st,Jmposium: sweeteners and sweetness theorv
Discovery of Highly Sweet Compounds from Natural Sources A. Douglas ~ i n ~ h o r and n ' Edward J. Kennelly University of Illinois at Chicago, Chicago, IL 60612 The organic constituents of plants may be divided into primary and secondary metabolites, with the former group having essential roles in cellular metabolism and the latter possessing more of a n ecological role, and being of value in defense against insects and other pathogens (1).Many plant secondary metabolites have important and varied uses a s pharmaceuticals, agrochemicals, and industrial materials (13).I t is from the plant secondary metabolite class that a number of highly sweet natural products have been found. Such compounds, in being so-called "high-potency sweeteners", are from about 50 to over 1,000 times the sweetness potency of sucrose and are represented by a variety of chemotypes. While there is every possibility that highly sweet compounds are produced by microorganisms, marine organisms, and the more primitive plant forms, thus far all of the highly sweet natural products discovered to date have been obtained from vascular plant sources (4). Efforts to find additional examples of highly sweet plant constituents have been stimulated both by a public demand for natural flavors, a s well as perceived problems with the toxicity, taste quality, stability, or price of existing synthetic high-potency sweeteners. By following up ethnobotanical leads to assist in the selection of candidate sweet-tasting plants, particularly those used medicinally by indigenous cultures, it is possible to discover new potently sweet natural product com~ounds.a s will be expl;~in(.din greater detad in t h ~rev~cw. i In :in t d i t , r art id^ In fhr\ .Journnl, the term ethnobotany \r,as dcfincd and interesting plant secondary metabolites with a wide range of biological activities were exemplified ( 5 ) . Importance of Plant-Derived Sweetening Agents Throughout the world the most widely used sweetener is sucrose, obtained from either the sugar cane (Saccharum officinarum L.) or the sugar beet (Beta uulgaris L.) plant (6). Sucrose is considered to be the quintessential sweet substance because the sweet taste it elicits in humans is clean and is not associated with any aftertaste. In addition to taste considerations, sucrose has many other desirable qualities as an ingredient in food: it is heat stable, water soluble, cheap to produce, and a good bulking agent (7). In medieval Europe this compound was considered a great luxury (81, but today humans consume over 100 million metric tons per year of sucrose on a worldwide basis (9). Consider that in the United States, a typical 12-02. can of soda alone contains about 39 grams of sugar. Despite its current widespread use in the human diet, the USDA has recommended in its latest dietary guidelines that sugar should be used sparingly (10). Of the reasons to lower human sucrose consumption, the most compelling one is its propensity to cause dental caries (11). Since the 1960's a significant section of the market for sugar substitutes in the United States has been focused on high-potency sweeteners. Presented before the Division of Chemical Education at the 206th National Meeting ofthe American Chemical Society. August 22. 1993. Chicago. IL. 'Author to whom correspondence should be addressed. 676
Journal of Chemical Education
Although a t one time saccharin was the only approved high-potency sweetener in the United States, this substance has been joined on the market more recently by aspartame and acesulfame K. Other synthetic compounds of commercial promise a s sucrose substitutes are alitame (12) and sucralose (121, and intensive efforts are being made to develop extremely sweet synthetic compounds, for example, of the guanidine and p-amino acid types (131.Despite the fact that sweet-tasting compounds appear to be much less frequently encountered than bitter plant constituents, the plant kingdom already has afforded several natural high-potency sweeteners that are commercially available in overseas countries. The number of different structural types of natural sweeteners relative to the few compounds of this type so far known represents a quite remarkable track record of success. Currently, there are about 75 plant constituents known to be potently sweet, which are representative of some 20 structural types, and several of these compounds have some commercial use, mainly in Japan. For example, stevioside (Fig. 1, 1) is the major ent-kaurene glycoside of the leaves of the South American herb, Steuia rebaudiana (Bertoni) Bertoni, and extracts of this plant containing stevioside and the related glycoside, rebaudioside A (Fig. 1, 2) have been used in Japan for sweetening foods and beverages for about 20 years. In 1988, S. rebaudiana sweeteners were estimated to possess a larger share of the high-potency sweetener market in Japan than either saccharin or aspartame (12). Stevioside also is a n approved sweetener in Brazil and Korea (12). "Glucosyl stevioside", which has a more pleasant taste than the parent compound and is also used a s a sweetener in Japan, is a mixture of products obtained when the sugar moieties of stevioside are transglycosylated using a bacterial enzyme (14).Another sweetener of considerable importance in Japan is the triteroene glycoside, glycyrrhizin (Fig. 1, 31, which is extracted from the roots of Glycyrrhizaglabra L. and other species of this genus. Arnmoniated glycyrrhizin, has been accorded "Generally Regarded a s Safe" (GRAS) status in the United States, and although sweet, is employed a s a flavoring agent (2, 12). Thaumatins I and I1 are small proteins obtained from the fruits of the West African plant, Thaumatococcus daniellii (Bennett) Benth., and a commercial preparation of a mixture of these proteins is used as a high-potency sweetener in Australia, J a p a n , and the United Kingdom (12). Extracts of other plants with more limited use for sweetening in Japan are obtained from the dried fruits of Siratiagrosuenorii (Swingle) C. Jeffrey lcontaining mogroside V (Fig. 1, 41 and other triterpene glycosidesl and from the fermented leaves of Hydrangea macrophylla Seringe var. thunbergri (Siebold) Makino (containingphyllodulcin (Fig. 1,5),a dihydroisocoumarin). I n addition to these natural sweeteners from plants, is neohesperidin dihydrochalcone (Fig. 1, 6), a semi-synthetic derivative of a flavonoid glycoside of the peels of Seville oranges (Citrus auranticum L.), which is approved for limited use for sweetening purposes in Belgium (12).
R1
1 Stevioside
p-glc
2 Rebaudioside A
P-glc
"P R2
p-glc
P-glc P-glc
5
Phyllodulcin
R 3 Glycyrrhizin
R
p-glcA L b l c ~ 6 Neohesperidin dihydrochalcone
Approaches to the Discovery of Highly Sweet Molecules from Higher Plants In the search for new sweeteners, one of the most demanding aspects is to find good candidate sweet-tasting plants, which might contain novel sweet compounds. Some types of bioactive phytochemicals occur in relatively restricted taxonomic groups, such as indole alkaloids or cardiac glycosides of medicinal use. However, potently sweet compounds are found in a wide variety of taxonomically unrelated plant families. When one examines sweet natural products in terms of the position of the families of angiosperm superorders (151, no pattern appears to exist to enable one to predict the occurrence of sweet compounds on a taxonomic basis. For example, as already mentioned, Steuia rebaudiana i s well-known for producing the po-
. "
over 100 herbarium leaf specimens in the genus Steuia, only one species other than S. rebaudiana was found to contain stevioside, namely, S. phlebophylla A. Gray, which was collected in Mexico in 1889 and is possibly now extinct (16, 17).Therefore, since chemotaxonomic methods are probably not very useful to discover new plant-derived sweeteners, we have had to use a variety of other methods to identify new plant leads. There are two major ways in which new plant leads have been identified in the past in this laboratory: through field investigations and through literature sources. Even though the distribution of plants that accumulate intensely sweet compounds appears to be random in the plant kingdom, we believe our work shows t h a t the judicious selection of candidate plant species,
p-glc :a-rha
rather than sheer luck, can result in the discovery of novel highly sweet compounds. Field investigations
In our investigations, we have found that one of the most successful ways to locate new sweet lead plants is to survey local populations, especially in marketplaces. Generally speaking, humans view sweet-tasting plants as safe to cor%me, while bitter-tasting plants are f;.&uently considered toxic. Therefore, one can often find one or two sweettasting plants in traditional food and drug marketplaces. Some plants i n these investigations have been used in the native culture a s sweeteners, per se, such a s the previously mentioned Steuia rebaudiana in Paraguay and Brazil, and Thaurnatococcus daniellii in tropical West Africa. Other plants m a y b e known as slightly sweet to the indigenous peoples, but are not used as sweeteners due to the low levels of the compounds, or the presence of bitter or bioactive
&
HOH2C
..
O-
a-ara
R
7 Gaudichaudioside A 8 Gaudichaudioside F
CHO (sweet) C=C-COMe (bitter) Figure 2. A sweet and a bitter labdane glycoside from Baccharis gaudichaudiana (ara = L-arabinopyranosyl). Volume 72 Number 8 Auaust 1995
677
compounds along with the sweet compound. For example, from the Paraguayan medicinal plant, Baccharis gaudichaudiana DC. (Comnositae). the sweet diteroene elvcoside, gaudichaudioside A ( ~ i 2,~ 7), . was isolated (%I. The same plant also afforded a new, bitter-tasting labdane arabinoside called gaudichaudioside F (Fig. 2, 8 ) (19). Therefore. this olant mav not necessarilv be identified a s distinctly sweet by local people.
model for human sweet taste, i t does respond well to many "bulk" (i.e., sugars and polyols) and "intense" sweeteners, and the assay is more economic to perform than other in uiuo options currently available.
Literature Sources
The most potently sweet compound thus far isolated in our laboratory came from the previously mentioned ethnobotanical lead, Lippia dulcis. The sweet-tasting bisabolane sesquiterpenoid (+I-hernandulcin (Fig. 3 , 9 ) was identified a s a minor constituent (0.004% wlw) of the aerial parts of the plant (20, 26), and rated about 1,000 times sweeter than sucrose on a molar basis, although its hedonic qualities were somewhat wanting. Attemots were made to product! ;I hrrnandul(:in drri\wtive u,irh more dcasant hfdon~cuual~tirso \ w thr rxirmt compound 121,. but no sweet-tast;ng substances wire obtained. we were able demonstrate that the C - l keto, the C-1' hydroxyl, and the double bond between C-4' and C-5' are involved in the mediation of sweetness by hernandulcin. Through the use of molecular modeling techniques, we have calculated that the C-1' hvdroxvl and the C-1 keto groups are located about 2.6 apart; and are, therefore, consistent with the AH (proton donor) and B electroneaative (proton acceptor) units i n the Shallenberger model for sweet compounds (271, a s discussed more fully in the introductory paper by Ellis and the modeling paper by Walters. The side-chain double bond represents a third binding site, that interacts with the receptor sites through dispersion or hydrophobic forces, a s postulated by authorities such as Hansch and Kier (28). Recent work with the recollection of the leaves and flowers ofL. dulcis from Panama afforded a second novel sweet substance, (+)-4P-hydroxyhernandulcin (Fig. 3, 101, in trace amounts. This compound is noteworthy because i t is only the second sweet bisabolane sesq u i t e r p e n e t o h a v e been discovered, a n d i t also demonstrates that a C-4 methvlene erouD i s not essential to elicit sweet taste in the h e ~ a n d ~ l c i n ' c ~ m ~ oclass, und and the C-4 OH group provides a potential point of attachment for sugars or other polar moieties, in order to generate more water-soluble hernandulcin analogs (21). Despite the potent sweetness of this compound, unless a derivative can be prepared with improved hedonic uualities as well a s better h a t e r so~ubiliobotanical iiterature sources. There is most likely a general understanding of sweet perception among all human cultures on earth. Therefore. a notation that a olant tastes sweet bv- a oar. ticular indigenous group of people, is frequently validatedsometimes by the discovery of new intensely sweet compounds, but more often by the identification of high levels of free sugars. One of our most successful uses of a n ethnobotanical lead came from Francisco Hernandez's monograph "Natural History of New Spain" written between 1570-1576. This Spanish physician noted that the Aztec people knew a plant called "Tzonpelic xihuitl" to be exin the monograph, as tremely swcct. ~ a s c OII d illustratio~;% urll .IS orhrr lirernrure sonrrru, the plnnr was idmrilied as Llnolo dulcri 'l'w\: \idx:nxt.a(~s.Tht: nlant w a i coll(.ctc,d i n Mexico, and its sweetness confirmed organoleptically. Activity-directed isolation led to the discovery of a n intensely sweet bisabolane sesquiterpene, which will be discussed i n more detail later i n this oaDer . . (20. 21). One shortcoming of t h ~ stvpc of ethnobotanml lead IS that "swcwt"m;~vnl.+orefer to the odor ot'the d:int. which is not related to skeet-taste perception. Another approach involved a search ofIndexKewensis, a repository of all of the published Latin binomials (scientific names) of seed plants, using specific epithets which might be indicative of sweetness, and yielded a number of new possible lead plants, as well as some well-known sweet plants. For example, a search of the epithet dulcis or dulcificum (Latin for sweet) in Iiulex Kewensis turned up well-known sweet plants such as Lippia dulcis andperiandra dulcis (the sources ofhernandulcin and periandrins I-V, respectively) (22).
..
Assays for Sweetness Alimitine-steo. i n our discoverv of new sweet comnounds is the ability to assay for sweetness. Currently, there is no reliable in uitro method: therefore. we must relv on more involved in uiuo models to evaluate potentially skeet compounds. I n order for humans to taste chromatographic fractions or pure isolates, preliminary safety testing comprised of mouse acute toxicity and bacterial mutaeeuicitv studies must be performed. These tests represent :sign& cant use of time and resources. Therefore, we have looked to possible animal models to aid i n the evaluation of sweetness. In the past few years, we have employed, with some success, electrophysiological and behavioral experiments using the Mongolian gerbil (23). In the electrophysiological assays, a potentially sweet plant extract, fraction, or pure compound is applied to the tongue of a n anesthetized gerbil, and electrophysiological recordings are taken from the chorda tympani nerve, evaluating up to 25 samples with a sinele gerbil. This is backed bv a conditioned-taste aversion assay using gerbils that are trained to avoid sucrose. The combination of these methods appears to be helpful in selecting extracts of different polarities from plants for the presence or absence of sweet constituents and about 213 of the pure compounds tested that were known to be sweet to humans were evaluated as "sweet" to the gerbil(24).Other researchers have had success with different animal models such a s primates (25). While the gerbil i s not a perfect
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678
Journal of Chemical Education
Examples of Recently Discovered Natural-Occurring Sweet Compounds Sesquiterpenes
ow ever;
A
Triterpene Glycosides Another sweet plant with a rich ethnobotanical background is Abrusprecatorius L. (Fabaceae).While the seeds of A. nrecatorius are notorious for containing abrin. one of the most potent proteinaceous poisons known to humans, its leaves and roots appear to be devoid of this protein, and have been used in many cultures as a licorice substitute. In fact, there have been a number of renorts that Abrus contains the sweet ssponm, glycyrrhw~n Fig.1,3,or1ginallv ~solatedfrom linnict,. 1niti;il studi(+ in this I:~hor.iitorydid not d(wct glgqrrluzin In the lcnvci ofA. p~,crrrorruscollcctcd in Flor~da.Further work in our hhoraron establ~shedthat 3 l-butanol-soluble extract of t h e l e a i e s contained four novel sweet cycloartane-type triterpene glywsides, named abmsosides AD (Fig. 3, 11-14) (29, 30). Their water-soluble ammonium salts were rated as being 3&100 times sweeter than sucrose.
-
R 9 Hernandulcin H 10 (+)-4P-Hydroxyhernandulcin OH
15 Dihydroquercetin 3-acetate
H
Other (2R, 3R)
16 5, 7, 3' -Trihydroxy-4'-methoxy-2,3-
CH3
racemate
R
dihydroflavonol3-acetate (synthetic)
11 12 13 14
Abrusoside A Abrusoside B Abrusoside C Abrusoside D
P-glc P-glcA-6-CH3cP-glc P-glc Lp-glc P-glcA LP-glc 17 Selligueain A
Figure 3. Examples of recently discovered plant-derived sweet compounds (glc = D-glucopyranosyl; glcA = D-glucuronopyranosyl). Since abrusosides A-D are heat stable, and the hedonic qualities of these compounds a r e pleasant, these compounds have been patented, and their commercial development is currently being pursued. Dihydroflavonols A new class of dihydroflavonol sweet compounds was found i n Tessaria dodoneifolia (Hook. & A m . ) Cabrera (Compositae) which was purchased in Asuncion, Paraguay, under the name "hierba duke", meaning "sweet herb." The young, growing shoots of the plant were extracted and subjected to a standard partition scheme; the sweetness was found to be most pronounced in the ethyl acetate fraction. The sweet principle was identified a s a compound of known structure, dihydroquercetin 3-acetate (Fig. 3, 151, and rated as about 50 times sweeter than sucrose. The introduction of a para-substituted methoxy group i n ring B of the compound produced the novel 5,7, 3'-trihydroxy-4'methoxy-2,3-dihydroflavonol3-acetate (Fig. 3, 16) which proved to be 400 times sweeter than sucrose, although the onset of the sweet taste was delayed i n comparison to sucrose (31).This represents an example of how the investigation of a plant can lead to the recognition of a whole new class of sweet compounds, and also how a simple synthetic modification of a n a t u r a l product lead structure may greatly increase the resultant sweetness. Proanthocyanidins Amost surprising class of sweet natural products are the proanthocyanidins, also known as "condensed tannins". Proanthocyanidins are a large group of polyphenolic compounds, well known for their astringent taste. Recent work in this laboratory resulted in the discovery of a sweet-tasting proanthocyanidin, selligueain A, from the rhizomes of a fern, Selliguea feel Bory (Polypodiaceae) collected on vol-
canic soil i n West Java, Indonesia (32). Selligueain A was rated about 35 times sweeter than a 2% sucrose solution. The structure elucidation of this compound involved peracetylation, thiolysis, and desulfurization steps to establish the compound a s a novel doubly-linked Aunit trimeric s u b s t a n c e , w i t h t h e s t r u c t u r e epiafzelechin-(4P-8, 2~+0+7)-epiafzelechin-(4a+3)-afzelechin (Fig. 3, 17). I t appears that there are stringent structural requirements for proanthocyandins to exhibit a sweet taste because compounds closely related in structure to selligueain A were astringent r a t h e r t h a n sweet. Sweet-tasting trimeric proanthocyanidins have been isolated from both ferns and higher plants (33, 34). We feel that due to the strict structural requirements needed to elicit sweetness, there may not be many other sweet-tasting proanthocyanidins in the plant kingdom. Summary Although sucrose continues to he the most commercially Important sweetener, thrre are a~mpellinghealth reasons to lower sucrose consumption by humans. Plants have provided a number of highly intense sweeteners, and although there appears to be no chemotaxonomic correlation that can be used to enhance the discovery of these compounds, we believe that through careful consideration of t h e ethnobotanical literature and surveys of medicinal plant marketplaces in countries that are not yet fully industrialized, the chances of finding new sweet compounds are greatly increased. For more than 10 years i n this laboratory, we have isolated several new intensely sweet compounds representing a variety of structural types of natural products. Such a pursuit of new sweet natural products has not only potential commercial use, but also can be of value scientifically to aid i n the better understanding of structure-sweetness relationships, which may ultimately be useful for the rational design of sweet molecules.
Volume 72 Number 8 August 1995
679
Acknowledgment
Studies mentioned in this article and performed at this institution were supported by the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, and by General Foods Corporation, White Plains, New York. We wish t o acknowledge the contributions to this project by many excellent graduate students and postdoctorals, and by faculty colleagues, and particularly wish to recognize the pioneering role played by D. D. Soejarto, in the discovery of highly sweet compounds from ethnobotanical leads. We also wish to thank J. Ellis and D. E. Walters for kindly reviewing this paper.
13. Walfers. D. E.; Orfhoefer F T:DuBois. G. E. SluraBnms:Discoueri MoiecuioiD~. s e n , a n d Chamorece~lion:Sympo.3ium Series No. 459; ACS: Washingon. DC, 1991; p333. 14. Ishliiawa, H.: Khshata, S.: Ohlani, K.: Tanaka, 0. Chem Pharm. Bali. 1991,39, 9"**
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