Structure-Activity Relationship of Highly Sweet Natural Products A

Home , Phyllodulcin

Structure-Activity Relationship of Highly Sweet Natural Products A. Douglas Kinghorn, Fekadu Fullas and Raouf A. Hussain

1. INTRODUCTION There is an insatiable desire by humans for sweet substances, more so for the hedonic delight of the sweet taste sensation rather than for caloric needs (1). In fact, evidence has been put forth that even a five-month-old human fetus has a liking for sweet substances (2). Sucrose, the most abundant of all sugars, has been known and used as a sweetener and food item since as far back as 2,000 B.C. It is one of 100 or so saccharides that have been demonstrated to exhibit a sweet taste, and is by far the most widely used sugar (3). Sugar cane (Saccharum officinarum L.) and sugar beet (Beta vulgaris L.) are the two major sources of sucrose. The world production of sucrose from these two sources exceeded 100 million metric tons in 1988 (4). High-fructose corn syrup (HFCS), commercially produced from corn starch, is a recently developed product used to replace sucrose in many food systems. HFCS production in the United States alone was over 11 billion pounds in 1988, with the world production of HFCS outside of the United States being 3.8 billion pounds in 1989 (5). Sucrose exhibits a clean sweetness that is unmasked by any other taste sensation. In addition to other properties, its high solubility in water, its stability under thermal and hydrolytic conditions, and its cheap cost of production make it a widely utilized sweetener (6). Thus far, no sweetener has been found, either of natural or synthetic origin, which fulfils all of the desirable properties of sucrose. Therefore, sucrose still enjoys wide popularity as a sweetener in foods, beverages and medicines. However, large amounts of sucrose are used to sweeten these products, a situation which creates consequent nutritional and medical problems. Sucrose consumption by humans has been shown definitively to be the major cause of dental caries (7), and has been associated with cardiovascular diseases, diabetes mellitus, obesity and micronutrient deficiency (8). There is an obvious need by diabetic patients to cut down on their sugar intake. Hence, there is a continuous and growing societal demand for highly sweet non-caloric and non-cariogenic sucrose substitutes. Any synthetic or natural sucrose substitute should have a sucrose-like taste profile, should have no toxic or cariogenic effects (either in the metabolized or unmodified form), should be odorless, should exhibit a liberal water solubility, and should be thermally and hydrolytically stable. A new commercially exploitable sucrose substitute should be economical to synthesize or to extract from a readily cultivable plant source. In addition to these diverse and demanding attributes, a new sweetener should be able to be easily incorporated into different food and beverage products. Finally, it should also be extremely sweet, usually at least 100 times the sweetness potency of sucrose (9). Such compounds are often referred to as "intense sweeteners", and are generally regarded as a separate category than the caloric or "bulk" sweeteners constituted by sugars and polyols (10). The market for non-nutritive, intensely sweet substances of use as sucrose substitutes in foods, beverages, and medicines is very large, and was estimated as $1.1 billion in the United States alone in 1989 (11). Although many synthetic (1,2,9,12-17) and natural compounds (1,2,12,17-21) have been found to be intensely sweet, only a handful have wide commercial use. The major potently sweet sucrose substitutes approved for use in countries in North America and Western Europe are synthetic compounds, inclusive of saccharin, cyclamate, aspartame and acesulfame-K (10). Saccharin is approved for use in over 90 countries around the world (7). However, in 1977, the United States Food and Drug Administration (FDA) proposed a ban on the use of saccharin largely because of findings of bladder tumors in rats fed with high doses of saccharin. This ban has been lifted by a U.S. Congressional moratorium and saccharin use has been extended five times since being first imposed. The compound has now been pronounced safe by numerous expert committees (22). Cyclamate has also been associated with the production of bladder cancer in laboratory animals, and, cyclohexylamine, one of its metabolites, has been linked to additional adverse effects. While this sweetener is still approved for use in more than 50 countries, it is presently banned in Canada, the United States and the United Kingdom (11,23). The dipeptide, aspartame, is used in about 75 countries in more than 500 different products and is regarded as a very pleasant-tasting substance (7), but is contraindicated for persons suffering from phenylketonuria (24). Acesulfame-K is now employed as a sweetener in about 40 countries, and although a stable substance, a bitter taste is sometimes perceived with this compound (25). The search for synthetic sweeteners with better temporal qualities, greater sweetness potency, and improved stability and safety is continuing, and among the most promising compounds are the dipeptide, alitame (26), and the chlorinated sucrose derivative, sucralose (27), which are awaiting approval in several countries. An example of a hyperpotent sweet compound is the N-cyclononyl guanidine derivative, sucrononic acid, the sweetest compound to have been reported in the literature to date, with a potency of some 200,000 times the sweetness of sucrose (28). Whether or not such types of extremely potent sweeteners will continue to show promise during further development, and eventually enter the market, remains to be seen. As will be seen from section 3 of this chapter, there are more than 70 known plant-derived potently sweet compounds, representing about 20 structural types of organic compounds, and these occur in species of over 20 families of higher plants. Presently, several plant-derived compounds are used as sweetening and/or flavoring agents for human consumption in one or more countries, namely, glycyrrhizin (Japan), phyllodulcin (Japan), mogroside V (Japan), stevioside (Japan, Brazil and Korea), rebaudioside A (Japan) and thaumatin (Japan and U.K.), with most of these being utilized in the form of plant extracts (29,30). Also commercially used are the semi- synthetic sweeteners, perillartine (Japan) and neohesperidin dihydrochalcone (Belgium and Argentina), which are based on natural products (14,31). In Japan, extracts from the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae), containing stevioside and rebaudioside A, have the largest share in the "intense" sweetener market (29). The plant-derived commercially available sweetening agents will be discussed briefly in the next three paragraphs. Glycyrrhizin is the sweet principle of the roots and rhizomes of Glycyrrhiza glabra L. (Leguminosae), as well as of other species in the same plant genus, and occurs as a mixture of potassium, calcium, and magnesium salts in over 10% w/w yield in the plant. This substance, an oleanane-type triterpene glycoside, is widely utilized for the sweetening of beverages, cosmetics, foods, medicines, and tobacco in Japan (32,33). Ammonium glycyrrhizin, the fully ammoniated salt of glycyrrhizic acid, is in the GRAS (Generally Recognized As Safe) list of approved natural flavoring agents of the U.S. Food and Drug Administration, and finds broad application as a foaming agent, flavor modifier, and flavorant (32,33). Extracts from Stevia rebaudiana leaves, with stevioside and rebaudioside A as the major sweet principles, have been used in Japan as a sucrose substitute for over 15 years, and are particularly advantageous because these diterpene glycosides are heat stable, nonfermentable, and suppress the pungency of sodium chloride which is used in many Japanese foods (34,35). Products made from S. rebaudiana are approved for use in the sweetening of dietetic foods, oral hygiene products, and soft drinks in Brazil (36,37), and also have minor use in South Korea (33). A third type of natural product sweetener with commercial use in more than one country is the protein, thaumatin, of which the major principles are thaumatins I and II, and is extracted from the fruits of the West African rain forest shrub, Thaumatococcus daniellii (Bennett) Benth. (Marantaceae). Although approved for use as a sweetening agent in Japan and the United Kingdom, thaumatin is used elsewhere as a flavor enhancer and palatability improver, including the United States (29,33). There are two other plants whose highly sweet-tasting extracts are utilized on a limited basis in Japan. The first of these is the Chinese plant "1o han kuo" [Siraitia grosvenorii (Swingle) C. Jeffrey] [synonyms Momordica grosvenorii Swingle; Thladiantha grosvenorii (Swingle) C. Jeffrey] (Cucurbitaceae), whose dried fruits contain the cucurbitane-type triterpene glycoside, mogroside V, as the most abundant sweet constituent (20,33). In addition, the crushed leaves of Hydrangea macrophylla Seringe var. thunbergii (Siebold) Makino (Saxifragaceae) are used at certain religious festivals, and their sweetness is attributed to dihydroisocoumarin, phyllodulcin (20,33). Perillartine is the semi-synthetic a-~yn-oxime of perillaldehyde, a major constituent of the volatile oil of Perillafrutescens (L.) Britton (Labiatae), and is used in Japan as a replacement for maple syrup or licorice in the sweetening of tobacco (33). Neohesperidin dihydrochalcone, prepared by the sequential alkaline hydrolysis and catalytic reduction of neohesperidin, a flavanone constituent of Citrus aurantium L. (Rutaceae), is permitted for use in chewing gum and certain beverages in Belgium and elsewhere (31).

2. THEORIES OF SWEET TASTE RESPONSE INITIATION Since sweet natural products often co-occur in complex mixtures with bitter- and/or neutral-tasting analogs, it is germane to mention briefly some of the presently held views on the mechanism of sweet taste reception. Although sweet taste chemoreception is thought to be mediated by proteinaceous receptor sites located on the microvillus membrane of taste cells of the tongue, such a receptor has not so far been isolated and characterized (38,39). However, indirect evidence has been provided for the involvement of proteinaceous receptors in the sweet taste response. Thus, when the rat tongue was treated with specific proteases, the response to the sweet taste of sugars but not to other tastes was selectively abolished (40). A prevailing theory put forward by Shallenberger and colleagues in the late 1960's refers to the fact that nearly all sweet compounds possess two electronegative groups designated as AH and B in their molecular structures, which act as an acid and a base, respectively. The atomic orbitals of these groups should be between 2.5 and 4.5 A apart (with 2.86 A being optimal) and be in the right spatial orientation. Such an AH,B "glucophore" is considered to form a double hydrogen-bonded complex with a reciprocal AH, B unit at the sweetness receptor sites on the papillae of the tongue (41,42). [Van der Wel and colleagues (16) point out that a sweet compound contains two units, a "glucophore" and an "auxogluc". The glucophore is defined as a group of atoms capable of forming a sweet compound when combined with any auxogluc, which would otherwise be tasteless]. Although one can often discern possible AH, B units in many sweet compounds, it is not always possible to do this reliably in more structurally complex natural products such as sweet glycosides. For potent sweeteners, a third lipophilic site (X) at distances of 3.5 and 5.5 A from the AH and B units, respectively, seems to be involved in the initiation of sweet response (43,44). However, to complicate this issue somewhat, it has been postulated that as many as eight binding sites are involved in the mediation of the sweet-taste of the exceptionally potent sweetener, sucrononic acid, which, as mentioned earlier, has been rated as being about 200,000 times sweeter than sucrose (28). It is well-known that some substances exhibit a bitter-sweet taste, while sweet substances such as saccharin have some intrinsic bitterness. It is not clear, however, how these molecules distribute themselves, either with some on sweet receptors and some on bitter receptors, or else as single molecules that can span both sweet and bitter receptor sites simultaneously. However, it appears for such molecules that the corresponding sweet and bitter receptor sites must at least be very close to each other (43). As will be seen from many examples later in this chapter, minor structural modification of highly sweet natural products frequently results in the production of either bitter or tasteless analogs. There has been considerable debate for some time as to whether a single receptor or multiple receptors is(are) responsible for the initiation of sweetness (39,41). Certain evidence with inhibitors and photoaffinity labeling ligands supports the single receptor notion (39). Other authorities have postulated the existence of multiple receptors, from evidence such as the structural diversity of sweet compounds thus far discovered, from single-nerve fiber electrophysiology data, from cross-adaptation experiments, and as a result of the demonstration of synergism in sweetener mixtures (39). The possibility of the occurrence of multiple receptors for sweet substances complicates the task of new sweetener design, as a different receptor might exist for each class of sweetener. Hence, the approach in synthetic sweetener design has been to modify structural features within a given class of compounds, acting at a common receptor (14). 3. STRUCTURE-SWEETNESS RELATIONSHIPS AMONG SOME NATURAL SWEETENERS In this section of the chapter, the presently known highly sweet substances of natural origin are listed in Table I, and new information on the known structure-sweetness relationships for each compound category is presented in the text. In order to focus attention on naturally occurring sweet substances, the only semi-synthetic compounds included in the table are those that represent prototype members of distinct structural types. We have reviewed the various types of natural product intense sweeteners in some detail previously (20,21), and only references published subsequently to these reviews will be provided in the table. The structures of the compounds will be interspersed in the text. The following abbreviations are used to designate the sugars present in the various glycosides included in these structures: api = D-apiofuranosyl; ara = L- arabinopyranosyl; glc = D-glucopyranosyl; glcA = D-glucuronopyranosyl; rha = L- rhamnopyranosyl; xyl = D-xylopyranosyl. There has been much activity in several laboratories in recent years leading to the isolation of many novel natural product sweeteners, and it is of interest that sweet compounds are now known in three new classes, namely, the proanthocyanidin, dibenz[b,d]oxocin, and amino acid classes. It is to be noted that to date, all of the natural product sweet substances have been found as constituents of higher plants, although it is conceivable that such compounds may also occur as constituents of microorganisms, lower plants, marine animals, or insects (21). While plants in restricted taxonomic groups often biosynthesize chemically similar secondary metabolites, the distribution of plants known to produce intensely sweet plants appears to be random throughout the angiosperms. However, there is some evidence of more than one species in the same genus producing the same sweet compounds, as in the case of Glycyrrhiza and Periandra species (21). In addition, in the last few years considerably more information has become available on this phenomenon. Prior to presenting data on the sweetness potency of each compound in Table I, it is pertinent to briefly mention how sensory data of this type are obtained in the laboratory. It is highly advisable to perform experiments using human taste panels only on compounds which are pure, and for which acute toxicity and bacterial mutagenicity studies have been performed (e.g., 48,57,76,77). Such preliminary safety testing will consume a minimum of several hundred milligrams of each sweet substance examined, which is often not available for minor sweet analogs present in plant extracts. It is mainly for this reason that several of the compounds listed in Table I are indicated as being sweet, but for which no quantitative data are available. A number of approaches to determining the sensory characteristics of sweet-tasting compounds have involved quite large numbers of human subjects, and enable hedonic attributes (indicating pleasant and unpleasant flavors) as well as sweetness intensity values relative to sucrose to be obtained (e.g., 78-80). However, in the last few years in our laboratory, we have performed sensory evaluations with small taste panels consisting of only three experienced staff personnel (48,57,76,77). In this manner, approximate threshold values of sweetness intensity for a compound under test can be determined by dilution until a sweetness level equivalent to that of an aqueous solution of 2% w/v sucrose is obtained. This method is very economical in the amount of each sample consumed, and TABLE I

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b

MONOTERPENE

Perillartine c (1) Perilla frutescens Britton 370 20 (Labiatae)

SESQUITERPENES

Hernandulcin (2) Lippia dulcis Trev. 1,500 20, 21 (Verbenaceae) 41]-Hydroxyhernandulcin L. dulcis N.S. d 48 (3)

DITERPENES

Diterpene acid

4~, 10o~-Dimethyl- 1,2,3,4,5,10 Pine tree f 1,300- 20 hexahydrofluorene-4ct,6ct- 1,800g dicarboxylic acid e (4) ent-Kaurene glycosides

Dulcoside A (5) Stevia rebaudiana (Bertoni) Bertoni 30 20 (Compositae) Rebaudioside A (6) S. rebaudiana 242 20 Rebaudioside B (7) S. rebaudiana 150 20 Rebaudioside C (8) S. rebaudiana 30 20 Rebaudioside D (9) S. rebaudiana 221 20 Rebaudioside E (10) S. rebaudiana 174 20 Stevioside (11) S. rebaudiana 210 20 Rubusoside (13) Rubus suavissimus S. Lee 114 20 (Rosaceae) Steviolbioside (12) S. rebaudiana 90 20 Steviol 13-O-13-D-glucoside Rubus suavissimus N.S. d 50 (14) Suavioside A (15) R. suavissimus N.S. d 49 Suavioside B (16) R. suavissimus N.S. d 50 Suavioside G (17) R. suavissimus N.S. d 50 TABLE I (continued)

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b ent-Kaurene glycosides (continued)

Suavioside H (19) R. suavissimus N.S. d 50 Suavioside I (18) R. suavissimus N.S. d 50 Suavioside J (20) R. suavissimus N.S. d 50

Labdane glycosides

Baiyunoside (21) Phlomis betonicoides Diels 5OO 20 (Labiatae) Phlomisoside I (22) P. betonicoides N.S. d 21 Gaudichaudioside A (23) Baccharis gaudichaudiana DC. 55 51 (Compositae)

TRITERPENES

Cucurbitane glycosides

Bryodulcoside h Bryonia dioica Jacq. N.S. d 20 (Cucurbitaceae) Bryoside (24) B. dioica N.S. d 52 Bryonoside (25) B. dioica N.S. d 52 Carnosifloside V (26) Hemsleya carnosi.flora 51 21, 53 C.Y. Wu et Z.L. Chen (Cucurbitaceae) Carnosifloside VI (27) H. carnosiflora 77 21 Scandenoside R6 (28) Hemsleya panacis-scandens 54 53, 54 C.Y. Wu et Z.L. Chen Mogroside IV (29) Siraitia grosvenorii i 233-392g 20, 54 (Swingle) C. Jeffrey (Cucurbitaceae) Mogroside V (30) S. grosvenorii 250-425g 20 11-Oxomogroside V (31) Siraitia siamensis Craib N.S. d 55 (Cucurbitaceae) S. grosvenorii 84 54 Siamenoside I (32) S. siamensis 563 54, 55 S. grosvenorii 10

TABLE I (continued)

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b

Cycloartane glycosides

Abrusoside A (33) A brus precatorius L.; 30 56-58 A. fruticulosus Wall et W. & A. (Leguminosae) Abrusoside B (34) A. precatorius; A. fruticulosus 100 57, 58 Abrusoside C (35) A. precatorius; A. fruticulosus 50 57, 58 Abrusoside D (36) A. precatorius; A. fruticulosus 75 57, 58

Oleanane glycosides

Glycyrrhizin (37) Glycyrrhiza glabra L. 93 20 (Leguminosae) Apioglycyrrhizin (38) Glycyt~hiza inflata Batal 180 59 Araboglycyrrhizin (39) G. #~ata 93 59 Periandrin I (40) Periandra dulcis Mart. 90 20 (Leguminosae) Periandrin II (41) P. dulcis 95 20 Periandrin III (42) P. dulcis 92 20 Periandrin IV (43) P. dulcis 85 20 Periandrin V (44) P. dulcis N.S. d 60

STEROIDAL SAPONINS

Osladin (45) Polypodium vulgare L. 500 20, 61 (Polypodiaceae) Polypodoside A (46) Polypodium glycylT"hiza DC. 600 62, 63 Eaton Polypodoside B (47) P. glycytT~hiza N.S. d 63, 64

PHENYLPROPANOIDS

trans-Anethole (48) Foenicuhtm vulgate Mill. 13 65 (Umbelliferae) Illicmm verum Hook. f. (Illiciaceae) Mytwhis odorata Scop. (Umbelliferae) TABLE I (continued)

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b

PHENYLPROPANOIDS (continued)

trans-Anethole (48) Osmorhiza longistylis DC. 13 65 (continued) (Umbelliferae) Piper marginatum Jacq. (Piperaceae) Tagetesfificifo#a Lag. (Compositae) trans-Cinnamaldehyde (49) Cinnamomum osmophloeum 50 21 Kanehira (Lauraceae)

DIHYDROISOCOUMARIN

Phyllodulcin (50)J Hydrangea macrophylla Seringe 400 20 var. thunbergii (Siebold) Makino (Saxifragaceae)

FLAVONOIDS

Dihydrochalcone glycosides

Naringin dihydrochalcone c Citrus paradisi Macfad. 300 20 (sl) (Rutaceae) Neohesperidin dihydro- Citrus aurantium L. 1,000 20 chalcone c (52) Glycyphyllin (53) Smilax glycyphylla Sm. N.S. d 20 (Liliaceae) Phlorizin (54) Symplocos lancifolia Sieb. et Zucc. N.S. d 20 (Symplocaceae) Trilobatin (55) Symplocos microcalyx Hayata N.S. d 20

Dihydroflavonols and Dihydroflavonol glycosides

Dihydroquercetin 3-0- Tessaria dodoneifo#a 400 21 acetate 4'-(methyl ether) c (Hook. & Arn.) Cabrera (s6) (Compositae) 12

TABLE I (continued)

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b

Dihydroflavonois and Dihydroflavonol glycosides (continued)

(2R,3R)-Dihydroquercetin T. dodoneifo#a; 80 21 3-O-acetate (57) Hymenoxys turneri K. Parker (Compositae) (2R,3 R )-2,3-Dihydro-5,7, 3 ', 4'- H. turneri 25 66 tetrahydroxy-6-methoxy-3-O- acetylflavonol (58) (2R,3R)-2,3-Dihydro-5,7,4'- H. tmTwri 20 66 trihydroxy-6-methoxy- 3-O-acetylflavonol (59) (2R,3R)-2,3-Dihydro- H. turned 15 66 5,7,3 ', 4'-t etrahydroxy- 6-methoxyflavonol (60) Neoastilbin (61) Engelhat'dtia cht),solepis Hance N.S. d 67 (Juglandaceae) Huangqioside E (62) E. chlysolepis N.S. d 68

PROANTHOCYANIDINS

Cinnamtannin B-1 (63) Cimlamomum sieboldii Meisner N.S. d 69 (Lauraceae) Cinnamtannin D- 1 (64) C. sieboldii N.S. d 69

Unnamed (65) Arachniodes sporadosora Nakaike; N.S. d 70 A. exifis Ching (Aspidiaceae) Unnamed (66) A. sporadosora; A. exifs N.S. d 70 Selligueain A (67) Selliguea feei 35 71 (Polypodiaceae)

BENZIbllNDENOII,2-d]PYRAN

Hematoxylin (68) Haematoxylon campechianum L. N.S. d 72 (Leguminosae) 13

TABLE I (continued)

PLANT-DERIVED HIGHLY SWEET COMPOUNDS

Compound type/name a Plant name Sweetness Reference potency b

AMINO ACID

Monatin (69) Schlerochiton ificifolius A. Meeuse 1,200- 73 (Acanthaceae) 1,400g

PROTEINS

Curculin Curculigo latoeolia Dryand. 550 74 (Hypoxidaceae) Mabinlin Capparis masaikai Levl. N.S. d 21 (Capparidaceae) Monellin Dioscoreophylhtm cumminsii 3,000 20 (Stapf) Diels. (Menispermaceae) Pentadin Pentadiplandra brazzeana 500 21, 75 Baillon (Pentadiplandraceae) Thaumatin Thaumatococcus daniellii 1,600 20 (Bennett) Benth. (Marantaceae) aStructures of compounds are shown in the text. bValues of relative sweetness on a weight comparison basis to sucrose (= 1.0) are taken from the respective literature data, or from ref. 20. Compounds may have been converted to more water- soluble salts, prior to sensory evaluation. CSemi-synthetic derivative of natural product. dSweetness potency not given. e Synthetic sweetener. fBinomial name not given. gRelative sweetness varied with the concentration of sucrose. hComplete structure and stereochemistry not yet determined. 1 Formerly named Momordica gT~osvenorii Swingle, and Thladiantha grosvenorii (Swingle) C. Jeffrey (33). JThe plant of origin has to be crushed or fermented in order to generate phyllodulcin. 14 provides at least some information on compound taste qualities other than sweetness. The sensory data for the sweet plant-derived compounds in Table I refer to sweetness intensity comparisons with sucrose on a weight basis. However, comparison of these data is most reliable for compounds in the same structural class that have been evaluated for sweetness intensity in the same laboratory. Also, sweetness intensity values tend to vary depending upon concentration of the tastant compound. For example, the sodium salt of the newly discovered amino acid, monatin (69) exhibited relative sweetness intensities to 5% and 10% w/v sucrose of 1,400 and 1,200, respectively (73). Therefore, for this compound, and for several others listed in Table I, sweetness intensity values are expressed as ranges. 3.1 Terpenoids and Steroids 3.1.1 Monoterpenoids. It was mentioned earlier in this chapter that the semi-synthetic oxime, perillartine (1), is sweet and has some commercial use in Japan. In experiments designed to optimize the sensory attributes of the oxime sweeteners, it was found that the introduction of ether groups was advantageous, while hydroxyl groups and ring oxygen atoms tended to lower sweetness intensity and to destroy the sweet taste, respectively (16). Despite the discovery of cyclic derivatives of perillartine that are sweeter than this lead compound (20), the further development of this class of sweet substances is limited by poor water solubility and inappropriate hedonic qualities (20,21).

OH I N )

~CH,2

3.1.2 Sesquiterpenoids. The bisabolane-type sesquiterpene alcohol, (+)-hernandulcin (2), has been rated by a taste panel as being about 1,500 times sweeter than 0.25 M sucrose on a weight basis, but has also some bitterness and distinct off- and attertastes (20,21). This novel compound was isolated in 1985 from Lippia dttlcis, a plant recognized as being sweet by the 16th century Spanish physician, Francisco HernS.ndez. Accordingly, the compound was named in Hern/mdez' honor (20,21). Both racemic hernandulcin and the (+)-isomer have been synthesized by several other groups, and the absolute configuration of the naturally occurring form has been shown as 6S,1'S (48,81). It has also been established that 6S, l'S-hernandulcin is the only one of the four possible diastereomeric forms of this substance to be intensely sweet (81). This sweet substance has been produced from both hairy root cultures and shoot cultures ofL. dulcis, with a 15 yield of as high as 2.9% w/w dry weight being obtained in the latter case (82,83). Although hernandulcin was obtained in very low abundance (0.004% w/w) when first isolated (80), this sweet sesquiterpenoid was afforded in a much higher yield (0.15% w/w) from the leaves and flowers of a sample of L. dulcis collected in Panama (48), thereby suggesting that it occurs at high levels when the plant is flowering.

0 OH

2 R=H

3 R =OH

Attempts were made in this laboratory to synthesize sweet-tasting analogs of hernandulcin with improved hedonic characteristics, but resulted in the production of no further derivatives with a sweet taste. Thus, acetylation of the tertiary alcohol unit at C-I' and reduction of the C-1 keto group abrogated any perception of sweetness in each case (76). A series of racemic hernandulcin analogs was prepared by directed-aldol condensation between appropriate starting ketones, according to reaction conditions worked out for the synthesis of the parent compound (77,80). It was found that, even when the C-1 keto and the C-1' hydroxyl groups were kept intact, removal of the double bonds between either C-2 and C-3, and C-4' and C-5', or the methyl groups attached to C-3 or C-5', or the lipophilic side-chain, or substitution of the cyclohexenone ring with a cyclopentenone ring, led to the generation of mainly bitter-tasting analogs (77). As a consequence of this work, and as a result of accompanying molecular modeling experiments, it was concluded that the C-1 keto group, the C-I' tertiary hydroxyl group, and the double bond between carbon atoms C-4' and C-5' are three structural units involved in the binding of hernandulcin to its putative receptor (77). A compound isolated and characterized from the Panamanian collection of L. dulcis referred to earlier is a second highly sweet bisabolane sesquiterpenoid, 413-hydroxyhernandulcin (3). The sweetness potency of this substance relative to sucrose was not determined because of the very small quantity isolated. However, this isolate is noteworthy since it demonstrates that a C-4 methylene unit is not essential for the mediation of the sweet taste of the hernandulcin-type natural product sweeteners, and also provides a possible point-of-attachment for sugars or other polar moieties, in order to render more water-soluble sweet hernandulcin analogs (48). Despite its high sweetness intensity, hernandulcin is limited as a potential sweetener because of its somewhat unpleasant hedonic attributes and its thermolability. In spite of this, a 16 dentifrice formulation containing menthol and some cyclic ketones has recently been developed to both mask the taste ofhernandulcin and to afford storage stability (84). 3.1.3 Diterpenoids. Perusal of Table I shows that the known sweet diterpenoids from plants can be classified into the tricyclic resin acid (4), and ent-kaurene (5-20) and labdane (21-23) glycosides. Despite being a very promising lead because of its sweetness potency (20,21), there appears to have been no further work performed on developing analogs of resin acid 4 in recent years. Therefore, this substance will not be discussed further in the present chapter. In the following paragraphs, the sweet-tasting ent-kaurene and labdane diterpene glycosides will be discussed in turn.

,, -',,.OO'H'COOH

Structurally closely related, potently sweet ent-kaurene glycosides are found in high concentration levels in the leaves of two taxonomically disparate species, Stevia rebaudiana (Compositae) and Rubus suavissimus (Rosaceae), which are native to the borders of Paraguay and Brazil, and the People's Republic of China, respectively (20,21). It is interesting to note that no other species in either of the genera Stevia or Rubus appear to accumulate sweet ent-kaurene glycosides in significant amounts, although these compounds have been detected in trace quantities in a Mexican species, Stevia phlebophylla A. Gray (85,86). Documentation has come to light recently indicating that the leaves of S. rebaudiana have been used by Guarani Indians, Mestizos, and local herbalists in Paraguay to sweeten beverages for at least 100 years (87). Chemical work to determine the structural nature of the sweet constituent or constituents of S. rebaudiana leaves began in the early years of the present century, but the structure of stevioside (11) was not correctly determined until some sixty years later (20,21,34). During the 1970's, additional sweet compounds were isolated and characterized from S. rebaudiana leaves by the Tanaka group at Hiroshima University in Japan, inclusive of rebaudioside A (6), which is sweeter and more pleasant-tasting than stevioside. The sweet ent-kaurene glycosides occur at remarkably high yields in dried S. rebaudiana leaves, with the four major glycosides being stevioside (5-10% w/w), rebaudioside A (2-4% w/w), rebaudioside C (9, 1-2% w/w), and dulcoside A (5, 0.4-0.7% w/w) (34). As noted earlier, S. rebaudiana extracts, as well as stevioside and rebaudioside A, have use in Japan for sweetening purposes, and are also commercially utilized in other countries (29,30,34,35). 17 [~"~OR2 -~ CH2

/+%H~FOOR1 __

R1 R2

5 I]-glc 13-glc~-rha

6 ]3-glc 13-glcZ---13-glc 13 [3-glc 7 H 13-glc~13-glc

-glc 8 13-glc ~3-glc~oc-rha 13 ~3-glc 9 13-glc2l]-glc [3-glc~13-glc 13 13-glc 10 13-glc~13-glc 13-gl cZ---13-glc

11 13-glc 13-glc~l]-glc

12 H 13-glc~13-glc

Sweetness potency figures for the eight individual sweet diterpene glycosides (5-12) so far isolated and characterized from S. rebaudiana leaves are shown in Table I. Thus, it may be observed that the more highly branched compound, rebaudioside A (6), is somewhat sweeter than stevioside (11), and that a similar relationship holds true for the minor S. rebaudiana constituents, rebaudiosides D (9) and -E (10), both of which are highly sweet. Removal of the C-19-affixed D- glucosyl groups of rebaudioside A and stevioside to produce rebaudioside B (7) and steviolbioside (12), respectively, which can be performed by alkaline hydrolysis, results in a diminution of sweetness potency in both cases. Substitution of one of the glucose units in the C-13-attached saccharide moiety by rhamnose of rebaudioside A and stevioside, as in rebaudioside C (8) and dulcoside A (5), results in even greater reduction in sweetness potencies (Table I). In addition, rebaudioside C has been demonstrated to exhibit pronounced bitter properties. While the other S. 18 rebaudiana sweeteners are less bitter than rebaudioside C, many of them have undesirable aftertastes (20,21). Rubusoside (13; = desglucosylstevioside) is found in the dried leaves of Rubus suavissimus in a yield of over 5% w/w, and has been rated as being about 115 times sweeter than sucrose, but, like stevioside, it has some bitterness and an at~ertaste. Recently, additional analogs of rubusoside have been isolated as minor constituents ofR. suavissimus leaves (49,50). One of these glycosides, suavioside A (15), was found to be sweet, while an analog with a keto group replacing the secondary alcohol group at the 3-position (sugeroside) was bitter (49). Compared with rubusoside and the sweet ent-kaurene glycosides from S. rebaudiana, suavioside A lacks an exomethylene functionality at C-16 and the position of the sugar moiety is translocated from C-13 to C-17 (49). Six additional minor diterpene glycoside constituents of R. suavissimus have been structurally determined recently, namely, steviol 13-O-]3-D-glucoside (14; = steviolmonoside) and suaviosides B, G, H, I and J (16-20) (50). Although quantitative sweetness potency values have not been determined for the minor sweet diterpene constituents ofR. suavissimus, suavioside B (16), which differs from rubusoside (13) only in the possession of a 913-hydroxyl group, is considerably less sweet (50). Several additional bitter- and neutral-tasting ent-kaurene diterpenoids were also obtained from R. suavissimus leaves (50).

/OR2 - 9 ~~1CH2

,,,, COOR1 ]9

R1 R2 R3

13 [3-glc ]3-glc

14 H ]3-glc H

16 [3-glc ]3-glc OH 19

~,,,,CH2OR /"'OH

HO ,,,,,,,

15 R = 13-glc

/OR2 ,~,,,, CH2R1 /"OH

R1 R2 R3

17 H 13-glc ]3-glc

18 OH H ]3-glc

There have been several attempts to improve the organoleptic properties of stevioside and rubusoside in the last ten years. In one such study, a disulfonic acid derivative of stevioside with increased hydrophobicity was found to be devoid of any bitter taste (88). The Tanaka group at Hiroshima, however, has improved the taste qualities of both stevioside and rubusoside by enzymic ~-(1-->4)-transglucosylation using cyclodextrin glucanotransferase (89-92). For example, 1,4-o~- mono-, di-, tri- and higher glucosylation occurred at both the 13-O-glucosyl and the 19-COO-[3- glucosyl moieties of stevioside, when treated with enzyme, leading to a mixture of glycosylated products consisting of nearly ten components (92). Most of these were less intensely sweet than stevioside, but more pleasant-tasting Sweetness intensity was optimal with three or four glucosyl units attached to the C-13 hydroxyl group (92). It appears that a sweet-tasting glycoside mixture obtained from stevioside in this manner now has commercial value in Japan, where it is known as "glucosyl stevioside" (92). 20

/OR2 : ..... /,~R1

R1 R2 R3

19 CHO 13-glc 13_glc

20 CH2OH 13-glc ~-glc

Baiyunoside (21) is a sweet constituent of the Chinese medicinal plant, Phlomis betonicoides Diels, that was first identified by the Tanaka group at Hiroshima. This substance has been rated as having a sweetness potency of about 500 times greater than that of sucrose, although it has a lingering aftertaste lasting for more than an hour (20,21). Phlomisoside I (22) is based on the same aglycone, baiyunol, as baiyunoside, and is also sweet-tasting. This compound bears a neohesperidyl saccharide unit, and may be contrasted with another P. betonicoides glycosidic constituent, phlomisoside II, which is bitter, and differs only from 22 in possessing a sophorosyl (13-D-glucopyranosyl-(2~ 1)-13-D-glucopyranosyl) sugar unit (20,21). Nishizawa and co-workers at Tokushima Bunri University in Japan have performed some substantial work on the preparation of analogs of baiyunoside, having initially prepared racemic baiyunol by the catalytic cyclization of 13-oxoambliofuran with a mercury (II) triflate/N,N-dimethylaniline complex (93), and then producing baiyunoside by a novel 2'-discriminated glucosidation procedure (94). Altogether, over 20 glycosides based on the baiyunoside parent molecule have been prepared, many of which were bitter while others were sweet. It was found that A7,8-baiyunoside was as sweet as baiyunoside itself, and, rather surprisingly that the sweetest compound of all derivatives made was the corresponding 13-D-glucopyranosyl-o~-D-glucopyranosyl analog of the previously mentioned bitter compound, phlomisoside II (95). The latter compound was reported as being very expensive to produce (95), which probably precludes its possible commercial development. 21

RO" ~,,,, v

21 R = ~-glc2~3-xyl

22 R = [3-glc2ct-rha

In recent work carried out at our institution, a novel labdane diterpene arabinoside, gaudichaudioside A (23) was obtained as the sweet principle of Baccharis gaudichaudiana DC. This observation is unexpected, since other species in the same genus taste very bitter rather than sweet. The plant was identified as being sweet-bitter-tasting after ethnobotanical inquiries at a medicinal plants' market at Asuncion, Paraguay, where it was referred to as "chilca melosa" and used traditionally as an antidiabetic remedy (51). The plant was collected in the field from a native population in eastern Paraguay, and found to exhibit a predominantly sweet taste, accompanied by some bitterness. In the laboratory, the sweet effect was traced to a 1-butanol extract, thereby suggesting the compound (or compounds) responsible was glycosidic. Gaudichaudioside A (Fig. 4, 23) was found to be based on a normal labdane skeleton, and to possess an L-arabinopyranosyl unit that is substituted equatorially, as well as having two double bonds, an unsaturated aldehyde affixed to C-8, and two hydroxymethyl groups attached to C-15 and C-19. Gaudichaudioside A exhibited about 55 times the sweetness intensity of a 2% w/w aqueous sucrose solution, when evaluated by a human taste panel. At the concentration at which it was tested, the compound gave only a very low perception of bitterness (51).

~ 15CH20H 2 ,~CHO

HOH2 " OR

23 R = o~-ara 22

Attempts to purify the aglycone of gaudichaudioside A (23) using mineral acids and various enzymes were not successful due to apparent lability. Therefore, it has not yet proven possible to synthesize potentially sweeter analogs of this parent compound with longer saccharide moieties, in an analogous manner to the work previously performed on baiyunoside (21) that has just been described. However, a series of five diterpene arabinosides closely related structurally to the parent compound has been isolated from ethyl acetate- and butanol-soluble extracts of B. gaudichaudiana, and were named gaudichaudiosides B through F (51,96). It was found that while only gaudichaudioside A was highly sweet, these other compounds demonstrated a range of taste effects. Thus, substitution of the C-8-affixed aldehyde of gaudichaudioside A with a hydroxymethyl group as in gaudichaudioside B (0.5% w/v in water) resulted in a sweetness sensation lasting for a few seconds followed by prolonged bitterness, when tasted (51). Substitution of gaudichaudioside B with an o~-substituted secondary hydroxyl group at C-2, as in gaudichaudioside C, resulted in a tasteless derivative. The other compounds in this series, inclusive of gaudichaudioside F, which is based on a novel trihomolabdane skeleton, gave either sweet-bitter or entirely bitter taste responses (51,96). Thus, the prototype labdane diterpenoid arabinoside, gaudichaudioside A (23) remains the only highly sweet compound in this class discovered to date. At one time, B. gaudichaudiana was taxonomically classified as a varietal form of Baccharis articulata (Lam.) Pers., a widely utilized medicinal plant in several South American countries. However, when B. articulata was examined for its constituents, neither gaudichaudiosides A-F nor any labdane diterpenoids were found to be present. These observation thereby offer chemotaxonomic substantiation for classifying these two taxa as separate species (97). 3.1.4 Triterpenoids. It may be seen from Table I that more highly sweet triterpenoids are now known than any other class of natural product. Furthermore, sweet compounds of this type are now based on three distinct triterpene carbon skeletons, namely, cucurbitane (24-32), cycloartane (33-36), and oleanane (37-44). In the latter category, two distinct groups are now evident, analogs of glycyrrhizin and of periandrin I. These groups of natural sweeteners will be discussed in turn. One of the most fascinating sweet-tasting species that has been encountered thus far is the Chinese medicinal plant "1o han kuo", which has been used for centuries for the treatment of colds, sore throats, and minor gastro-intestinal complaints. The fruits of this vine are dried in large ovens before being used in commerce, a fact which testifies to the thermal stability of the sweet components. However, this plant was not studied botanically until the 1930's, and proved to be a new species when first examined in 1937 (20). Originally called Momordica grosvenorii, then later Thadiantha grosvenorii, the plant is now correctly referred to as Siraitia grosvenorii (20). Phytochemical work commenced on S. grosvenorii in the 1970's and the structures of the two major sweet constituents of "1o han kuo" as mogroside IV (29) and V (30) were determined by Takemoto and Arihara and colleagues at Tokushima Bunri University in Japan (20,21). However, the major sweet constituent is mogroside V, and it occurs in over 1% w/w yield in S. grosvenorii fruits (20). This compound is one of the sweetest natural products, and has been rated in a range of 256-425 times the sweetness potency of sucrose (20,54). Mogroside IV, with one D-glucose unit 23 less in its structure than mogroside V, is slightly less potently sweet.

OH H o"%0 25 R2 O P _

RiO" 7"%,,,

R1 R2

24 13-glcLo~-rha 13-glc

25 13-glcLo~-rha 13-glcLI3-glc

~176 ~ 92

HOoso 0

o i R1 ,.,.,.,

R1 R2 R3

26 13-glc CH20-l]-glc2--13-glc CH3

27 13-glc CH20-I]-glc613-glc CH3

28 13@c CH3 CH20-13-glc213-glc 24

OR2

H R

R1o ....,,,

R1 R2 R3

29 13-glc613-glc 13-glc~13-glc oc-OH, [~-H

30 13-glc613-glc [3-glc~13-glc c~-OH, I3-H 16 13-glc 31 13-glc6--13-glc I3-glc~13-glc =o 16 13-glc 32 13-glc 13-glc~13-glc c~-OH, I3-H

13-glc

Following the discovery of the mogrosides IV and V, the cucurbitane triterpenes have emerged as a large group of natural sweeteners. Phytochemical investigation of the roots of Bryonia dioica has led to the isolation and characterization of two sweet compounds, bryoside (24) and bryonoside (25). Both of these compounds were isolated and structurally determined earlier by Hylands and Kosugi (98), but the structure of the sugar unit attached to C-25 in bryonoside was revised (52). No information was provided about the relative sweetness potencies of these substances (52). Three sweet natural product cucurbitane glycosides have been purified from two species in the genus Hemsleya by the Tanaka group at Hiroshima (26-28), and were found to co-occur with several analogs that were either bitter-or neutral-tasting (21,53,54). However, none of these compounds, nor several sweet semi-synthetic analogs were found to be of very high sweetness potency (53,54). Two minor sweet cucurbitane glycosides were isolated from the fruits of Siraitia grosvenorii, namely, l l-oxomogroside V (31) and siamenoside I (32). Siamenoside I, which is identical in structure to mogroside V (30), except for being only monoglucosylated at the C-3 position, is the sweetest compound among the cucurbitane glycosides discovered so far, in having a sweetness potency rated as 563 times that of sucrose (54). Compounds 29 through 32 were earlier isolated from Siraitia siamensis by Tanaka and co-workers (55). It has been determined that in order to exhibit a sweet taste in this class of compounds, at 25 least three sugar units must be present in the molecule, and glycosides of 1 l o~-hydroxy, 1113- hydroxy, and 11-keto compounds are, respectively, highly sweet, neutral-tasting, and less highly sweet or bitter (53,54). Abrusosides A-D (33-36) are a group of recently discovered cycloartane-type triterpene sweeteners, that were first isolated from the leaves of Abrus precatorius (56,57). Although the seeds of this species are well-known to produce the ribosome-inactivating protein toxin, abrin, the leaves of A. precatorius do not appear to be poisonous, and are ingested without apparent harm in systems of traditional medicine in certain southeast Asian countries. The well-defined sweetness of the leaves has been frequently documented in the scientific literature as being due to the presence of the oleanane-type triterpene sweetener, glycyrrhizin (see below). However, analysis of a sample of the leaves ofA. precatorius collected in Florida did not reveal the presence of glycyrrhizin, but the new compounds abrusosides A-D were found to occur. These compounds are similar in polarity to glycyrrhizin, and were extracted from a 1-butanol extract of A. precatorius leaves. Abrusoside A (33), the least polar representative of this series, was found to possess a cyclopropyl ring and an tx,[3-unsaturated 8-1actone ring, as well as an unsubstituted carboxylic acid unit at C- 29, and was glucosylated at the C-3 position. Abrusoside A was shown to be based on a new carbon skeleton, and the structure of its aglycone was confirmed atter the performance of single- crystal X-ray crystallography on the methyl ester (56). The three other analogs isolated from A. precatorius leaves, abrusosides B-D (34-36), all possess a disaccharide unit affixed to C-3 and are sweeter than abrusoside A.

0 " I

H _

RO "'COOH 29

33 13-glc

34 13-glcA-6-CH3~13-glc

35 [3-glc2--13-glc

36 J3-glcALf3-glc 26

The two sweetest compounds in this series are those possessing glucuronic acid units, namely, abrusoside B and abrusoside D, with the former compound with one D-glucuronic acid methyl ester being more potent. Abrusoside C, with a sophorosyl sugar unit, was intermediate in sweetness potency between abrusoside D and abrusoside A. In A. precatorius leaves, the most abundant of these compounds was abrusoside D (57). All four compounds have also been detected in the leaves of a second species, A. fruticulosus, with abrusoside B being the most abundant representative (58). The abrusosides are very stable to heat, and can be made water-soluble by conversion to their ammonium salts, and do not seem to have a bitter taste accompanying their sweetness (57). As mentioned earlier, the oleanane triterpene diglucuronate, glycyrrhizin (37) and its ammonium salt, are widely used for sweetening and flavoring purposes (20,21). While two previous semi-synthetic studies that modified the saccharide substitution of glycyrrhizin to improve its sweetness potency were inconclusive (99,100), a more highly sweet analog has recently been isolated from Glycyrrhiza #~ata roots (59). Thus, apioglycyrrhizin (38) was rated as exhibiting about twice the sweetness potency of the parent substance, and a further isolate from this plant source, araboglycyrrhizin (39), exhibited comparable sweetness intensity to glycyrhizin itself (59). The periandrins are a further group of oleanane triterpenes, and were first isolated from Periandra dulcis, and they are present in P. mediterranea (20). Periandrins I-IV (40-43) were characterized in the early 1980's by Hashimoto and colleagues at Kobe Women's College of Pharmacy in Japan, and all have about the same sweetness potency of glycyrrhizin (Table I). Recently, periandrin V (44) was obtained as a further sweet constituent of P. dulcis roots, and it was found that the terminal D-glucuronic acid sugar residue of periandrin I was replaced by D-xylose. However, the sweetness intensity of compound 44 relative to sucrose has not yet been determined (60).

% COOH

RO %

37 13-glcALI3-glcA

38 13-glcA213-api

39 13-glcALot-ara 27

HOOC, %

R1 R2

40 13-glcALl3-glcA CHO

42 I]-glcALl]-glcA CH20H

44 13-glcA2 13-xyl CHO

HOOC, "s

RiO

R1 R2

41 ]3-glcA213-glc CHO

43 ~3-glcALl3-glcA CH2OH

3.1.5 Steroidal Saponins The fern genus, Polypodium, has so far yielded three sweet steroidal saponins, namely, osladin (45) and polypodosides A and B (46, 47). The first-named of these compounds was structurally determined without full stereochemistry as an isolate of P. 28 vulgare by Herout and co-workers at the Czechoslovak Academy of Sciences in Prague in 1971, with the configuration of the aglycone later determined by partial synthesis from solasodine (20,21). However, Nishizawa and co-workers have recently established the correct structure of osladin as 45, after isolation from the plant and single-crystal X-ray diffraction, thereby reversing the stereochemistry from that originally proposed at positions C-22, C-25, and C-26. In addition, the configuration of the C-26-affixed rhamnose unit was assigned for the first time for osladin (61). The same group has also established that the actual sweetness potency of osladin relative to sucrose is 500 times (61), and not the higher figure of 3,000 as widely quoted in the literature (20,21). The compound has been produced from a steroidal aldehyde by total synthesis, using a triflic acid-catalyzed 2'-discriminated and [3-selective glucosylation in addition to an a-selective thermal rhamnosylation (101). At this institution, we have examined the rhizomes of the North American fern, Polypodium glycyl~hiza, and isolated three novel steroidal saponins, which have been called polypodosides A-C (62-64). The major sweet-tasting constituent of P. glycyrrhiza rhizomes is polypodoside A, which is based on a known aglycone, polypodogenin, a compound previously assigned by Czechoslovak workers as the A7-8-derivative of the aglycone of osladin (62). However, in view of the recent structural revision for osladin, it has been necessary to revise the stereochemistry of polypodoside A at the three asymmetric centers in the pyran ring in the aglycone (46). This was done on the basis of comparing the ]3C-NMR spectrum of polypodoside A with that of authentic osladin (63), and similar reasoning has been used to revise the structure of a second sweet constituent ofP. glycyrrhiza, polypodoside B (47).

O R2 y- H RIO 0

R] R2 Other

45 13-glc~ot-rha c~-rha 7,8-dihydro

46 ]3-glc~c~-rha ot-rha

47 [3-glc ot-rha -- 29

Polypodoside A was rated as exhibiting 600 times the sweetness intensity of a 6% w/w aqueous sucrose solution, but it revealed a licorice-like offiaste and a lingering attertaste (62). Although the quantitative sensory evaluation of polypodoside B was not carried out, it was somewhat less intensely sweet than polypodoside A. It was found that polypodoside C, a compound which only differs structurally from polypodoside B in having an L-acofriopyranosyl (3-O-methylrhamnosyl) unit affixed to C-26 in place of an L-rhamnosyl residue, was devoid of sweetness. Alter investigating several other compounds in this series, it was concluded that steroidal saponins of this type must be bidesmosidic with saccharide substitution at both C-3 and C-26, in order to exhibit a sweet taste (64). 3.2 Phenylpropanoids In an earlier study in this laboratory, trans-cinnamaldehyde (49) was found to be responsible for the sweet taste exhibited by the leaves of Cinnamomum osmophloeum, and was rated as being 50 times sweeter than sucrose by a taste panel (21). As a result of the investigation of six plants either collected in the field in Costa Rica or cultivated at the Pharmacognosy Field Station, University of Illinois at Chicago, their sweet taste was attributed to high levels of trans- anethole (48), as listed in Table I. Of these species, Myrrhis odorata is documented as being a sweet-tasting plant (102), but its sweet constituent had hitherto been unknown. When purified, trans-anethole was judged by a taste panel to exhibit a sweetness intensity of nearly 13 times that of sucrose (10,000 ppm) (65). Both cinnamaldehyde and anethole are used at low concentrations as flavoring agents in foods in the United States and elsewhere, but they both possess undesirable hedonic attributes which do not merit their further development as sweeteners (21,65). However, the realization that trans-anethole is potently sweet, and can occur commonly in plants is important from the point-of-view of selecting candidate sweet plants for study in the field. Consequently, it is necessary to use analytical methods for the dereplication of phenylpropanoids as well as sugars and polyols when working on the isolation of potently sweet natural products, so that time and resources are not wasted on re-isolating these sweet compounds (65,103).

R2~R1

R1 R2

48 CH 3 OCH 3

49 CHO H 30

3.3 Dihydroisocoumarin Phyllodulcin (50) is released when the newly harvested leaves of Hydrangea macrophylla var. thunbergii are crushed or fermented, and this dihydroisocoumarin is the sweet principle of a ceremonial tea called "amacha" that is used in Japan (20,21). While highly sweet (400 x the sweetness of 3% sucrose), the compound is limited as a sweetener by its almost total insolubility in water, and unpleasant hedonic attributes, such as a lingering aitertaste (21). Although no further sweet-tasting natural product analogs appear to have been isolated, there is a vast literature on attempts to improve the sweetness characteristics of phyllodulcin, by producing synthetic derivatives. This has been summarized recently by van der Wel and colleagues, who also describe the various hypotheses on the structural elements of the phyllodulcin and other dihydroisocoumarins that are necessary to exhibit a sweet taste (16).

OCH3 ~ t I II OH OH 0

3.4 Flavonoids Flavonoids are usually regarded as bitter- or neutral-tasting plant constituents. However, there are flavonoids in two structural classes for which sweet representatives are known, namely, the dihydrochalcones (51-55) and the dihydroflavonols (56-62), and these will be discussed in turn. 3.4.1 Dihydrochalcones The semi-synthetic dihydrochalcone glycosides, naringin dihydrochalcone (51) and neohesperidin dihydrochalcone (52), are produced from widely available by-products of the citrus industry, and compound 52, the sweeter of the two, has agreeable hedonic properties, with a lack of bitterness, although it has a slow onset of sweetness (20,21). There have been many attempts to produce dihydrochalcone analogs with taste qualities more like those of sucrose, and it is clear from these studies that highly sweet compounds in this series require a 3-hydroxy-4-alkoxy substitution in ring B (16). The effects of varying the substituents on ring A and in the pyran ring of the dihydrochalcones have been summarized by van der Wel and colleagues (16). The only dihydrochalcone currently in use as a sweetener is neohesperidin dihydrochalcone, which has particular use in chewing gum, candies, and oral hygiene products because of its long-lasting sweetness (31). 31

93 R10 OR 2

OR 40

R1 R2 R3 114 R5

51 [3-glc~c~-rha CH3 H H H

52 [3-glc2---o~-rha CH3 OH H H

53 H H H (x-rha H

54 H H H H [3-glc

55 ~3-glc H H H H

One of the first plant constituents to be recognized as being sweet was the dihydrochalcone glycoside, glycyphyllin (53), which was isolated in 1886 from the Australian species, Glycyphylla smilax (20,21). Related sweet-tasting compounds are phlorizin (54) and trilobatin (5fi), although it is not apparent how potently sweet any of these three naturally occurring dihydochalcones is relative to sucrose (20,21). 3.4.2 Dihydroflavonols Sweet-tasting representatives of the dihydroflavonol class of compounds were first isolated in 1988, in independent studies by Tanaka and co-workers from Engelhardtia chrysolepis (67) and in our laboratory from Tessaria dodoneifolia (21,104). However, Delaveau and colleagues had earlier pointed to the sweetness and astringency of the dihydroflavonol constituents in the bark of Glycoxylon huberi Ducke (21,105). In a phytochemical study on T. dodoneifofia, the previously known (+)-dihydroquercetin 3-O-acetate (57) was isolated as a sweet constituent of the young shoots of this plant. This compound was rated as having 80 times the sweetness potency of sucrose. The introduction of a 4'-methyl ether group in ring B, as in synthetic (racemic) dihydroquercetin 3-acetate 4'-(methyl ether) (56), greatly increased the sweetness potency to 400 times that of sucrose. Compound 56 was synthesized from 2,4-bis(benzyloxy)-6-(methoxymethoxy)acetophenone and 3-(benzyloxy)-4-methoxybenzaldehyde, according to a known method for producing dihydroflavonols, and remains the sweetest member of the class so far found. It contains ring B 3-hydroxy-4-alkoxy substitution, like the more highly sweet dihydrochalcones, a functionality which also confers greater stability to the molecule relative 32 to 57 (105).

2' 3' R3

R2~~,i~~ "OR1 OH O

R1 R2 R3 R4 Other

56 Ac H OH CH3

57 Ac H OH H 2R, 3R

58 Ac CH30 OH H 2R, 3R

59 Ac CH30 H H 2R, 3R

60 H CH30 OH H 2R, 3R

61 cz-rha H OH H 2S, 3S

62 ct-rha~- 13-glc H OH H 2R, 3R

However, removal of the acetoxyl group of 56, as in the synthetic (racemic) compound, dihydroquercetin 4'-(methyl ether), reduced the sweetness potency to a tenth of its former level. Replacement of both the 3- and 4'-substituents by hydroxyl groups leads to the tasteless compound, (+)-dihydroquercetin (105). In work performed in collaboration with Mabry and co- workers at the University of Texas, a series of sweet dihydroflavonols was reported from the above-ground parts of Hymenoxys turneri (57-60) (66). One of the isolates, (2R,3R)-2,3-dihydro- 5,7,3',4'-tetrahydroxy-6-methoxy-3-O-acetylflavonol (58), with a 6-methoxy substituent, was less than half as sweet as compound 57. As a result of evaluating the sweetness of additional isolates from this plant (Table I), it was concluded that in the dihydroflavonol series of sweeteners a ring-B catechol unit is not mandatory for the exhibition of sweetness, since the 3-O-acetate unit also appears to have a role in mediating the sweet effect of these compounds (66,105). Also, Kasai and colleagues have shown that naturally occurring taxifolin glycosides (61,62) with both 2S,3S- and 2R,3R- stereochemistry may exhibit a sweet taste (67,68). 33

3.5 Proanthocyanidins The proanthocyanidins (formerly known as "condensed tannins") have emerged as a rather unlikely group of sweet-tasting compounds in recent years, since this group of polyphenols and the polyesters based on gallic and/or hexahydroxydiphenic acid ("hydrolyzable tannins") are much better known for the harsh, astringent taste they produce in the mouth (a feeling of constriction, roughness and dryness) (106). However, two of twelve proanthocyanidins obtained from the roots of Cinnamomum sieboldii were reported by Nishioka and colleagues at Kyushu University in Japan to be sweet-tasting in 1985 (69). Subsequently, in a review article, Tanaka accorded these sweet compounds the trivial names cinnamtannin B-1 (63) and cinnamtannin D-1 (64), respectively (107). More recently, N. Tanaka and co-workers at the Science University of Tokyo demonstrated that a pair of proanthocyanidins in the acid (65) and the corresponding lactone (66) form were sweet-tasting These compounds were isolated from two fern species in the same genus, namely, Arachniodes sporadosora and A. exilis (70). None of the four sweet-tasting proanthocyanidins 63- 66 appears to have been evaluated for its sweetness intensity relative to sucrose. In very recent work carried out at the University of Illinois at Chicago, selligueain A (67), a further sweet-tasting proanthocyanidin, has been isolated from the rhizomes of a fern, SelligT~ea feel, collected in Indonesia. Like compounds 63-66, selligueain A possesses a doubly-linked ring A, and is trimeric. However, selligueain A differs from 63-66 in having an epiafzelechin C-unit rather than an epicatechin unit in this part of the molecule. Compound 67 has been assessed for sweetness potency, and was found to exhibit about 35 times the sweetness intensity of a 2% w/v sucrose solution. At a concentration of 0.5% w/v in water, selligueain A was judged as being pleasant-tasting rather than astringent. It may be anticipated, however, that sweet-tasting proanthocyanidins are rare, because of the stringent structural requirements necessary to elicit sweetness (71 ).

HO O ( IH OH

R1 R2

OH 63 OH ~-OH

64 OH t~-OH

67 H ~-OH Ik 35 *OH'...... J"S HO" "~ HOA.I.x.,,OH FII 34

...... //" , OH

3.6 Benz[b]indeno[1,2-d]pyran derivative Tanaka and co-workers at Hiroshima University have followed up on the observation that an extract of the heartwood of Haematoxylon campechiamim is known to taste sweet, and the sweet principle turned out to be hematoxylin (68), which is a well-known microscopical staining reagent (72). The sweetness potency of this compound relative to sucrose was not determined. By 35 reference to several tasteless compounds also obtained in the same investigation, it was concluded that the structural requirements necessary for the attribution of sweetness of 68 are the C-4 hydroxyl group, and the cis-stereochemistry (13-OH at C-6a and 13-H at C-12) linking the pyran and cyclopentene rings (72).

OH HO~) H Hf

/ HO OH

3.7 Amino acid Monatin [4-hydroxy-4-(indol-3-ylmethyl)glutamic acid; 69] has recently been obtained as a very sweet constituent of the root bark of the South African plant, Schlerochiton ilicifolius, by Vleggaar and co-workers at the University of Pretoria in South Africa (73). This compound was purified by ion-exchange chromatography and gel filtration. The compound was obtained in this manner as a mixture of salts, with the sodium salt predominating, and the free amino acid was produced by treatment of the salt mixture with glacial acetic acid (73). The trivial name of this compound is based on a local Sepedi name, "monate", meaning "nice". Monatin is of comparable sweetness to the synthetic amino acid, 6-chloro-D-tryptophan (1,300 times sweeter than sucrose) (16). It is of interest to note that the plant of origin of monatin was described taxonomically for the first time as recently as 1965. Furthermore, in a lengthy description of the plant, there was no mention of any sweet characteristics, which is rather surprising in view of the sweetness potency of monatin (108).

HO2/~~ CO2H ~H,~..~ ~N OH N 2 I H 69 36

3.8 Protein and Peptide Sweeteners and Taste Modifiers Sweet-tasting proteins have attracted wide attention, and thaumatin, monellin, mabinlin, and pentadin have been subjected to review previously (20,21,109,110). The five presently known highly sweet proteins and their plants of origin are shown in Table I. Of these, only thaumatin is used commercially as a sweetening agent and flavor enhancing agent (21,33,111). The complete amino acid sequence of monellin was reported recently (112), and the solid-phase synthesis of this sweet protein has been undertaken (113). Attempts are continuing to delineate the sweet receptor binding sites of thaumatin and monellin, although it has been found that the two compounds have no apparent similarities in either their amino acid sequences or their backbone three-dimensional structures (114). The structure of curculin, obtained from the fruits of Curculigo latifolia collected in Malaysia, was recently reported by Kurihara and co-workers at Yokohama National University in Japan. The compound possesses 114 amino acid residues, and, in addition to eliciting a sweet taste itself, it induces the sour-tasting substance citric acid to taste sweet (74). Another taste-modifying protein is miraculin, from Richardella dulcifica (Schumach. & Thonning) DC. (Sapotaceae), a West African plant (20,21). In contrast, gurmarin, a sweet-taste-suppressing peptide of 35 amino acids has been isolated from the leaves of Gymnema sylvestre R. Br. (Asclepiadaceae), although this has a very weak effect in humans while strongly suppressing the sweet taste responses in the rat (115). 3.9 Non-Protein Sweetness-Modifying Natural Products A number of non-proteinaceous substances of plant origin are known that induce or inhibit the sensation of sweetness. Sweetness inducers and enhancers from plants include cynarin, chlorogenic acid, caffeic acid, and arabinogalactin (larch gum) (33). A synthetic compound, 2-(4- methoxyphenoxy)propanoic acid, which is also a constituent of roasted coffee beans, is currently on the market as a sweetness inhibitor (33). Several oleanane-type triterpene esters with sweetness-inhibitory activity occur in Gymnema ~ylvestre leaves (33,116,117), with dammarane- type saponins with similar effects having been reported recently from the leaves ofHovenia dulcis Thunb. (Rhamnaceae) (118) and Ziziphusjujuba Mill. (Rhamnaceae) (33,119,120).

4. CONCLUSIONS It may be seen from this chapter that ongoing research activities on the isolation and characterization of naturally occurring sweet principles have continued to afford many novel molecules in several structural classes. These compounds occur in species representing a taxonomically wide range of plant families. The fact that many of these compounds are highly sweet-tasting might well be of curiosity value only, were it not for the fact that several of the known naturally occurring intense sweeteners have important commercial uses, particularly in Japan. Methodology has been developed in terms of candidate plant selection, dereplication of sugars, polyols and sweet phenylpropanoids, and in other phytochemical procedures, so that significant progress can be made in the elucidation of further highly sweet-tasting molecules with only a modest investment of capital (121). In addition to being used in an unmodified form as 37 sweeteners, plant-derived sweet-tasting molecules can serve as useful lead molecules for synthetic optimization. Therefore, a knowledge of structure-sweetness relationships of plant sweeteners, and their naturally occurring congeners and semi-synthetic analogs is of use in assisting with the rational design of new sweeteners based on natural product leads.

ACKNOWLEDGMENTS Certain of the work carried out at the University of Illinois at Chicago described in this chapter was supported by grant R01-DE08937, funded by the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland. We are very grateful to many capable postdoctorals and graduate students, as well as faculty colleagues, who have participated in our sweetener research, and whose names are indicated in the bibliography section. Prof. D.D. Soejarto of this institution is thanked for providing valuable taxonomic information for some of the plants mentioned in this chapter. We thank Prof. M. Nishizawa, of Tokushima Bunri University, Tokushima, Japan, for kindly providing the 13C-NMR spectrum of osladin. We wish to acknowledge Drs. I.-S. Lee and M.-S. Chung, and Mr. R. Suttisri for helpful suggestions.

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