- Email: [email protected]
Selective suppression of judged sweetness by ziziphins
Physiology&Behavior, Vol. 30, pp. 867-874. PergamonPress Ltd., 1983. Printedin the U.S.A.
Selective Suppression of Judged Sweetness by Ziziphins V I V I A N V. S M I T H z A N D B R U C E P. H A L P E R N 3
Department o f Psychology and Division o f Biological Sciences Section o f Neurobiology and Behavior, Cornell University, Ithaca, N Y 14853 R e c e i v e d 7 July 1983 SMITH, V. V. AND B. P. HALPERN. Selective suppress!on of judged sweetness by ziziphins. PHYSIOL BEHAV 30(6) 867-874, 1983.--The effects of ziziphins and of control treatments upon judgments by human adults of the sweetness, sourness, bitterness, and saltiness of American apple cider or apple juice wdre measured with a category estimation method during repeated trials before, during (90 sec treatment duration only), and after, treatment. Sweetness was reduced after either a 10 sec or a 90 sec whole mouth treatment with ziziphins, but not after quinine sulfate or apple juice control treatment. No differences in after-treatment sourness, bitterness, or saltiness occurred between treatments. The reduction in sweetness was weak with 10 sec 3.5% W/V ziziphins treatment, but strong after 90 sec 0.88% W/V ziziphins treatment; duration of suppression was ca. 70 sec. The mechanism was identified as taste modification since adaptation, crossadaptation, and mixture suppression were eliminated by control treatments and by post-treatment rests and rinse. Comparisons with known gymnemic acids effects suggest that net dissociation of ziziphins from taste receptor membranes and/or inactivation in the membrane may be much faster than with gymnemic acids. Taste modifiers
Ziziphins
Sweetness
Apple cider
ZIZIPHINS are taste modifying saponins in the leaves of the Chinese jujube tree, Ziziphus jujuba P. Miller (Rhamnaceae) [7]. Extracts containing these saponins (ZjE) reversibly depress for human adults the judged sweetness of sucrose solutions [5, 7, 16]. In the only published test of ZjE selectivity [16], the extracts produced no change in which taste quality category the judges assigned to solutions of NaCI, HCI, sucrose, or quinine hydrochloride, and depressed the perceived sweetness intensity of sucrose solutions without changing the saltiness o f a NaC1 solution or the bitterness of a quinine hydrochloride solution. Meiselman et al. did find that the sourness of a HC1 solution was judged to increase after treatment with any one of several ZjE fractions derived from an aqueous-alcohol extract of Z. jujuba, but indepdently of the sweetness modifying ability of that fraction [16]. Thus, the question of ZjE selectivity is unresolved, because effects on both judged sweetness and sourness have been reported. Recovery of judged sweetness after ZjE treatment is though to be more rapid than recovery after a gymnemic acids treatment [8]. However, effects of ZjE treatment duration have not been examined, and selectivity during the recovery process has not been studied. Further, although a model has been developed which attempts to described the general nature of ZjE actions on gustatory receptor cell membranes [8], no analysis of the initial interaction at the receptor cell membrane has been made. The present experiments were designed to clarify the degree of ZjE selectivity, both during the time of preak suppression and during re-
Apple juice
covery. In addition, an initial examination was made of competitive inhibition as the mechanism of action of ZjE. To evaluate the taste-modifying selectivity of ZjE, we chose apple cider and apple juice as stimulus liquids. These unfermented natural beverages [20,21], which are approximately 12% sugar (fructose, dextrose, and sucrose: personal communication, V. L. Bump, Seneca Brand Products), elicit a range of taste qualities, with sweetness and sourness consistently reported. There is little bitterness (in American apple cider or juice) and no saltiness. Repeated, four-tastequality category scale intensity judgments of these complex stimulus liquids were made before and after ziziphins and control treatment in order to both reveal and track over time ziziphins-dependent taste changes. METHOD Participants were non-smoking paid volunteers who passed a screening test for consistency and sensitivity of gustatory judgments [12]. All were Cornell University students or staff. For Experiment 1 (effect of brief treatment) the ages of the six participants ranged from 19 to 21 years; four were female. In Experiment 2 (competition and mixture effects), the eight participants ranged in age from 19 to 26 years; three were female. Green Ziziphus jujuba leaves were gathered in October, 1978, directly from a 59 year old tree (H-124) at the Monroe County Arboretum of Highland Park, Rochester, NY. They were freeze-dried and then stored in sealed plastic containers
1Supported by NSF Grant BNS-8014148 to B.P.H. and College of Arts and Sciences Undergraduate Research Grant to V.V.S. 2Present address: 34 Chestnut Drive, Hastings-on-Hudson, NY 10706. 3Requests for reprints should be addressed to Bruce P. Halpern.
CoDvri~ht © 1983 Pergamon Press Ltd.--0031-9384/83/060867-08503.00
868
SMITH AND HALPERN
Experiment Two
Experiment One Adult Human Who Has Passed Screening Test
Adult Human Who Has Passed
Screening Test __~
Hold 10 ml Apple ~lCider in Mouth Move Tongue
YES
J Ho'dtom' AOO'e L
and
-
\ s0it0u,/
Juicein Mouth
l and Move Tongue r NO
ES
9 yESNO "= I Rinse Mouth
I
with 5 ml water ] Judge Intensity for Four
Vigorously with 10 m[
nse
YES
Judge / Intensity for Four Qualities /
\spi, f out/ H~_,
Samples of Apple Cider
NO =-
\App,,
YES /"
Treatment"'-,..,NO
=
=
Hold 10 ml Treatment Liquid in Mouth and
YES
~
Judge Intensity / for Four / Qualities /
Move Tongue
~'ES
J Hold 10 ml of Treatment -J Liquid in Mouth
NO~E}even .Samples~
V o-i-~ppl, Juice ">
FIG. 1. Flow diagram for experiment one. Six participants were tested in each of four separate sessions. The first session was a practice session; its data were not used. Sessions one and two used the control treatment liquid: 24/xM quinine sulfate in distilled water. Sessions three and four used the experimental treatment liquid: 3.5% W/V aqueous extract of Z.jujuba leaves (ZjEw). Here, and in Fig. 2, the "seconds counters" reset to zero seconds counted after a YES output.
FIG. 2. Flow diagram for experiment two. Eight participants were tested in each of four separate sessions. The first session was a practice session; its data were not used. Sessions one and two used the apple juice control treatment liquid. Session three used the 0.88% W/V ZjEw in apple juice experimental treatment liquid. Session four used the quinine control treatment liquid: 66/xM quinine sulfate in apple juice.
at room temperature. A 3.5% W/V aqueous extract (ZjEw) was prepared from the leaves [6]. F o r both experiments, the distilled w a t e r ' s refractive index (n) was 1.3330; its conductivity was less than or equal to 1.8/xS. The quinine sulfate was U . S . P . ; the apple concentrate, which was Seneca frozen CT 28, no preservatives, no sugar added (the same product was designated as either apple cider or apple juice (V. L. Bump, S e n e c a Brand Products, personal communication)), was reconstituted with distilled water according to directions supplied with the product; the p r u n e iuice was S u n s w e e t bottled. All stimulus, treatment, and rinse liquids were administered at 22---I°C. Participants were requested not to eat or drink anything in the hour prior to each session. Participants were instructed
to " P l e a s e m o v e your tongue around while tasting the liqu i d s " . . . Sweetness, sourness, bitterness, and saltiness intensity were m e a s u r e d at each j u d g m e n t using a category estimation method [4,17]. For e a c h j u d g m e n t , participants w e r e instructed to " . . . . indicate on a scale from 1 to 10 how sweet, sour, salty and bitter the liquid is (I means the liquid has none o f that taste quality and 10 means it is extrmely high in that quality). E a c h liquid may have more than one t a s t e . " On each j u d g m e n t sheet there were four 13 cm lines marked " S w e e t n e s s , " " S o u r n e s s , " " S a l t i n e s s , " or " B i t t e r n e s s , " each labeled " N o n e " at the left end, " E x t r e m e " at the far right. The lines w e r e n u m b e r e d 1 (none) to 10 (extreme) with equidistant arabic numerals. The participants marked the lines after the stimulus liquid was spit out
S U P P R E S S I O N O F S W E E T N E S S BY Z I Z I P H I N S but prior to the next stimulus, before and after treatments in both experiments, and also during treatment, i.e., with treatment liquid in the mouth, in Experiment 2. A separate judgment sheet was used for each judgment. Statistical analysis was done separately for each quality category. Evaluation of successive pairs of trials within each treatment condition was done for Experiment 1 only, using the Wilcoxon Matched Pairs Signed Rank Test. Overall results of each experiment were evaluated with the Kramer Test [10] across all trials, both between treatment conditions and within each treatment condition. The within treatment Kramer Test [10] analysis permitted identification of trials on which judged intensity was greater or less than that expected from a chance distribution.
869 Extreme
10-
8
E
Experiment Two The stimulus liquid and the solvent for the ZjEw and quinine control treatment liquids was Seneca Natural Style apple juice (Fig. 2). F o r the ZjEw treatment, 3.5% W/V ZjEw was prepared and immediately freeze-dried until needed. To make the ziziphins treatment liquid, the freeze-dried extract was reconstituted to its original concentration with distilled water, and then diluted 3:1 with apple juice, giving a final ZjEw treatment concentration of 0.88% W/V. The quinine control liquid, which allowed mixture suppression effects to be identified, was 66/~M quinine sulfate in apple juice:prune juice, 9:1. Each participant made judgments in tour sessions. The first two sessions used an apple juice only treatment (for control of adaptation and sugar effects) the third, the ZjEw treatment liquid; the fourth, the quinine control treatment liquid. The first session was a practice session. In each session, a total of eleven 10 ml samples of apple juice were held in the mouth for three sec, then spit out and judged. Another apple juice sample was presented 10 see later, with no rinses. Ten sec after the third apple juice sample was spit out, 10 ml of a treatment liquid was taken into the mouth and held for 90 sec. Judgments continued every 10 see during the treatment. The relevant instructions were " . . . you will be given a liquid to hold in your mouth, this time for 90 second~. During the time it is in your mouth, you will be asked to judge the taste of the liquid 9 times (every 10 seconds, as I signal you) using the same type of scoring sheet." After the treatment
-
m
6
w
o
..
T -1i i I Sour I h' I I ~,~" I
Experiment One The stimulus liquid was Seneca Apple Barrel apple cider (Fig. 1). The ziziphins treatment was freshly prepared 3.5% W/V ZjEw [6]; the control treatment was 24 /~M quinine sulfate in distilled water: prune juice, 4:1. Each participant made judgements in four sessions. The first two sessions used the control treatment; the last two, the ZjEw treatment. The first session was a practice session. In each session, a total of eight 10 ml samples of apple cider were held in the mouth for 3 sec, then spit out and judged. Another apple cider sample was presented 40 sec after spitting out the preceding sample, with no rinses. Forty seconds after the third apple cider sample, 10 ml of treatment liquid was presented. It was held in the mouth, with tongue motion encouraged, for 10 sec. The treatment liquid was then spit out. After a 5 sec rest, participants vigorously rinsed with 10 ml distilled water, spit out, rested for 10 sec, and then began a series of five post-treatment judgments of apple cider samples. After all four sessions had been completed, each participant made one four-quality judgment of the ZjEw and of the 24 /~M quinine control treatment liquids.
;~
Sweet
~
g 2
Q ....
I
I
I
I
J-
Nonq
Q-
!
I
:1,+, ,,V' \
+__,__+/
-v-
I ',
k~"
', !
~-
•
I
:
*
.
: ;
' '
\'* - - + <,,B i t t e r ±I "
i l
_L
I. . . .
,"
~1
II I
•
Salty
~.£ I
i
i
~
i
I
i
I
I
I
2
3
I.
5
6
7
8
Triols ' , ' 58 101 I~ & 187 Time After Treotment (sec)
IJ5
FIG. 3. Median category scale ( l = n o n e of that quality, 10=extremely high intensity of that quality) intensity judgments, +_standard error of the median, made by 6 participants for sweet, sour, bitter, and salty taste quality categories, before and after a whole mouth l0 ml treatment, are shown here and in Fig. 4. Sweetness judgements are shown by squares and solid connecting lines; sourness, solid circles and broken lines; bitterness, diamonds and heavy broken lines; saltiness, stars. Standard errors are indicated by thin vertical lines (terminated with short horizontal lines for sourness), solid for sweet; broken for sour; dashed for bitter; and dotted for salty; with errors less than 0.2 not plotted. Treatment for Fig. 3 was 3.5% W/V ZjEw.
liquid was spit out, there was a 10 sec rest, a 5 sec rinse with l0 ml distilled water, and another l0 sec rest. Stimulus liquid presentations then resumed for the remaining eight presentations. A total of 20 four-quality intensity judgments were made in each session. RESULTS
Experiment One Between conditions, i.e., control versus ZjEw treatment conditions, there were no statistically significant differences (Kramer Test, p >0.05) over all 16 trials, although the differences between conditions was greatest immediately after treatment for sweetness on trial 4, with the sum of ranks one less than that required for significance. Within treatment conditions (8 trials each), the ZjEw treatment condition produced a significant decrease (Kramer Test, p~<0.05) in judged sweetness of apple cider only on the trial immediately following the 10 second treatment (Fig. 3). No significant deviations from a chance distributions (Kramer Test, p >0.05) of intensity judgments occurred across all 8 trials for sourness, bitterness, or saltiness with either treatment, or for any quality category with the quinine control treatment (Fig.
870
SMITH AND HALPERN 4). The successive pairs of trials analysis with the Wilcoxon Signed Rank test showed no significant differences for any taste quality for the quinine control treatment condition (p>0.09, one-tailed). However, for both ZjEw treatment condition sessions there was a significant change in sweetness between trials 3 and 4 (Wilcoxon, p<~0.05, one-tailed) (Fig. 3), but not between preceding or following pairs of trials (p>0.09, one-tailed). No other comparisons of pairs for the ZjEw treatment condition reached statistical significance (p~>0.09), except for bitterness between trials 3 and 4 of the first ZjEw treatment session (p=0.05). The ZjEw treatment liquid received a median quality profile of sweetness 1, sourness 2, saltiness 1, and bitterness 8.5; the 24/zM quinine sulfate control, 2.5, 2, 1, and 4, respectively.
Extreme 10-
8 o
~
E
6 i I
w
t Sour t
g
I i
~ ~"~
I
,
/
ul
& ..
I
,, t
±
",
I E
T I
I
g
~-. T ,/ i " ' ~ , . . . . +"
IE
I d.
¢.s=., i
: Bitter'ff
I
2
I
3
Experiment Two
i
I I I .1_
i e. ,,
Non
I
+
°
o
Between treatment comparisons. On before-treatment trials (trials 1-3), no significant differences occurred between the three treatment conditions for any of the four taste quality categories on which intensity judgments of the apple juice samples were made (p>0.05, Kramer Test) (Fig. 5). During treatment, the ZjEw produced bitterness judgments larger than those during the apple juice treatment but less than those during the quinine control treatment. In addition, sweetness intensity during ZjEw treatment was less than that during apple juice treatment (Fig. 5). The quinine control treatment produced bitterness judgments which exceeded those with apple juice treatment for almost the entire 90 sec treatment period (trials 4--11, p<0.05, Kramer Test). Differences in judged sourness between quinine control and apple juice treatments (p~<0.05, Kramer Test) occurred on two during-treatment trials (4 and 5), while quinine control
+
i
i
i
I
i
4
5
6
7
8
Triats i
i
i
i
15
58
I01
144
Time
After
Treotment
187 (sec)
FIG. 4. Median category scale intensity judgements, _+standard error of the median, before and after treatment with 24 I~M quinine sulfate control liquid. Symbols, etc. as in Fig. 3, except that the bitterness data points are not connected.
TABLE 1 WITHIN-TREATMENTCONDITIONKRAMERTEST STATISTICALLYSIGNIFICANT(p<0.05) DIFFERENCESIN DISTRIBUTIONOF JUDGED INTENSITYACROSSALL 20 TRIALSOF APPLE JUICE Trials Treatment Condition
Taste Quality Category
3.5% W/V Ziziphins in Apple Juice
Sweet Sour Salty Bitter
Apple Juice
Sweet Sour Salty Bitter
66 tzM Quinine Sulfate in Apple Juice
Sweet Sour Salty Bitter
During Treatment 1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17 18 19 20
:~ :1: ¢
+
+
+
+
+
+
+
$
+
÷
+ +
+
+
+
+
+
+
+
+
+
+
+
+
Plus symbols (+) indicate trials on which, for that quality category, intensity was greater (p~0.05) than expected from a chance distribution of judged intensities across the 20 trials; ¢p~<0.01. Minus symbols (-) indicate trials on which, for that quality category, intensity was less (p~<0.05) than that expected from a chance distribution of judged intensity across the 20 trials; =p~<0.0l.
SUPPRESSION OF SWEETNESS BY ZIZIPHINS
L
Trea
871
t me n t
Apple
Juice
Jk LL L
4 _~
2 I
I
I
I
I
I
,
,
I
I
8-
1
i
Ziziphins
,-
m
2~ r
1
I
1
'
'
r
I
1 r 2 , 3 14
/
Control
, 5'
6
7 ' 8'
'
'
'
•
•
•
'
I
I
I
I
1
9 '10
11
12113
14
15
16
Trials FIG. 5. Median category scale intensity judgements, _+standarderror of the median (solid vertical bars; errors <0.1 are not shown), made by 8 participants for sweet (clear columns), sour (upward diagonal columns), bitter (dotted columns), and salty (downward diagonal columns) taste qualities, l=none of that quality; 10=extremely high intensity of that quality. Statistically significant (p<0.05, Kramer Test) differences between 3.5% ziziphins (ZjEw) treatment condition (trials 1-20) and apple juice or 66/zM quinine sulfate control treatment trials are shown by an asterisk for differences in sweetness intensity; by an encircled asterisk, for differences in bitterness intensity. No significant differences 'between ZiEw treatment condition trials and the apple juice or quinine control treatment (trials 1-20) were found for sourness or saltiness.
treatment judged sweetness was less than apple juice treatment judged sweetness (p~<0.05, Kramer Test) on most during-treatment trials (trials 4--8 and 10). After treatment, only differences in sweetness intensity occurred (except for one apple juice treatment condition versus quinine control treatment condition difference in apple juice sourness on trial 20). The judged sweetness of apple juice after the ZjEw treatment was significantly less (p ~<0.05, Kramer Test) than the sweetness of apple juice after the apple juice treatment on after-treatment trials 14 and 15, and was significantly less than the sweetness intensity after the quinine control treatment on the first four after-treatment trials (trials 13-16) (Fig. 5). Within treatment effects. Within each treatment condition, separate Kramer Tests for each quality category were done to identify trials on which intensity was greater or less than that expected from a chance, i.e., p >0.05, distribution of judged intensities across the 20 trials. All intensity judgments which significantly departed from a chance level are indicated in Table 1.
DISCUSSION Sweetness of apple cider/apple juice was reduced following whole mouth, sip and spit exposure to ZjEw. For a 10 sec treatment with 3.5% W/V ZjEw (Experiment 1), judgments of taste intensity of apple cider were made before and after treatment. The sole statistically significant effect across all eight trials of a condition was decreased sweetness on the first trial after the rest and water rinse which followed the brief ziziphins treatment. This sweetness reduction after ZjEw but not after quinine control treatment was confirmed by comparison of successive pairs of trials. Median sweetness intensity was depressed through 101 sec after the 10 sec ZjEw treatment, but a statistically significant reduction occurred only at 15 sec after treatment, and not at the 58 sec or 101 sec after treatment measurement times. The relatively high variability of the data from experiment one could have masked significant reductions at these two latter judgment times. This problem can be clarified by a comparison with the data of Experiment 2, in which less
872 variability occurred. For Experiment 2, taste modification was also of relatively short duration. That is, sweetness was significantly depressed only through 65 sec after the ZjEw treatment of Experiment 2. In this case, median sweetness intensities at 75 sec after treatment and at subsequent judgment times after the 90 sec ZjEw treatment were at pretreatment levels. Since brief actions of ZjEw are indicated in both Experiments 1 and 2, it may be correct to expect that the 10 sec ZjEw treatment of experiment one would produce a somewhat shorter period of sweetness reduction than the 90 sec ZjEw treatment duration of Experiment 2. A potential weakness of Experiment 1 was the greater bitterness of 3.5% W/V ZjEw in comparison with the 24 ~M quinine control treatment liquid. Although compounds judged bitter do not generally cross-adapt upon judged sweetness [1], a control treatment liquid with a judged bitterness at least as high as that of the ZjEw treatment would have been desirable. No across-all-trials of each condition significant differences occurred in judged sourness, bitterness, or saltiness in Experiment 1. For sourness and saltiness, absence of both quinine control treatment and ZjEw treatment condition effects was confirmed by the successive pairs of trials analysis, but bitterness did show one significant change in the first ZjEw session (Fig. 1). A between-treatments comparison of the ZjEw treatment conditions with the 24 p~M quinine sulfate control condition showed a trend towards reduced sweetness after the ziziphins treatment, but statistical significance was not reached. The weakness of the rather selective effect of ZjEw on sweetness in Experiment 1 may be due to the limited time during which the ziziphins could interact with taste receptor cell membranes [3,7] and/or it may be related to the proportion of receptor cells reached during the brief treatment. However, a similar treatment with another taste modifier, gymnemic acids, with 5 ml of liquid held in the mouth for only 3 sec (in a mixture with sucrose), reduced the sweetness of sucrose by approximately 60% when measured 3 min after the brief gymnemic acids treatment [14]. No rinse was used, but the sweetness suppression was significantly greater than that due to a control treatment. This powerful and long lasting effect of gymnemic acids on sweetness [14] indicates that limited access to and contact time with human taste receptors does not necessarily produce a weak and transient taste modification of sweetness. Rather, differences between gymnemic acids and ziziphins are indicated. Other factors in the present ZjEw experiment (Experiment 1) may also have contributed to the difference. Such factors might include the absence during treatment of sugars which, if present, could have permitted a use-dependent binding of ZjEw to membrane sites; complex effects of the several different sugars contained in apple cider; the mixture interaction possibility of apple cider, and perhaps a lack of similarity of active taste modifier concentrations. In general, this selective but relatively weak sweetness suppression by a brief ZjEw treatment supports previous reports ot Oltterences between ZjEw and gymnemic acids [6, 7, 16]. On the other hand, the selective taste modification produced by a brief treatment of ZjEw followed by a rinse is compatable with an action at the taste receptor cell membrane [8]. A clear, powerful (ca. 50% reduction of judged sweetness), but still selective and relatively brief taste modification by ZjEw appeared after the 90 sec treatment of Experi= ment 2. It is interesting that, although the ZjEw concentration was 0.88% W/V rather than the 3.5% W/V of Experiment
SMITH AND HALPERN 1, a more prolonged reduction of sweetness occurred. That is, sweetness was reduced re: the 66 ~M quinine sulfate control on the four successive trials which occurred after the rests and H20 rinse which followed the 90 sec ZjEw treatment. Since the rinse and rests occupied 25 sec, while judgments were made every 10 sec, sweetness was attenuated for one minute after the treatment. In contrast, the sweetness suppression of Experiment 1 (10 sec treatment) was seen at 15 sec but not at 58 sec post-treatment. The greater sweetness suppression produced by a longer (90 sec vs. 10 sec) but lower concentration (0.88% versus 3.5%) ZjEw treatment suggests that the rate at which ziziphins associate with the taste receptor cell membrane such that sweetness modification occurs is relatively low. The comparison in a preceding paragraph between gymnemic acids and ZjE indicated that a brief gymnemic acids treatment was much more effective in sweetness suppression than a brief ZjE treatment. This might suggest either that the rate at which gymnemic acids associate with the taste receptor cell membrane to produce taste modification is faster than that of ZjE, or that gymnemic acids' dissociation from or inactivation in the membrane is much slower than ZjE, giving a greater net taste modification. The long duration of taste modification following gymnemic acids treatment (e.g., [14,15]) supports a slow dissociation and/or inactivation of gymnemic acids. However, ZjEw could have a nonmonotonic dose-response function in humans, as ZjE have in the blowfly [9]. This would cause ZjEw concentrations above some level to be less effective than lower concentrations. The observed suppression of the sweetness of apple cider/ apple juice after the ZjEw treatments of the present study is briefer than the previously reported 5-6 min reduction in judged sweetness of 80 mM sucrose by ZjE [5,7]. These earlier studies used a 3 min treatment duration, which may have been long enough to permit penetration by ziziphins into taste receptor cell membranes. If such penetration occurred, it could alter membrane structure and produce nonselective changes in taste intensity [3,8]. However, only judged sweetness, rather than the intensity of several taste quality categories, was measured. One earlier investigation which had a 3 min ziziphins treatment [16] did use NaC1, HC1, and quinine hydrochloride, as well as sucrose, to evaluate the selectivity of the treatment. However, posttreatment effects were measured at only one time: less than one minute after the end of the treatment. Maximum effect upon membrane structure may require a longer total action time once penetration has begun 13, 8, 9]. Consequently, the relative importance for the present brief sweetness suppression of factors such as the sweet and sour taste of apple cider/apple juice, the 90 sec versus 3 min treatment duration, and/or the possible interactions between the ZjEw and the sugars, etc. present during treatment, can not now be evaluated. A study which employed ziziphins treatments of 3 min or more, and measured the post-treatment effects for at least 10 rain, using a mutliple quality category technique and suitable stimuli, could clarify this question. With gymnemic acids, a study similar to that just proposed, but with a treatment duration of only 60 sec, found effects only on sweetness [19]. Taste modification is clearly identified only when alternative processes which can reduce taste intensity are eliminated. With prolonged treatment, two such processes are adaptation and mixture suppression. Apple juice was both the liquid judged before and after treatment and the solvent for the 90 sec treatments of Experiment 2 (with prune juice
S U P P R E S S I O N O F S W E E T N E S S BY Z I Z I P H I N S added for the quinine control treatment). Consequently, direct adaptation by the sugars, acids, and other constitutents of apple juice would be comparable across treatments, and would be mathematically cancelled out by between treatment comparisons. In addition, the absence of reduced sweetness or sourness after the apple juice treatment demonstrates that the post-treatment rests and rinse were sufficient to remove its adaptation effects. The potential for cross-adaptation differs between treatments. Both the ZjEw and quinine control treatments are more bitter than the apple juice treatment, with the quinine control treatment more bitter than the ZjEw. However, available data do not indicate cross-adaptation of compounds judged bitter upon judged sweetness [1]. If mixture suppression produced during treatment continued after treatment, a reduction in post-treatment sweetness proportional to the bitterness, sourness, and/or saltiness of the treatment would be expected [2]. There was no saltiness during the treatments (or before treatment), no significant differences in sourness between ZjEw and control treatments, and no increased sourness during any treatment. It was already noted that both the ZjEw and the quinine control treatments were more bitter than the apple juice, with the quinine control treatment the most bitter. Therefore, if mixture suppression due to the taste of the treatments were not eliminated by the rests and rinse, but instead continued into the post-treatment trials, the greatest sweetness suppression would be expected after the quinine control treatment. The present data showed no reduction in sweetness after the quinine control treatment. Indeed, sweetness after the quinine control treatment was not significantly different from sweetness after the apple juice treatment. It appears that neither adaptation nor mixture suppression can account for the reduced sweetness after the ZjE treatment. Taste modification is thus demonstrated. In contrast, an apparent release from the mixture suppression is suggested after the ZjEw treatment. Sweetness is significantly reduced on several post-treatment trials by the taste modification action of ZjEw, as shown by between treatment measures. Bitterness and saltiness tend to increase on these post-treatment trials, although not statistically significant when compared to other treatments, and then return to pretreatment levels (ca. 1 =none of that quality) as sweetness returns to pre-treatment levels. It is perhaps not surprising that no trend for increase in judged sourness occurred, since sourness is resistant to mixture suppression during whole mouth stimulation [2]. A more recent report of suppression of judged sourness when a sucrose-citric acid mixture is applied to a single fungiform papilla [ l l ] might be interpreted to suggest that some increase in sourness should have occurred during those trials of the present experiment on which sweetness modification was present. However, the same report [l l] demonstrated that the sucrose-citric acid mixture judged sourness is unchanged when judged sweetness, per se, is blocked by gymnemic acids. Thus, the absence of enhanced sourness in the present study is compatible with the proposal that the ziziphins and gymnemic acids have similar actions [8]. The trend for increased saltiness during the sweetness modification is particularly interesting, since apple juice is typically 0.0021% W/V sodium [13]. This is approximately equivalent to 0.36 mM NaC1, which would be well below the lowest reported human thresholds for NaC1 detection [18]. It appears that taste quality judgments during release from mixture suppression will be assigned to the available quality
873 categories. This could yield spurious indications of enhancement of some quality categories during taste modification. Such release-produced judgments as an explanation for apparent facilitation of taste qualities during taste modification by gymnemic acids has been previously noted by Meiselman and Halpern [15] and by DeSimone et al. [3]. Of course, such explanations are made at the level of the perceptual data upon which they are based. Independent measures with appropriate manipulations are required to permit the positing of neurophysiological mechanisms. The within-treatments Kramer Test analyses of Experiment 2 pinpoint effects on taste judgments which are related to the taste of a treatment liquid per se, as well as effects related to both taste modification and to direct taste effects. For example, with the apple juice treatment condition, suppression of judged sourness occurred during the latter part of the treatment. Some of the post-treatment trials showed an elevated sourness. The general pattern is of opposite effects during treatment in comparison with judgments made before and/or after treatment. This pattern was also found for judged sweetness in the quinine control and ZjEw treatment conditions. Thus, sweetness was elevated (compared to a chance distribution of intensities across all trials) after, or before and after, either the quinine control or the ZjEw treatment, while there was a reduction in judged sweetness during the time when one of these two treatment liquids was being held in the mouth. Precisely the opposite pattern occurs with judged bitterness, where the bitterness intensity during ZjEw or quinine control treatment increases beyond a random distribution level, while before and after the treatment, judged bitterness of apple juice alone is lower than chance would predict. The importance of appropriate control treatments is indicated by the interpretation which can be proposed for the judgments made during the 90 sec duration treatments. A statistically significant reduction in judged sweetness intensity started at 20 sec after the beginning of the 90 sec ZjEw treatment, if comparisons are made with the 90 sec duration apple juice control treatment judgments. Therefore, it might have been concluded that a taste modification induced by ZjEw is observed during treatment, that this taste modification reduces sweetness more rapidly and strongly than does adaptation by the apple juice, and that this sweetness modification is not prevented by competition. Of course, the correlated statistically significant increases in bitterness intensity during the ZjEw treatment would have questioned this interpretation. However, a comparison with the judgments made during the 90 sec duration quinine control treatment demonstrates that during treatment, the quinine control is as effective as the ZjEw treatment in reducing sweetness. Consequently, this experiment does not reveal the time course of taste modification by ZjEw during treatment. As previously noted, the use of the apple juice and quinine control treatments does permit an unequivocal identification of selective sweetness taste modification after the ZjEw treatment. The overall outcome of the present studies indicates that ziziphins produce relatively brief, very selective taste modification of sweetness. The reduction in judged sweetness occurs when the modifier is presented simultaneously with the mixture of sugars present in apple cider/apple juice. Thus, in common with the gymnemic acids [14], taste modification by ziziphins is not blocked by the competition for the receptor cell membrane produced when the sugar or sugars which are the stimuli for the sweetness judgments are present during the modifier treatment. These several
874
SMITH AND HALPERN
p s y c h o p h y s i c a l similarities b e t w e e n g y m n e m i c acids and ziziphins are c o m p a t i b l e with the p r o p o s a l t h a t b o t h p r o d u c e q u a l i t a t i v e l y similar a l t h o u g h t e m p o r a l l y d i s t i n g u i s h a b l e effects at the r e c e p t o r cell m e m b r a n e [8]. ACKNOWLEDGEMENTS We thank Mr. V. L. Bump (Seneca Brand Products) for informalion on apple cider and apple juice; P. J. Canney (Section of
Neurobiology and Behavior) for assistance with ziziphins preparation; Mrs. P. A. Halpern (Graduate Field of Education) for assistance with the flow diagrams; Dr. P. A. Hyypio (L. H. Bailey Hortorium, Cornell) for confirming the identification of leaves; J. W. Kelly, Plant Taxonomist (Department of Parks, Monroe County, Rochester, NY), for assistance in securingZ, jujaba leaves: and Mr. P. J. Canney, Dr. Linda M. Kennedy, and Dr. Aileen Sontag Trant for comments on the manuscript.
REFERENCES
1. Bartoshuk, L. M. Gustatory system. In: Handbook o f Behavioral Biology. Volume I. Sensory. Integration, edited by R. Bruce Masterton. New York: Plenum, 1978, pp. 528-529. 2. Bartoshuk, L. M. Taste mixtures: Is suppression related to compression? Physiol Behav 14: 643-649, 1975. 3. DeSimone, J. A., G. L. Heck and L. M. Bartoshuk. Surface active taste modifiers: a comparison of the physical and psychological properties of gymnemic acid and sodium lauryl sulfate. Chem Senses 5: 317-330, 1980. 4. Engen, T. Psychophysics. II. Scaling methods. In: Woodworth and Scholsberg's Experimental Psychology. Third Edition. edited by J. Kling and L. A. Riggs. New York: Holt, Rinehart and Winston, 1971, pp. 64-65. 5. Halpern, B. P. and C. Singer-Granich. Recovery of human sweetness judgments after oral treatment with ziziphins, lnt Cong Physiol Sci 275h Proc 13: 833, 1977. 6. Kennedy, L. M. and B. P. Halpern. Fly chemoreceptors: A model system for the taste modifier Ziziphin. Physiol Behav 24: 135-143, 1980. 7. Kennedy, L. M. and B. P. Halpern. Extraction, purification and characterization of a sweetness-modifying component from Ziziphus jujuba. Chem Senses 5: 137%1384, 1980. 8. Kennedy, L. M. and B. P. Halpern. A biphasic model for action of the gymnemic acids and ziziphins on taste receptor cell membranes. Chem Senses 5: 14%158, 1980. 9. Kennedy, L. M. and B. P. Halpern. Action of gymnemic acids and ziziphins on chemoreceptor cells: dose-effect and time course relationships. Assoc" Chemo Sei 3: 1981. 10. Kramer, A., G. Kahan, D. Cooper, A. Papavasilliou. A nonparametric ranking method for the statistical evaluation of sensory data. Chem Senses Flavour 1: 121-133, 1974. 11. Kuznicki, J. T. and N. B. McCutcheon. Cross-enhancement of the sour taste on single human taste papillae. J Exp Psych (Gen) 108: 68-89, 1979.
12. Lester, B. and B. P. Halpern. Effect of stimulus presentation duration on gustatory reaction time. Physiol Behav 22:31%324, ! 979. 13. Marsh, A. C., R. N. Klippstein and S. D. Kaplan. The Sodium Content o f Your Food. Washington, DC: U.S. Department of Agriculture, 1980. 14. Meiselman, H. L. and B. P. Halpern. Human judgments of Gymnema syh,estre and sucrose mixtures. Physiol Behav 5: 945-948, 1970. 15. Meiselman, H. L. and B. P. Halpern. Effects ofGymnema sylvestre on complex tastes elicited by amino acids and sucrose. Physiol Behav 5: 137%1384, 1970. 16. Meiselman, H. L., B. P. Halpern and G. P. Dateo. Reduction of sweetness judgments by extracts from the leaves of Ziziphus jt~iuha. Physiol Behav 17: 313-317, 1976. 17. Moskowitz, H. R. Sensations, measurements and pleasantness: confessions of a latent introspectionist. In: Taste atzd Development. Genesis o f Sweet Preference, edited by J. M. Weiffenbach. Bethesda, MD: National Institutes of Health, Public Health Service, DHEW Publication No. (N1H) 77-1068, 1977, pp. 284-286. 18. O'Mahony, M. Salt taste adaptation: the psychophysical effects of adapting solutions and residual stimuli from prior tastings on the taste of sodium chloride. Perception 8: 441-476, 1979. 19. Riskey, D. R., J. A. Desor and D. Vellucci. Effects of gymnemic acid concentration and time since exposure on intensity of simple tastes: A test of the biphasic model for the action of gymnemic acid. Chem Sense.~ 7: 143-152, 1982. 20. Thorner, M. E. and R. J. Herzberg. Non-alcohalic Food Service Beverage Handbook. Second edition. Westport, CT: AVI Publishing Co., 1978, pp. 191-192. 21. Wells, P. Apple juices: popular, but are they real? The New York 77mes. October 31, 1979, pp. CI and C6.
Selective Suppression of Judged Sweetness by Ziziphins V I V I A N V. S M I T H z A N D B R U C E P. H A L P E R N 3
Department o f Psychology and Division o f Biological Sciences Section o f Neurobiology and Behavior, Cornell University, Ithaca, N Y 14853 R e c e i v e d 7 July 1983 SMITH, V. V. AND B. P. HALPERN. Selective suppress!on of judged sweetness by ziziphins. PHYSIOL BEHAV 30(6) 867-874, 1983.--The effects of ziziphins and of control treatments upon judgments by human adults of the sweetness, sourness, bitterness, and saltiness of American apple cider or apple juice wdre measured with a category estimation method during repeated trials before, during (90 sec treatment duration only), and after, treatment. Sweetness was reduced after either a 10 sec or a 90 sec whole mouth treatment with ziziphins, but not after quinine sulfate or apple juice control treatment. No differences in after-treatment sourness, bitterness, or saltiness occurred between treatments. The reduction in sweetness was weak with 10 sec 3.5% W/V ziziphins treatment, but strong after 90 sec 0.88% W/V ziziphins treatment; duration of suppression was ca. 70 sec. The mechanism was identified as taste modification since adaptation, crossadaptation, and mixture suppression were eliminated by control treatments and by post-treatment rests and rinse. Comparisons with known gymnemic acids effects suggest that net dissociation of ziziphins from taste receptor membranes and/or inactivation in the membrane may be much faster than with gymnemic acids. Taste modifiers
Ziziphins
Sweetness
Apple cider
ZIZIPHINS are taste modifying saponins in the leaves of the Chinese jujube tree, Ziziphus jujuba P. Miller (Rhamnaceae) [7]. Extracts containing these saponins (ZjE) reversibly depress for human adults the judged sweetness of sucrose solutions [5, 7, 16]. In the only published test of ZjE selectivity [16], the extracts produced no change in which taste quality category the judges assigned to solutions of NaCI, HCI, sucrose, or quinine hydrochloride, and depressed the perceived sweetness intensity of sucrose solutions without changing the saltiness o f a NaC1 solution or the bitterness of a quinine hydrochloride solution. Meiselman et al. did find that the sourness of a HC1 solution was judged to increase after treatment with any one of several ZjE fractions derived from an aqueous-alcohol extract of Z. jujuba, but indepdently of the sweetness modifying ability of that fraction [16]. Thus, the question of ZjE selectivity is unresolved, because effects on both judged sweetness and sourness have been reported. Recovery of judged sweetness after ZjE treatment is though to be more rapid than recovery after a gymnemic acids treatment [8]. However, effects of ZjE treatment duration have not been examined, and selectivity during the recovery process has not been studied. Further, although a model has been developed which attempts to described the general nature of ZjE actions on gustatory receptor cell membranes [8], no analysis of the initial interaction at the receptor cell membrane has been made. The present experiments were designed to clarify the degree of ZjE selectivity, both during the time of preak suppression and during re-
Apple juice
covery. In addition, an initial examination was made of competitive inhibition as the mechanism of action of ZjE. To evaluate the taste-modifying selectivity of ZjE, we chose apple cider and apple juice as stimulus liquids. These unfermented natural beverages [20,21], which are approximately 12% sugar (fructose, dextrose, and sucrose: personal communication, V. L. Bump, Seneca Brand Products), elicit a range of taste qualities, with sweetness and sourness consistently reported. There is little bitterness (in American apple cider or juice) and no saltiness. Repeated, four-tastequality category scale intensity judgments of these complex stimulus liquids were made before and after ziziphins and control treatment in order to both reveal and track over time ziziphins-dependent taste changes. METHOD Participants were non-smoking paid volunteers who passed a screening test for consistency and sensitivity of gustatory judgments [12]. All were Cornell University students or staff. For Experiment 1 (effect of brief treatment) the ages of the six participants ranged from 19 to 21 years; four were female. In Experiment 2 (competition and mixture effects), the eight participants ranged in age from 19 to 26 years; three were female. Green Ziziphus jujuba leaves were gathered in October, 1978, directly from a 59 year old tree (H-124) at the Monroe County Arboretum of Highland Park, Rochester, NY. They were freeze-dried and then stored in sealed plastic containers
1Supported by NSF Grant BNS-8014148 to B.P.H. and College of Arts and Sciences Undergraduate Research Grant to V.V.S. 2Present address: 34 Chestnut Drive, Hastings-on-Hudson, NY 10706. 3Requests for reprints should be addressed to Bruce P. Halpern.
CoDvri~ht © 1983 Pergamon Press Ltd.--0031-9384/83/060867-08503.00
868
SMITH AND HALPERN
Experiment Two
Experiment One Adult Human Who Has Passed Screening Test
Adult Human Who Has Passed
Screening Test __~
Hold 10 ml Apple ~lCider in Mouth Move Tongue
YES
J Ho'dtom' AOO'e L
and
-
\ s0it0u,/
Juicein Mouth
l and Move Tongue r NO
ES
9 yESNO "= I Rinse Mouth
I
with 5 ml water ] Judge Intensity for Four
Vigorously with 10 m[
nse
YES
Judge / Intensity for Four Qualities /
\spi, f out/ H~_,
Samples of Apple Cider
NO =-
\App,,
YES /"
Treatment"'-,..,NO
=
=
Hold 10 ml Treatment Liquid in Mouth and
YES
~
Judge Intensity / for Four / Qualities /
Move Tongue
~'ES
J Hold 10 ml of Treatment -J Liquid in Mouth
NO~E}even .Samples~
V o-i-~ppl, Juice ">
FIG. 1. Flow diagram for experiment one. Six participants were tested in each of four separate sessions. The first session was a practice session; its data were not used. Sessions one and two used the control treatment liquid: 24/xM quinine sulfate in distilled water. Sessions three and four used the experimental treatment liquid: 3.5% W/V aqueous extract of Z.jujuba leaves (ZjEw). Here, and in Fig. 2, the "seconds counters" reset to zero seconds counted after a YES output.
FIG. 2. Flow diagram for experiment two. Eight participants were tested in each of four separate sessions. The first session was a practice session; its data were not used. Sessions one and two used the apple juice control treatment liquid. Session three used the 0.88% W/V ZjEw in apple juice experimental treatment liquid. Session four used the quinine control treatment liquid: 66/xM quinine sulfate in apple juice.
at room temperature. A 3.5% W/V aqueous extract (ZjEw) was prepared from the leaves [6]. F o r both experiments, the distilled w a t e r ' s refractive index (n) was 1.3330; its conductivity was less than or equal to 1.8/xS. The quinine sulfate was U . S . P . ; the apple concentrate, which was Seneca frozen CT 28, no preservatives, no sugar added (the same product was designated as either apple cider or apple juice (V. L. Bump, S e n e c a Brand Products, personal communication)), was reconstituted with distilled water according to directions supplied with the product; the p r u n e iuice was S u n s w e e t bottled. All stimulus, treatment, and rinse liquids were administered at 22---I°C. Participants were requested not to eat or drink anything in the hour prior to each session. Participants were instructed
to " P l e a s e m o v e your tongue around while tasting the liqu i d s " . . . Sweetness, sourness, bitterness, and saltiness intensity were m e a s u r e d at each j u d g m e n t using a category estimation method [4,17]. For e a c h j u d g m e n t , participants w e r e instructed to " . . . . indicate on a scale from 1 to 10 how sweet, sour, salty and bitter the liquid is (I means the liquid has none o f that taste quality and 10 means it is extrmely high in that quality). E a c h liquid may have more than one t a s t e . " On each j u d g m e n t sheet there were four 13 cm lines marked " S w e e t n e s s , " " S o u r n e s s , " " S a l t i n e s s , " or " B i t t e r n e s s , " each labeled " N o n e " at the left end, " E x t r e m e " at the far right. The lines w e r e n u m b e r e d 1 (none) to 10 (extreme) with equidistant arabic numerals. The participants marked the lines after the stimulus liquid was spit out
S U P P R E S S I O N O F S W E E T N E S S BY Z I Z I P H I N S but prior to the next stimulus, before and after treatments in both experiments, and also during treatment, i.e., with treatment liquid in the mouth, in Experiment 2. A separate judgment sheet was used for each judgment. Statistical analysis was done separately for each quality category. Evaluation of successive pairs of trials within each treatment condition was done for Experiment 1 only, using the Wilcoxon Matched Pairs Signed Rank Test. Overall results of each experiment were evaluated with the Kramer Test [10] across all trials, both between treatment conditions and within each treatment condition. The within treatment Kramer Test [10] analysis permitted identification of trials on which judged intensity was greater or less than that expected from a chance distribution.
869 Extreme
10-
8
E
Experiment Two The stimulus liquid and the solvent for the ZjEw and quinine control treatment liquids was Seneca Natural Style apple juice (Fig. 2). F o r the ZjEw treatment, 3.5% W/V ZjEw was prepared and immediately freeze-dried until needed. To make the ziziphins treatment liquid, the freeze-dried extract was reconstituted to its original concentration with distilled water, and then diluted 3:1 with apple juice, giving a final ZjEw treatment concentration of 0.88% W/V. The quinine control liquid, which allowed mixture suppression effects to be identified, was 66/~M quinine sulfate in apple juice:prune juice, 9:1. Each participant made judgments in tour sessions. The first two sessions used an apple juice only treatment (for control of adaptation and sugar effects) the third, the ZjEw treatment liquid; the fourth, the quinine control treatment liquid. The first session was a practice session. In each session, a total of eleven 10 ml samples of apple juice were held in the mouth for three sec, then spit out and judged. Another apple juice sample was presented 10 see later, with no rinses. Ten sec after the third apple juice sample was spit out, 10 ml of a treatment liquid was taken into the mouth and held for 90 sec. Judgments continued every 10 see during the treatment. The relevant instructions were " . . . you will be given a liquid to hold in your mouth, this time for 90 second~. During the time it is in your mouth, you will be asked to judge the taste of the liquid 9 times (every 10 seconds, as I signal you) using the same type of scoring sheet." After the treatment
-
m
6
w
o
..
T -1i i I Sour I h' I I ~,~" I
Experiment One The stimulus liquid was Seneca Apple Barrel apple cider (Fig. 1). The ziziphins treatment was freshly prepared 3.5% W/V ZjEw [6]; the control treatment was 24 /~M quinine sulfate in distilled water: prune juice, 4:1. Each participant made judgements in four sessions. The first two sessions used the control treatment; the last two, the ZjEw treatment. The first session was a practice session. In each session, a total of eight 10 ml samples of apple cider were held in the mouth for 3 sec, then spit out and judged. Another apple cider sample was presented 40 sec after spitting out the preceding sample, with no rinses. Forty seconds after the third apple cider sample, 10 ml of treatment liquid was presented. It was held in the mouth, with tongue motion encouraged, for 10 sec. The treatment liquid was then spit out. After a 5 sec rest, participants vigorously rinsed with 10 ml distilled water, spit out, rested for 10 sec, and then began a series of five post-treatment judgments of apple cider samples. After all four sessions had been completed, each participant made one four-quality judgment of the ZjEw and of the 24 /~M quinine control treatment liquids.
;~
Sweet
~
g 2
Q ....
I
I
I
I
J-
Nonq
Q-
!
I
:1,+, ,,V' \
+__,__+/
-v-
I ',
k~"
', !
~-
•
I
:
*
.
: ;
' '
\'* - - + <,,B i t t e r ±I "
i l
_L
I. . . .
,"
~1
II I
•
Salty
~.£ I
i
i
~
i
I
i
I
I
I
2
3
I.
5
6
7
8
Triols ' , ' 58 101 I~ & 187 Time After Treotment (sec)
IJ5
FIG. 3. Median category scale ( l = n o n e of that quality, 10=extremely high intensity of that quality) intensity judgments, +_standard error of the median, made by 6 participants for sweet, sour, bitter, and salty taste quality categories, before and after a whole mouth l0 ml treatment, are shown here and in Fig. 4. Sweetness judgements are shown by squares and solid connecting lines; sourness, solid circles and broken lines; bitterness, diamonds and heavy broken lines; saltiness, stars. Standard errors are indicated by thin vertical lines (terminated with short horizontal lines for sourness), solid for sweet; broken for sour; dashed for bitter; and dotted for salty; with errors less than 0.2 not plotted. Treatment for Fig. 3 was 3.5% W/V ZjEw.
liquid was spit out, there was a 10 sec rest, a 5 sec rinse with l0 ml distilled water, and another l0 sec rest. Stimulus liquid presentations then resumed for the remaining eight presentations. A total of 20 four-quality intensity judgments were made in each session. RESULTS
Experiment One Between conditions, i.e., control versus ZjEw treatment conditions, there were no statistically significant differences (Kramer Test, p >0.05) over all 16 trials, although the differences between conditions was greatest immediately after treatment for sweetness on trial 4, with the sum of ranks one less than that required for significance. Within treatment conditions (8 trials each), the ZjEw treatment condition produced a significant decrease (Kramer Test, p~<0.05) in judged sweetness of apple cider only on the trial immediately following the 10 second treatment (Fig. 3). No significant deviations from a chance distributions (Kramer Test, p >0.05) of intensity judgments occurred across all 8 trials for sourness, bitterness, or saltiness with either treatment, or for any quality category with the quinine control treatment (Fig.
870
SMITH AND HALPERN 4). The successive pairs of trials analysis with the Wilcoxon Signed Rank test showed no significant differences for any taste quality for the quinine control treatment condition (p>0.09, one-tailed). However, for both ZjEw treatment condition sessions there was a significant change in sweetness between trials 3 and 4 (Wilcoxon, p<~0.05, one-tailed) (Fig. 3), but not between preceding or following pairs of trials (p>0.09, one-tailed). No other comparisons of pairs for the ZjEw treatment condition reached statistical significance (p~>0.09), except for bitterness between trials 3 and 4 of the first ZjEw treatment session (p=0.05). The ZjEw treatment liquid received a median quality profile of sweetness 1, sourness 2, saltiness 1, and bitterness 8.5; the 24/zM quinine sulfate control, 2.5, 2, 1, and 4, respectively.
Extreme 10-
8 o
~
E
6 i I
w
t Sour t
g
I i
~ ~"~
I
,
/
ul
& ..
I
,, t
±
",
I E
T I
I
g
~-. T ,/ i " ' ~ , . . . . +"
IE
I d.
¢.s=., i
: Bitter'ff
I
2
I
3
Experiment Two
i
I I I .1_
i e. ,,
Non
I
+
°
o
Between treatment comparisons. On before-treatment trials (trials 1-3), no significant differences occurred between the three treatment conditions for any of the four taste quality categories on which intensity judgments of the apple juice samples were made (p>0.05, Kramer Test) (Fig. 5). During treatment, the ZjEw produced bitterness judgments larger than those during the apple juice treatment but less than those during the quinine control treatment. In addition, sweetness intensity during ZjEw treatment was less than that during apple juice treatment (Fig. 5). The quinine control treatment produced bitterness judgments which exceeded those with apple juice treatment for almost the entire 90 sec treatment period (trials 4--11, p<0.05, Kramer Test). Differences in judged sourness between quinine control and apple juice treatments (p~<0.05, Kramer Test) occurred on two during-treatment trials (4 and 5), while quinine control
+
i
i
i
I
i
4
5
6
7
8
Triats i
i
i
i
15
58
I01
144
Time
After
Treotment
187 (sec)
FIG. 4. Median category scale intensity judgements, _+standard error of the median, before and after treatment with 24 I~M quinine sulfate control liquid. Symbols, etc. as in Fig. 3, except that the bitterness data points are not connected.
TABLE 1 WITHIN-TREATMENTCONDITIONKRAMERTEST STATISTICALLYSIGNIFICANT(p<0.05) DIFFERENCESIN DISTRIBUTIONOF JUDGED INTENSITYACROSSALL 20 TRIALSOF APPLE JUICE Trials Treatment Condition
Taste Quality Category
3.5% W/V Ziziphins in Apple Juice
Sweet Sour Salty Bitter
Apple Juice
Sweet Sour Salty Bitter
66 tzM Quinine Sulfate in Apple Juice
Sweet Sour Salty Bitter
During Treatment 1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17 18 19 20
:~ :1: ¢
+
+
+
+
+
+
+
$
+
÷
+ +
+
+
+
+
+
+
+
+
+
+
+
+
Plus symbols (+) indicate trials on which, for that quality category, intensity was greater (p~0.05) than expected from a chance distribution of judged intensities across the 20 trials; ¢p~<0.01. Minus symbols (-) indicate trials on which, for that quality category, intensity was less (p~<0.05) than that expected from a chance distribution of judged intensity across the 20 trials; =p~<0.0l.
SUPPRESSION OF SWEETNESS BY ZIZIPHINS
L
Trea
871
t me n t
Apple
Juice
Jk LL L
4 _~
2 I
I
I
I
I
I
,
,
I
I
8-
1
i
Ziziphins
,-
m
2~ r
1
I
1
'
'
r
I
1 r 2 , 3 14
/
Control
, 5'
6
7 ' 8'
'
'
'
•
•
•
'
I
I
I
I
1
9 '10
11
12113
14
15
16
Trials FIG. 5. Median category scale intensity judgements, _+standarderror of the median (solid vertical bars; errors <0.1 are not shown), made by 8 participants for sweet (clear columns), sour (upward diagonal columns), bitter (dotted columns), and salty (downward diagonal columns) taste qualities, l=none of that quality; 10=extremely high intensity of that quality. Statistically significant (p<0.05, Kramer Test) differences between 3.5% ziziphins (ZjEw) treatment condition (trials 1-20) and apple juice or 66/zM quinine sulfate control treatment trials are shown by an asterisk for differences in sweetness intensity; by an encircled asterisk, for differences in bitterness intensity. No significant differences 'between ZiEw treatment condition trials and the apple juice or quinine control treatment (trials 1-20) were found for sourness or saltiness.
treatment judged sweetness was less than apple juice treatment judged sweetness (p~<0.05, Kramer Test) on most during-treatment trials (trials 4--8 and 10). After treatment, only differences in sweetness intensity occurred (except for one apple juice treatment condition versus quinine control treatment condition difference in apple juice sourness on trial 20). The judged sweetness of apple juice after the ZjEw treatment was significantly less (p ~<0.05, Kramer Test) than the sweetness of apple juice after the apple juice treatment on after-treatment trials 14 and 15, and was significantly less than the sweetness intensity after the quinine control treatment on the first four after-treatment trials (trials 13-16) (Fig. 5). Within treatment effects. Within each treatment condition, separate Kramer Tests for each quality category were done to identify trials on which intensity was greater or less than that expected from a chance, i.e., p >0.05, distribution of judged intensities across the 20 trials. All intensity judgments which significantly departed from a chance level are indicated in Table 1.
DISCUSSION Sweetness of apple cider/apple juice was reduced following whole mouth, sip and spit exposure to ZjEw. For a 10 sec treatment with 3.5% W/V ZjEw (Experiment 1), judgments of taste intensity of apple cider were made before and after treatment. The sole statistically significant effect across all eight trials of a condition was decreased sweetness on the first trial after the rest and water rinse which followed the brief ziziphins treatment. This sweetness reduction after ZjEw but not after quinine control treatment was confirmed by comparison of successive pairs of trials. Median sweetness intensity was depressed through 101 sec after the 10 sec ZjEw treatment, but a statistically significant reduction occurred only at 15 sec after treatment, and not at the 58 sec or 101 sec after treatment measurement times. The relatively high variability of the data from experiment one could have masked significant reductions at these two latter judgment times. This problem can be clarified by a comparison with the data of Experiment 2, in which less
872 variability occurred. For Experiment 2, taste modification was also of relatively short duration. That is, sweetness was significantly depressed only through 65 sec after the ZjEw treatment of Experiment 2. In this case, median sweetness intensities at 75 sec after treatment and at subsequent judgment times after the 90 sec ZjEw treatment were at pretreatment levels. Since brief actions of ZjEw are indicated in both Experiments 1 and 2, it may be correct to expect that the 10 sec ZjEw treatment of experiment one would produce a somewhat shorter period of sweetness reduction than the 90 sec ZjEw treatment duration of Experiment 2. A potential weakness of Experiment 1 was the greater bitterness of 3.5% W/V ZjEw in comparison with the 24 ~M quinine control treatment liquid. Although compounds judged bitter do not generally cross-adapt upon judged sweetness [1], a control treatment liquid with a judged bitterness at least as high as that of the ZjEw treatment would have been desirable. No across-all-trials of each condition significant differences occurred in judged sourness, bitterness, or saltiness in Experiment 1. For sourness and saltiness, absence of both quinine control treatment and ZjEw treatment condition effects was confirmed by the successive pairs of trials analysis, but bitterness did show one significant change in the first ZjEw session (Fig. 1). A between-treatments comparison of the ZjEw treatment conditions with the 24 p~M quinine sulfate control condition showed a trend towards reduced sweetness after the ziziphins treatment, but statistical significance was not reached. The weakness of the rather selective effect of ZjEw on sweetness in Experiment 1 may be due to the limited time during which the ziziphins could interact with taste receptor cell membranes [3,7] and/or it may be related to the proportion of receptor cells reached during the brief treatment. However, a similar treatment with another taste modifier, gymnemic acids, with 5 ml of liquid held in the mouth for only 3 sec (in a mixture with sucrose), reduced the sweetness of sucrose by approximately 60% when measured 3 min after the brief gymnemic acids treatment [14]. No rinse was used, but the sweetness suppression was significantly greater than that due to a control treatment. This powerful and long lasting effect of gymnemic acids on sweetness [14] indicates that limited access to and contact time with human taste receptors does not necessarily produce a weak and transient taste modification of sweetness. Rather, differences between gymnemic acids and ziziphins are indicated. Other factors in the present ZjEw experiment (Experiment 1) may also have contributed to the difference. Such factors might include the absence during treatment of sugars which, if present, could have permitted a use-dependent binding of ZjEw to membrane sites; complex effects of the several different sugars contained in apple cider; the mixture interaction possibility of apple cider, and perhaps a lack of similarity of active taste modifier concentrations. In general, this selective but relatively weak sweetness suppression by a brief ZjEw treatment supports previous reports ot Oltterences between ZjEw and gymnemic acids [6, 7, 16]. On the other hand, the selective taste modification produced by a brief treatment of ZjEw followed by a rinse is compatable with an action at the taste receptor cell membrane [8]. A clear, powerful (ca. 50% reduction of judged sweetness), but still selective and relatively brief taste modification by ZjEw appeared after the 90 sec treatment of Experi= ment 2. It is interesting that, although the ZjEw concentration was 0.88% W/V rather than the 3.5% W/V of Experiment
SMITH AND HALPERN 1, a more prolonged reduction of sweetness occurred. That is, sweetness was reduced re: the 66 ~M quinine sulfate control on the four successive trials which occurred after the rests and H20 rinse which followed the 90 sec ZjEw treatment. Since the rinse and rests occupied 25 sec, while judgments were made every 10 sec, sweetness was attenuated for one minute after the treatment. In contrast, the sweetness suppression of Experiment 1 (10 sec treatment) was seen at 15 sec but not at 58 sec post-treatment. The greater sweetness suppression produced by a longer (90 sec vs. 10 sec) but lower concentration (0.88% versus 3.5%) ZjEw treatment suggests that the rate at which ziziphins associate with the taste receptor cell membrane such that sweetness modification occurs is relatively low. The comparison in a preceding paragraph between gymnemic acids and ZjE indicated that a brief gymnemic acids treatment was much more effective in sweetness suppression than a brief ZjE treatment. This might suggest either that the rate at which gymnemic acids associate with the taste receptor cell membrane to produce taste modification is faster than that of ZjE, or that gymnemic acids' dissociation from or inactivation in the membrane is much slower than ZjE, giving a greater net taste modification. The long duration of taste modification following gymnemic acids treatment (e.g., [14,15]) supports a slow dissociation and/or inactivation of gymnemic acids. However, ZjEw could have a nonmonotonic dose-response function in humans, as ZjE have in the blowfly [9]. This would cause ZjEw concentrations above some level to be less effective than lower concentrations. The observed suppression of the sweetness of apple cider/ apple juice after the ZjEw treatments of the present study is briefer than the previously reported 5-6 min reduction in judged sweetness of 80 mM sucrose by ZjE [5,7]. These earlier studies used a 3 min treatment duration, which may have been long enough to permit penetration by ziziphins into taste receptor cell membranes. If such penetration occurred, it could alter membrane structure and produce nonselective changes in taste intensity [3,8]. However, only judged sweetness, rather than the intensity of several taste quality categories, was measured. One earlier investigation which had a 3 min ziziphins treatment [16] did use NaC1, HC1, and quinine hydrochloride, as well as sucrose, to evaluate the selectivity of the treatment. However, posttreatment effects were measured at only one time: less than one minute after the end of the treatment. Maximum effect upon membrane structure may require a longer total action time once penetration has begun 13, 8, 9]. Consequently, the relative importance for the present brief sweetness suppression of factors such as the sweet and sour taste of apple cider/apple juice, the 90 sec versus 3 min treatment duration, and/or the possible interactions between the ZjEw and the sugars, etc. present during treatment, can not now be evaluated. A study which employed ziziphins treatments of 3 min or more, and measured the post-treatment effects for at least 10 rain, using a mutliple quality category technique and suitable stimuli, could clarify this question. With gymnemic acids, a study similar to that just proposed, but with a treatment duration of only 60 sec, found effects only on sweetness [19]. Taste modification is clearly identified only when alternative processes which can reduce taste intensity are eliminated. With prolonged treatment, two such processes are adaptation and mixture suppression. Apple juice was both the liquid judged before and after treatment and the solvent for the 90 sec treatments of Experiment 2 (with prune juice
S U P P R E S S I O N O F S W E E T N E S S BY Z I Z I P H I N S added for the quinine control treatment). Consequently, direct adaptation by the sugars, acids, and other constitutents of apple juice would be comparable across treatments, and would be mathematically cancelled out by between treatment comparisons. In addition, the absence of reduced sweetness or sourness after the apple juice treatment demonstrates that the post-treatment rests and rinse were sufficient to remove its adaptation effects. The potential for cross-adaptation differs between treatments. Both the ZjEw and quinine control treatments are more bitter than the apple juice treatment, with the quinine control treatment more bitter than the ZjEw. However, available data do not indicate cross-adaptation of compounds judged bitter upon judged sweetness [1]. If mixture suppression produced during treatment continued after treatment, a reduction in post-treatment sweetness proportional to the bitterness, sourness, and/or saltiness of the treatment would be expected [2]. There was no saltiness during the treatments (or before treatment), no significant differences in sourness between ZjEw and control treatments, and no increased sourness during any treatment. It was already noted that both the ZjEw and the quinine control treatments were more bitter than the apple juice, with the quinine control treatment the most bitter. Therefore, if mixture suppression due to the taste of the treatments were not eliminated by the rests and rinse, but instead continued into the post-treatment trials, the greatest sweetness suppression would be expected after the quinine control treatment. The present data showed no reduction in sweetness after the quinine control treatment. Indeed, sweetness after the quinine control treatment was not significantly different from sweetness after the apple juice treatment. It appears that neither adaptation nor mixture suppression can account for the reduced sweetness after the ZjE treatment. Taste modification is thus demonstrated. In contrast, an apparent release from the mixture suppression is suggested after the ZjEw treatment. Sweetness is significantly reduced on several post-treatment trials by the taste modification action of ZjEw, as shown by between treatment measures. Bitterness and saltiness tend to increase on these post-treatment trials, although not statistically significant when compared to other treatments, and then return to pretreatment levels (ca. 1 =none of that quality) as sweetness returns to pre-treatment levels. It is perhaps not surprising that no trend for increase in judged sourness occurred, since sourness is resistant to mixture suppression during whole mouth stimulation [2]. A more recent report of suppression of judged sourness when a sucrose-citric acid mixture is applied to a single fungiform papilla [ l l ] might be interpreted to suggest that some increase in sourness should have occurred during those trials of the present experiment on which sweetness modification was present. However, the same report [l l] demonstrated that the sucrose-citric acid mixture judged sourness is unchanged when judged sweetness, per se, is blocked by gymnemic acids. Thus, the absence of enhanced sourness in the present study is compatible with the proposal that the ziziphins and gymnemic acids have similar actions [8]. The trend for increased saltiness during the sweetness modification is particularly interesting, since apple juice is typically 0.0021% W/V sodium [13]. This is approximately equivalent to 0.36 mM NaC1, which would be well below the lowest reported human thresholds for NaC1 detection [18]. It appears that taste quality judgments during release from mixture suppression will be assigned to the available quality
873 categories. This could yield spurious indications of enhancement of some quality categories during taste modification. Such release-produced judgments as an explanation for apparent facilitation of taste qualities during taste modification by gymnemic acids has been previously noted by Meiselman and Halpern [15] and by DeSimone et al. [3]. Of course, such explanations are made at the level of the perceptual data upon which they are based. Independent measures with appropriate manipulations are required to permit the positing of neurophysiological mechanisms. The within-treatments Kramer Test analyses of Experiment 2 pinpoint effects on taste judgments which are related to the taste of a treatment liquid per se, as well as effects related to both taste modification and to direct taste effects. For example, with the apple juice treatment condition, suppression of judged sourness occurred during the latter part of the treatment. Some of the post-treatment trials showed an elevated sourness. The general pattern is of opposite effects during treatment in comparison with judgments made before and/or after treatment. This pattern was also found for judged sweetness in the quinine control and ZjEw treatment conditions. Thus, sweetness was elevated (compared to a chance distribution of intensities across all trials) after, or before and after, either the quinine control or the ZjEw treatment, while there was a reduction in judged sweetness during the time when one of these two treatment liquids was being held in the mouth. Precisely the opposite pattern occurs with judged bitterness, where the bitterness intensity during ZjEw or quinine control treatment increases beyond a random distribution level, while before and after the treatment, judged bitterness of apple juice alone is lower than chance would predict. The importance of appropriate control treatments is indicated by the interpretation which can be proposed for the judgments made during the 90 sec duration treatments. A statistically significant reduction in judged sweetness intensity started at 20 sec after the beginning of the 90 sec ZjEw treatment, if comparisons are made with the 90 sec duration apple juice control treatment judgments. Therefore, it might have been concluded that a taste modification induced by ZjEw is observed during treatment, that this taste modification reduces sweetness more rapidly and strongly than does adaptation by the apple juice, and that this sweetness modification is not prevented by competition. Of course, the correlated statistically significant increases in bitterness intensity during the ZjEw treatment would have questioned this interpretation. However, a comparison with the judgments made during the 90 sec duration quinine control treatment demonstrates that during treatment, the quinine control is as effective as the ZjEw treatment in reducing sweetness. Consequently, this experiment does not reveal the time course of taste modification by ZjEw during treatment. As previously noted, the use of the apple juice and quinine control treatments does permit an unequivocal identification of selective sweetness taste modification after the ZjEw treatment. The overall outcome of the present studies indicates that ziziphins produce relatively brief, very selective taste modification of sweetness. The reduction in judged sweetness occurs when the modifier is presented simultaneously with the mixture of sugars present in apple cider/apple juice. Thus, in common with the gymnemic acids [14], taste modification by ziziphins is not blocked by the competition for the receptor cell membrane produced when the sugar or sugars which are the stimuli for the sweetness judgments are present during the modifier treatment. These several
874
SMITH AND HALPERN
p s y c h o p h y s i c a l similarities b e t w e e n g y m n e m i c acids and ziziphins are c o m p a t i b l e with the p r o p o s a l t h a t b o t h p r o d u c e q u a l i t a t i v e l y similar a l t h o u g h t e m p o r a l l y d i s t i n g u i s h a b l e effects at the r e c e p t o r cell m e m b r a n e [8]. ACKNOWLEDGEMENTS We thank Mr. V. L. Bump (Seneca Brand Products) for informalion on apple cider and apple juice; P. J. Canney (Section of
Neurobiology and Behavior) for assistance with ziziphins preparation; Mrs. P. A. Halpern (Graduate Field of Education) for assistance with the flow diagrams; Dr. P. A. Hyypio (L. H. Bailey Hortorium, Cornell) for confirming the identification of leaves; J. W. Kelly, Plant Taxonomist (Department of Parks, Monroe County, Rochester, NY), for assistance in securingZ, jujaba leaves: and Mr. P. J. Canney, Dr. Linda M. Kennedy, and Dr. Aileen Sontag Trant for comments on the manuscript.
REFERENCES
1. Bartoshuk, L. M. Gustatory system. In: Handbook o f Behavioral Biology. Volume I. Sensory. Integration, edited by R. Bruce Masterton. New York: Plenum, 1978, pp. 528-529. 2. Bartoshuk, L. M. Taste mixtures: Is suppression related to compression? Physiol Behav 14: 643-649, 1975. 3. DeSimone, J. A., G. L. Heck and L. M. Bartoshuk. Surface active taste modifiers: a comparison of the physical and psychological properties of gymnemic acid and sodium lauryl sulfate. Chem Senses 5: 317-330, 1980. 4. Engen, T. Psychophysics. II. Scaling methods. In: Woodworth and Scholsberg's Experimental Psychology. Third Edition. edited by J. Kling and L. A. Riggs. New York: Holt, Rinehart and Winston, 1971, pp. 64-65. 5. Halpern, B. P. and C. Singer-Granich. Recovery of human sweetness judgments after oral treatment with ziziphins, lnt Cong Physiol Sci 275h Proc 13: 833, 1977. 6. Kennedy, L. M. and B. P. Halpern. Fly chemoreceptors: A model system for the taste modifier Ziziphin. Physiol Behav 24: 135-143, 1980. 7. Kennedy, L. M. and B. P. Halpern. Extraction, purification and characterization of a sweetness-modifying component from Ziziphus jujuba. Chem Senses 5: 137%1384, 1980. 8. Kennedy, L. M. and B. P. Halpern. A biphasic model for action of the gymnemic acids and ziziphins on taste receptor cell membranes. Chem Senses 5: 14%158, 1980. 9. Kennedy, L. M. and B. P. Halpern. Action of gymnemic acids and ziziphins on chemoreceptor cells: dose-effect and time course relationships. Assoc" Chemo Sei 3: 1981. 10. Kramer, A., G. Kahan, D. Cooper, A. Papavasilliou. A nonparametric ranking method for the statistical evaluation of sensory data. Chem Senses Flavour 1: 121-133, 1974. 11. Kuznicki, J. T. and N. B. McCutcheon. Cross-enhancement of the sour taste on single human taste papillae. J Exp Psych (Gen) 108: 68-89, 1979.
12. Lester, B. and B. P. Halpern. Effect of stimulus presentation duration on gustatory reaction time. Physiol Behav 22:31%324, ! 979. 13. Marsh, A. C., R. N. Klippstein and S. D. Kaplan. The Sodium Content o f Your Food. Washington, DC: U.S. Department of Agriculture, 1980. 14. Meiselman, H. L. and B. P. Halpern. Human judgments of Gymnema syh,estre and sucrose mixtures. Physiol Behav 5: 945-948, 1970. 15. Meiselman, H. L. and B. P. Halpern. Effects ofGymnema sylvestre on complex tastes elicited by amino acids and sucrose. Physiol Behav 5: 137%1384, 1970. 16. Meiselman, H. L., B. P. Halpern and G. P. Dateo. Reduction of sweetness judgments by extracts from the leaves of Ziziphus jt~iuha. Physiol Behav 17: 313-317, 1976. 17. Moskowitz, H. R. Sensations, measurements and pleasantness: confessions of a latent introspectionist. In: Taste atzd Development. Genesis o f Sweet Preference, edited by J. M. Weiffenbach. Bethesda, MD: National Institutes of Health, Public Health Service, DHEW Publication No. (N1H) 77-1068, 1977, pp. 284-286. 18. O'Mahony, M. Salt taste adaptation: the psychophysical effects of adapting solutions and residual stimuli from prior tastings on the taste of sodium chloride. Perception 8: 441-476, 1979. 19. Riskey, D. R., J. A. Desor and D. Vellucci. Effects of gymnemic acid concentration and time since exposure on intensity of simple tastes: A test of the biphasic model for the action of gymnemic acid. Chem Sense.~ 7: 143-152, 1982. 20. Thorner, M. E. and R. J. Herzberg. Non-alcohalic Food Service Beverage Handbook. Second edition. Westport, CT: AVI Publishing Co., 1978, pp. 191-192. 21. Wells, P. Apple juices: popular, but are they real? The New York 77mes. October 31, 1979, pp. CI and C6.