, salivary concen tration and plaque-inhibiting effect in the oral cavity after m outh rinsing. Arch Oral Biol 23:289-294,1978. 8. Rolla, G.; Loe, H.; and Schiott, C.R. T he affinity of chlorhexidine for hydroxyapatite and salivary m u cins. J Periodont Res 5:90-95, 1970. 9. Rolla, G.; Loe, H.; and Schiott, C.R. Retention of chlorhexidine in the hum an oral cavity. A rch Oral Biol 16:1109-1116,1971. 10.Ramjford, S.P. Indices for prevalence and inci dence of periodontal disease. J Periodontol 30:51-59, 1959. 11. Turesky, S.; Filmore, N.D.; and Glickman, I. R educed p laq u e form ation by th e c h lo ro m eth y l analogue of victam ine C. J P eriodontol 41:41-43, 1970. 12. Yankell, S., and others. Overnight plaque for m ation. J Prev Dent 6:313-315, 1980. 13. Waler, S.M., and Rolla, G. Plaque inhibiting ef fect of com binations of chlorhexidine and the m etal ions zinc and tin. Acta Odontol Scand 38:213-217, 1980. 14. Boulware, R.T., and others. Effects of sangui n arine oral rinses on saliva glycolysis. European Or ganization for Caries Research, XXX Congress, July 1983.
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