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Researchers Unlocking Potential Of Diverse, Widely Distributed Saponins • Growing knowledge about biological effects of these complex natural products sets stagefor research into more potent analogs \ . Maureen Rouhi, C&EN Washington A. Maureen Rouhi, C&EN Washington
210th ACS National Meeting 210th ACS National Meeting
cientists all over the world are scrutinizing saponins because of the host of biological effects they exert. The diversity of structures, the challenges of isolation, the pharmacological and biological activities still to be discovered, and the promise of commercialization—these all are driving the study of this group of natural products. Saponins are complex molecules made up of sugars linked to a triterpene or a steroid. They are widely distributed in plants, including some foods (for example, beans, spinach, tomatoes, and potatoes) and animal feed (alfalfa and clover). And some toxins produced by marine organisms are, in fact, saponins. Last month, researchers from more than 20 countries presented their work at a symposium organized by the Division of Agricultural & Food Chemistry. The symposium was like an oasis for saponin scientists who were thirsting for a forum to swap information. ''Usually in meetings one hears maybe one or two papers about saponins/7 according to one scientist from Germany. By contrast, this ambitious five-day gathering was devoted almost entirely to saponins. "Saponins have not been the subject of an ACS symposium before/' said symposium coorganizer George
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SEPTEMBER 11,1995 C&EN
R. Waller, professor of biochemistry at Oklahoma State University, Stillwater. "Researchers in this field have not had a chance to consult with each other [at this level] and to develop plans for future work." Waller proudly noted the strong international participation. From Japan alone, symposium coorganizer Kazuo Yamasaki, professor of pharmaceutical sciences at Hiroshima University School of Medicine, brought a contingent of scientists for 14 presentations. That's really "quite a congregation of Japanese people coming for one symposium," said Waller. The symposium was well attended to the last day, with a mU-morning poster session buzzing up to the final bell. The attendees clearly were excited to exchange information, and for good reason. There was much to learn about new plant sources, new compounds, new biological activities, and better techniques for isolation and structural elucidation.
Panax notoginseng produces potent anti-tumor-promoting saponins.
And there were calls for collaboration to establish a database of mass spectral information, for example, or to gain access to pure compounds. The symposium underscored the many years scientists have devoted to an "inventory and data-gathering phase" in saponin research, according to Manuel F. Balandrin, a research scientist at NPS Pharmaceuticals, Salt Lake City. "I think the area now is poised to fuse what it has learned with classical principles of medicinal chemistry to move forward to designer compounds." Koji Nakanishi, chemistry professor at Columbia University, also foresees a new direction. "Saponins have been rather a neglected area among organic chemists," he said. "Up to now, these compounds have been mostly targets for structure determination. The varied modes of action are just starting to be clarified on the molecular structural basis. This is going to be an exciting future." Techniques for structure analysis figured prominently in the sympo| sium. Structure determination is a 1 major challenge because of the diver£ sity and complexity of saponins. A 1 saponin molecule has two parts. The !z aglycone (or sapogenin) is the non^ saccharide portion, and it can be a 1 triterpene, a steroid, or even a steroi^ dal alkaloid. Attached to the aglycone | are one or more sugar chains. These can be linear or branched and can have up to 11 monosaccharide units. Structure analysis becomes even more complicated when only minuscule amounts of pure compounds are available for study. Nakanishi described a versatile but still underused technique for microgram analysis of absolute configuration, called excitoncoupled circular dichroism (CD). The technique is based on interactions of two or more chromophores of the molecule under study in solution. When the chromophores are close enough to interact, the energy level of the excited state of the mol-
ecule splits, giving rise to CD curves split into a positive and a negative component. The sign of the first half of the curve corresponds with the absolute sense of the twist between the interacting groups. Nakanishi, whose research is supported by the National Institutes of Health (NIH), started applying this technique to various natural products in 1969 with then-graduate-student Nobuyuki Harada, who is now a professor at Tohoku University, Sendai, Japan. At Columbia, Nakanishi and long-time collaborator and senior research scientist Nina Berova have continued to expand the exciton-coupled CD technique to challengIndonesian farmers tend a field of Abrus precatorius, which produces sweet saponins. ing cases, including molluscicidal and insect antifeedant saponins, sugars, sphingosines, and acyclic polyols. The active constituents of Quil A as potential a fatty acid residue attached to fucose, the first residue of the sugar group technique requires no reference material adjuvants for human vaccines. for assignments of absolute configuraSeveral scientists examining Quillaja linked as an ester at C-28. In the Netherlands, a group headed tion, Nakanishi said. saponins discussed their recent results The trick is to convert interacting at the symposium. The researchers are by research scientist Gerrit van de groups to highly ultraviolet-absorbing gleaning different information from Werken at the National Institute of Pubchromophores, Berova said. The re- their studies, suggesting that they are lic Health & Environmental Protection, searchers use anthroylation, naphthoyla- looking at very similar but not identical Bilthoven, has been characterizing Quil tion, and cinnamoylation, among other compounds. Although some structures A saponins as part of efforts to isolate an techniques, to boost the absorbance of are closely related to those of com- active substitute for the Quil A crude chromophores. By doing so, even a pounds isolated in the late 1980s by mixture. His group has separated up to small amount of material will have an Ryuichi Higuchi and coworkers at Kyu- 23 saponin fractions, varying primarily intense absorption in the CD spectrum. shu University, Fukuoka, Japan, others in the composition of one of the sugar groups. Using an unorthodox techRecently, Nakanishi, Berova, and post- show substantial differences. doctoral fellow Stefan Matile described The aglycone in Quil A saponins is nique—called monomer mapping—they porphyrins as even more powerful chro- quillaic acid. It is linked to sugar have established that the saponin fracmophores for structural studies by exci- groups at C-3 and C-28 and has an al- tions have at least one nonsaccharide ton-coupled CD [/. Am. Chem. Soc, 117, dehyde at C-4. Some components have monomer in the sugar group at C-28, usually 3,5-dihydroxy-6-methyloctanoic 7021 (1995)]. With porphyrins, stereoacid. Recently, the Dutch scientists decenters as far apart as 50 A can still comtected a new nonsaccharide, correspondmunicate and produce a split CD specing to C 8 H 12 0 5 . They are working on trum, according to Nakanishi. And in elucidating its structure. collaboration with scientists at RhoneAt the symposium, Dirk C. van SetPoulenc Rorer, Collegeville, Pa., Nakaniten, a graduate student of van de Wershi, Berova, and graduate student Ning ken's, explained their monomer mapZhao have extended the technique to ping technique. A computer program tertiary amines like quinuclidines by use generates possible compositions for the of quaternary ammonium salts as chrosugar portion of the saponin based on mophores [/. Am. Chem. Soc, 117, 7844 the experimentally determined masses (1995)]. of the sugar moieties and the assumpStructural determination indeed can tion that the sugar chains are made up be confounding, as shown by structural exclusively from monomers known to studies of Quillaja saponaria saponins. As be present. With this approach, mixtures early as 1951, scientists knew that sacan be analyzed without prior separaponins from the bark of Q. saponaria, a tion of components. South American tree, make animal vacAt Boston University School of Medicines more effective. And an extract cine, biophysics professor Catherine E. called Quil A is used in veterinary vacCostello uses tandem mass spectrometry cines. Its use for human vaccines, howto probe biologically active compounds, ever, is not desirable because the materiincluding constituents of Quil A that al is poorly defined chemically and prohave been purified at the Wistar Instiduces some toxic effects. Thus, various An African villager prepares a saponin tute, Philadelphia. With advances in iongroups are isolating and characterizing mixture for control of schistosomiasis. SEPTEMBER 11, 1995 C&EN
29
SCIENCE/TECHNOLOGY
Saponin symposium participants included (clockwise from top left) Lemmich, Hostettmann, Nakanishi, Kinghorn, Balandrin, Sashida, Kensil, and Costello.
ization methods and analyzer techniques, it is now possible to get information from subpicomole amounts of material and to characterize components in mixtures, she told the symposium. In work with Quil A saponins begun while she was at Massachusetts Institute of Technology and funded by NIH, Costello's group has used a variety of ionization techniques: liquid secondary ionization, matrix-assisted laser desorption/ionization, and electrospray ionization. They establish structural details by using both collision-induced dissociation on four-sector and triple-quadrupole tandem mass spectrometers and postsource decay on a reflectron time-offlight instrument. For the Quillaja work, simpler saponins characterized in earlier studies served as models. The Boston scientists have confirmed the general features of the structure proposed by Higuchi and coworkers. In addition, they have shown that, even for highly purified fractions of Quil A saponins, structural heterogeneity is encountered at several points. For example, the C-3 glycosyl may include either xylose or rhamnose. Sometimes xylose is absent from the C-28 oligosaccharide. And the arabinose attached to the fatty acyl moiety may be substituted with a deoxyhexose residue. In one minor component, the glucuronic acid linked to C-3 is esterified by a pentosyl group. 30
SEPTEMBER 11, 1995 C&EN
Meanwhile, at Cambridge Biotech Corp., Worcester, Mass., Charlotte R. Kensil, senior director for adjuvant and drug delivery research, has been carrying out structure/activity studies of a Quil A fraction called QS-21. This fraction makes up about 5 to 10% of the saponins in Quil A, Kensil said. "What we have is a purified adjuvant-active saponin that has higher activity and lower toxicity [than Quil A] that is now being tested in clinical trials." Earlier, Kensil and coworkers prepared and tested the adjuvant properties of QS-21 derivatives modified at the carboxyl group of glucuronic acid or at the aldehyde of quillaic acid. They found that the aldehyde group is required for adjuvant activity, whereas the glucuronic acid moiety is not (Vaccine, in press). The Cambridge Biotech scientists next turned to the fatty acid domain of QS-21. Using mild alkaline hydrolysis conditions, they split QS-21 into three components: two fatty acid residues and the deacylated saponin. None of the hydrolysis products has adjuvant activity. "Our conclusion is that acylation is important. However, we have not defined the minimal critical acyl group required for activity," Kensil told C&EN. This finding is significant.
It means that vaccine formulations and storage conditions should be carefully selected to prevent deacylation and the consequent loss of adjuvant potency, she explained. Symposium participants heard numerous reports of other saponin activities, ranging from antitumor, antimicrobial, and allelopathic (plant-protecting) effects to shark-repellent properties. Two European groups described how some African countries are capitalizing on the molluscicidal properties of some saponins as one way to control the parasitic disease schistosomiasis: eliminate the snails that act as intermediate hosts. "Schistosomiasis is endemic in 76 countries and affects over 200 million people, mainly in tropical and subtropical regions," said Kurt Hostettmann, director of the Institute of Pharmacognosy & Phytochemistry at the University of Lausanne, Switzerland. "It has been known for a long time that saponin-containing plants are toxic to snails," Hostettmann said. His research group has been characterizing saponins from two African trees— Swartzia madagascariensis and Tetrapleura tetraptera. "Fruits of these trees were known locally in Africa to be toxic to snails," he said, and they are very rich in saponins, up to 10% by weight. Aqueous extracts of the fruits are effective even at concentrations as low as 2.5 mg per L. And field trials in Tanza-
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Making beer in the jungle is easy Beer lovers stranded in the Amazon jungle of Brazil need not worry about the supply of their favorite drink, thanks to saponins. With a knife, water, and the bark of Ampelozizyphus amazonica, anyone can make beer there. Used medicinally as a treatment for the common cold, the bark also provides an instant drink very much like commercial beer, said Isao Kubo, professor of natural products chemistry at the department of environmental science, policy, and management of the University of California, Berkeley. Kubo has been combing the rain forests for bitter compounds. These
nia and Nigeria have proven that aqueous mixtures of molluscicidal saponins can effectively eliminate the host snails that infest water holes that often are the sole sources of drinking, cooking, washing, and bathing water in some villages in Africa. Involving the local people is crucial in efforts to halt the spread of schistosomiasis. With molluscicidal saponins, villagers using local technology can prepare and apply the snail-killing mixtures. Swartzia is a good candidate source of molluscicides, Hostettmann said. Because it grows wild, the local people have a plentiful supply. "Local people won't plant something they can't eat," he explained. "Especially during the dry season, it is a competition between giving water to this type of plant to kill the snail or to plants that give food." Else Lemmich, associate professor in the department of medicinal chemistry at the Royal Danish School of Pharmacy, Copenhagen, also has been involved in schistosomiasis control in Africa, particularly in Zimbabwe as part 32
SEPTEMBER 11,1995 C&EN
usually protect a plant from being eaten by herbivores. They can thus be used as alternative insect control agents. Another application is in food flavoring. "Only a few bitter substances—such as caffeine and quinine—are useful as food additives," said Kubo. "And their use, particularly of quinine, is beginning to be restricted because of toxicity concerns. The food industry is interested in other good, bitter-tasting substances." Making beer in the jungle is easy, says Kubo. Freshly collected bark, which is extremely bitter, is washed with water. The outer layer is shaved off with a knife, and the shavings are soaked in water. The resulting extract is brown and bitter. (The color and bitterness depend on the amount of shavings and the length of soaking.) Before the beer is poured, the liquid is shaken well to form a layer of foam. "It tastes like commercial beer but without the alcohol. You can drink as much as you like without getting intoxicated," said Kubo. Using fractionation guided by the bitter taste, Kubo has established that the bitter foam-forming constituent is a novel saponin, which has been named ampelozin.
The assay is based on hemolysis of red blood cells. A sample of water treated with saponin extract is mixed with the cell suspension, and the saponins cause the blood cells to burst. The extent of hemolysis, which is correlated to saponin concentration, is determined by measuring turbidity with a colorimeter. "If you find the concentration is too low, you can add a little more immediately, rather than wait for a day," Lemmich explained. Local control of schistosomiasis snails requires no more than a crude extract. But isolating and characterizing the active saponins is important "to ensure that the extract used is the same—contains the same chemical compounds—as has been tested toxicologically," she said. "In our studies, we have seen that different strains or cultivars of the plant may contain different saponins. For example, the saponins in the Phytolacca strain from Ethiopia and those in one from Zimbabwe are rather different." Both Hostettmann and Lemmich emphasized the need for thorough toxicity and environmental impact studies. "It's not yet been done; it's very expensive," said Lemmich. The delegation of Japanese scientists gave the symposium an overview of the diversity of Japanese research in the saponin field—ranging from phytochemical screening for myriad biological effects to elucidation of actions at chemical
of a Danish-Zimbabwean collaboration to develop molluscicides. This program has identified an Ethiopian strain of the Phytolacca dodecandra tree as one of the most promising sources of snail-killing saponins. "It can be grown very fast. You can cultivate from cuttings and harvest berries after nine months. When fully grown but not yet ripe, the berries of this tree contain up to 22% saponins," said Lemmich. "That's a very high percentage. We have estimated a maximum yield of 1.4 kg of saponins per tree or 7 tons per hectare under the best conditions." Another advantage to this strain, Lemmich said, is that toxicity studies already have been done on it. "It's best to continue with this plant because . . . we feel safe." Lemmich's group is developing a field assay that will estimate saponin concentrations within 30 minutes. Compared with 24 hours needed for testing with live snails or six hours with another field assay described earlier by Hostettmann and coworkers, the assay is very fast.
CD technique establishes absolute configuration CD spectrum
f\ Wavelength, nm
Exciton-coupled circular dichroism (CD) is based on interacting chromophores. When the two chromophores are oriented with positive chirality (clockwise movement from front to back) the first half (I) of the split CD curve is positive. With negative chirality, the first half of the split CD curve is negative.
Saponin derivative is about 150 times sweeter than sugar levels. Among the delegates was Osamu Tokyo University of Tanaka, professor at the department of Pharmacy & Life food and nutrition at Suzugamine Science, have been Women's College, Hiroshima City. surveying plants Tanaka is interested in saponins that that are not part of can protect processed foods from deteri- traditional medioration by yeasts. Recently, he and co- cine. Looking only workers at Maruzen Pharmaceuticals, at plants used in Onomichi City, have been studying sa- folk medicine leaves ponins of Yucca schidigera, a plant native out many potential to North America and known for its me- compounds, they dicinal use by Native Americans. Tana- said. ka says the plant is recognized by the One of their most Food & Drug Administration (FDA) as promising discoverCH3OOC o Abrusoside E safe for human use, and an extract from ies comes from Or6"-methyl ester its rhizomes is used as a foaming agent nithogalum saunderfor carbonated beverages and as a flavor siae, a member of HO enhancer. the lily family with OH Six saponins with antifungal activity no medicinal folkhave now been isolated from Y. schidi- loric background. Note: In the natural product abrusoside E, the group in color is a carboxyl group. gera by Tanaka's group. The compounds From the bulb of are active against Saccharomyces cerev- this plant, they have isiae, or brewers' yeast; Candida albicans, isolated a saponin tentatively named more potent than anticancer agents now a dermatophyte yeast; and Hansenula OSW-1. The compound has little toxicity in clinical use, such as mitomycin C, adanomala, a food-deteriorating yeast. Be- to normal human cells but is remarkably riamycin, cisplatin, camptothecin, and cause of the desirable properties of the toxic to malignant tumor cells. In vitro taxol. yucca extract, the yucca saponins now assays show it to be up to 100 times Meanwhile, at Tohoku University, Kaare under development at Maruzen as preservatives for processed foods, Tanaka told C&EN. II Cancer prevention by saponins is the • focus of work by Takao Konoshima, an 11 • assistant professor at Kyoto Pharmaceu1 1 tical University. At the Laboratory of • Pharmaceutical Sciences of Natural Re11 • sources, Konoshima and coworkers 1 1 have been testing saponins for anti• tumor-promoting activity, including 11 • those from Panax notoginseng, a ginseng 11 species. Konoshima says this particular • ginseng plant is beginning to be cultivat11 • ed in China as a source of hemostatic 1 1 and antihepatitis drugs. Their recent re• sults indicate the plant also is a potential 11 • source of antitumor promoters. 11 The root of P. notoginseng contains sa• ponins and acetylene compounds, which 1 When it comes to Claisen condensations, DBE-4 (dimethyl succinate) is a •1 have been characterized before. One of natural Which is one reason why it's finding wide usage as a starting mate 11 the saponins, ginsenoside-Rgl, is present • in this plant at levels that are about 10 rial for quinacridone pigments. It's also an important intermediate for 11 • times higher than those in other Panax insecticides, pharmaceuticals and specialty chemicals. In fact, the list of 11 species. Using an assay based on activapatents just keeps on growing. Could yours be next? There's only one way • tion of the Epstein-Barr virus by tumor 11 O • promoters and a two-stage carcinogenesis to find out. Call 1-800-231-0998 for a sample. O II II 11 test on mouse skin tumor, Konoshima • CH30-C-
