Case Study of the Swiss Flora for Prior Phytochemical and Biological

Jan 29, 2013 - Estimates in the literature as to what extent the world's higher plant species have been studied chemically or for bioactivity are cont...
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Case Study of the Swiss Flora for Prior Phytochemical and Biological Investigations Michael Adams, Magalie Chammartin, Matthias Hamburger, and Olivier Potterat* Department of Pharmaceutical Sciences, Division of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland S Supporting Information *

ABSTRACT: Estimates in the literature as to what extent the world’s higher plant species have been studied chemically or for bioactivity are contradictory and range from 0.5% to >12%. In this survey, a model to make credible estimates of the extent of their study is proposed and is exemplified by applying it in a case study of plants native to Switzerland. Using a widely available database (SciFinder Scholar), 454 535 literature references for the 2677 native Swiss plant species were retrieved. It was determined that 55% of these species have been investigated phytochemically and 28% for biological activity. The influence of factors such as commonness, growth form, habitat, medicinal use, and reported toxicity on the extent to which different plant groups have been studied is analyzed. The predictive value of random sampling of subsets of plants is compared to the study of the entire Swiss flora, to show that a credible estimate of the extent of prior studies can be achieved with just 5% of these species.

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the WWF stated that 5% of tropical plant species have been examined for their medicinal values.12 Verpoorte et al. in 1998,13 relying on data taken from the scientific database NAPRALERT,14 showed that 36 446 (12%) of higher plants had been studied phytochemically and 13 795 (4.6%) for biological activity. Cragg et al. pointed out that between 1960 and 1981 the National Cancer Institute (NCI) had screened 114 000 extracts from 35 000 plants, showing that this one institution alone had surveyed 12% of all plants for at least one activity.15 This list could be continued, but it is already clear that there seems to be no general consensus on this issue. There are no universal definitions of “thoroughly studied”, “chemical composition”, or “medicinal value”; so the controversial numbers in the literature may all have some veracity. Tobacco, for instance, is probably the most exhaustively studied plant chemically, as more than 2500 compounds from it have been identified.16 By this standard, only a few plant species would be “exhaustively” studied. Besides, any phytochemical investigation reveals only a narrow spectrum of the overall profile of the secondary metabolites a plant may produce,17 and even tobacco will continue to yield more compounds in the future. The Dictionary of Natural Products18probably the most comprehensive databank of natural productscontains 243 000 entries and is updated with about 10 000 new compounds every year. However, due to the way data are organized, this

he number of known plant species in the world, without all the synonyms used, has been estimated to be about 300 000.1,2 About 3% of all plants are thought to have been utilized as foods. Yet 90% of foods are derived from just 20 species, and more than 50% are from rice, maize, and wheat.3 Plant biodiversity is more significant for medicinal reasons. About 70% of the world’s population rely on traditional medicines,4,5 which are predominantly plant based.4 According to the International Union for Conservation of Nature (IUCN),6 there is well documented evidence for the medicinal use of 28 000 plant species, which would be 9.4% if the number of 298 000 species from www.theplantlist.org is accepted.2 However, the estimate of medicinal plant numbers by IUCN6 may be too low, as other authorities have shown that in many countries the percentages of medicinally used plants are several times higher.7 A quarter of all registered drugs in 2001 were shown to be of plant origin,8 with 80% of all plant-derived registered drugs used for indications comparable to the plants from which they were derived in traditional medicine.4 Despite their importance as foods and medicines and in drug discovery, it is unclear how many plants have actually ever been studied chemically and for their bioactivities, and such estimates are contradictory. Cox and Balick in 1994 stated that “less than half of 1% (of plants) have been studied exhaustively for their chemical composition and medicinal value”.9 A brochure from the Missouri Botanical Garden claims that “Less than 2 percent of all plants have been thoroughly tested for medicinal applications”.10 A University of Michigan Web site indicates that “Fewer than 1% of tropical plants have been screened for possible use to medicinal science”.11 Stolton and Dudley from © 2013 American Chemical Society and American Society of Pharmacognosy

Received: October 2, 2012 Published: January 29, 2013 209

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have been tested for antimicrobial properties, and 4% have been studied clinically. The level of investigation varies greatly between plants, and only a very few species have been studied extensively, as defined by more than 10 studies in all categories and subcategories. These plants are Cannabis sativa L., Digitalis purpurea L., Hippophaë rhamnoides L., Hypericum perforatum L., Petasites hybridus (L.) Gaertn., Rhodiola rosea L., Secale cereale L., Silybum marianum (L.) Gaertn., and Viscum album L. About 13%/7% of plants have been documented in more than 10 studies in the main categories phytochemistry/bioactivity, whereas 15%/7% were reported on in just one study each. The remainder of the studied plants have between two and 10 reports (Figure 1a and b).

databank has no complete listing of all biological sources, which makes it inappropriate to extract a list of studied plants. The database NAPRALERT,14 on which Verpoorte13 relied, claims to cover literature comprehensively from at least 1975 to 2003, but only about 15% of the literature from 2004 onward. This makes it insufficient for an up-to-date statistical analysis, because of the significant increase of phytochemical and bioactivity studies on plants in the past decade. The number of publications searchable on SciFinder Scholar (Chemical Abstracts Services, Columbus, OH, USA),19 which contains search terms indicating phytochemical studies, has doubled since 2004. For example, of the 49 547 references containing the search term “flavonoid”, 28 867 were published since 2003; for “essential oil” it is 22 001 out of 41 411 (data retrieved in September 2012). To establish a scientifically sound estimate of how many plants have been studied (and to what extent), a direct approach has been taken in the present investigation, in which a complete literature search was undertaken of every single plant species, to retrieve all the literature and to analyze this as to whether it refers to the phytochemistry or bioactivity, or is not relevant in this context. The literature search was performed using SciFinder Scholar,19 which is the largest scientific database currently available, with more than 33 million references. To do this on a global scale would have been hardly feasible. Instead, a case study for the flora of one countrySwitzerlandwas performed. Despite its tiny size (41 285 km2) Switzerland is botanically quite diverse. On just 0.4% of the European landmass, it harbors about 20% of the continent’s plant biodiversity.20 Endemism levels are low (just two species, Draba ladina Braun-Blanq. and Arenaria bernenis Favarger),20 and there are large overlaps in the species composition with the other countries in the region, a result of relative recent colonization after the last Ice Age. Switzerland is also a suitable country for this kind of survey, because botanical mapping data and data concerning plant distribution, commonness, toxicity, growth forms, and other factors were readily available and complete.20−23 The present survey of Swiss plants should serve also as a case study to demonstrate that analyzing a randomly selected subset from plant lists may be suitable to predict the outcome of a comprehensive study of the global flora.

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RESULTS AND DISCUSSION All 2677 higher plants native to Switzerland as described by the national institution responsible for the mapping of the Swiss flora24 were searched via SciFinder Scholar. A total of 454 535 literature references were retrieved, sighted, and categorized as to whether they dealt with the plant’s chemistry or its biological activities, or neither of these. Within these categories, the references were assigned to subcategories. In the following analysis it is considered mostly whether or not plant taxa have ever been studied at all and only briefly to what extent. This information can be retrieved from the Supporting Information (Table S1). Where pairs of numbers are given (such as 55%/ 28%), the first number refers to phytochemical studies and the second to bioactivity studies. In summary, 55% of Swiss plants have been studied phytochemically, 42% have had isolated compounds, 19% have been studied for their fatty acid composition, 15% have had their essential oils analyzed, and 9% have been studied for phytosterols. About 28% have been tested for bioactivity, 24% have been studied using in vitro pharmacological methods, 17% have been studied in vivo, 16%

Figure 1. Number of species listed according to how many studies (0, 1−5, 5−9, and ≥10) were found on their phytochemistry, and the subcategories of phytochemistry (a), and for bioactivity and the subcategories of bioactivity (b).

About 3.2% (87) of native Swiss plant species are ferns (Pteridophyta).20 Of these, 46 (53%) have been studied phytochemically and 19 (22%) for bioactivity. Among the 2589 seed plants (Spermatophytina) all 10 species of gymnosperms (Pinophyta) have been studied phytochemically and seven for bioactivity. Of the 2580 angiosperms 1401 (54%) have been 210

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numbers of species studied phytochemically. Figure 2b presents the same analysis for bioactivities. The 45 most species-rich plant families are shown with their residual values (positive or negative) in Table 1. The floristic guide Flora Helvetica20 lists 169 (6.3%) plant species as being toxic. Of these, 146 (86%) have been studied phytochemically and 61 (36%) for bioactivity. From the remaining 2508 species not listed as toxic, 1312 (52%) have been studied for their phytochemical constituents and 629 (25%) were studied for bioactivity. The Flora Helvetica20 also differentiates three levels of toxicity: toxic (109), more toxic (28), and highly toxic (32). Of these, 91 (83%), 28 (100%), and 27 (84%), respectively, were studied phytochemically and 63 (58%), 23 (82%), and 20 (63%) were studied for bioactivity. Just a small number (n = 45, 1.7%) of native plants are listed in the Swiss Pharmacopoeia (Pharmacopoeia Helvetica 1995)26 as medicinal plants. Ten (19%) medicinal plants are categorized as toxic, whereas 159 (6.4%) of nonmedicinal plants are listed as toxic. All medicinal plants have been studied phytochemically, and 41 (91%) were studied for bioactivity. Plants were classified for their commonness in five categories according to Aeschlimann and Burdet,23 which distinguishes the categories “very rare” (n = 149), “rare” (n = 602), “not common” (n = 784), “common” (n = 925), and “very common” (n = 84). The percentages of species studied (phytochemistry/ bioactivity) were 30%/16%, 43%/20%, 52%/22%, 63%/33%, and 91%/63%, respectively (Figure 3), and thus correlated positively with the degree of commonness. The Flora Helvetica20 offers a habitat description for all species in the following eight categories: fertilized meadow plants (n = 72), cultured plants (n = 41), weeds or ruderal plants (n = 552), forest plants (n = 473), dry meadow plants (n = 328), marsh plants (n = 312), water plants (n = 112), pioneer plants (n = 134), and mountain plants (n = 622). The percentages of species studied in the different habitats were as follows: fertilized meadows, 87%/51%; cultured plants, 80%/ 63%; weeds and ruderal plants, 70%/42%; woodland plants, 60%/36%; dry meadows, 54%/22%; marshlands, 51%/24%; water plants, 51%/26%; pioneer plants, 50%/22%; mountain plants, 34%/9.6%, respectively. The category “mountain plants” from Lauber and Wagner20 was found to accommodate too many plants (n = 622) for a differentiated analysis. In order to distinguish between plants growing generally in mountain areas and those that occur only in high altitude habitats, the group was subdivided according to the temperature values in Lauber and Wagner.20 In this way, an additional category of highaltitude plants (n = 201), which all had a temperature value of 1 (indicating that they thrive only at high altitudes above the tree line), was formed. For high-altitude plants, the percentages of species studied were 24%/2%, and thus were lower than for mountain plants in general. Native plants were analyzed according to their growth forms as listed in Lauber and Wagner.20 The largest group was herbs (n = 2327), followed by shrubs (131), trees (88), aquatic plants (74), and dwarf shrubs (54). Trees (86%/73%) were the beststudied group, followed by shrubs (57%/34%) and dwarf shrubs (59%/31%), herbs (53%/26%), and finally aquatic plants (47%/19%). Once a full data set for all plant species was assembled, subsets of plants were randomly selected, and the analysis was repeated. This was done three times for every 10th (267 species), 20th (133), and 40th (67) plant in the list, starting once at number one, once at three, and once at five. Figure 4

studied phytochemically and 709 (27%) for bioactivity. Among the 385 (14%) native species of grassy plantsmembers of the families Cyperaceae, Juncaceae, and Poaceae105 (27%) have been studied phytochemically and 33 (8.6%) for bioactivity. In contrast, “nongrassy” plants [2292 (85.6%)] have been more studied [1353 (59%) phytochemically and 704 (31%) biologically]. No grassy plants were considered to be toxic,20 and only one (Triticum sp.) was a medicinal plant. A total of 162 (6.3%) angiosperms, a gymnosperm (10%) (Taxus baccata L.), and four ferns (4.6%) were considered toxic.20 For categorizing the extent of study, the use of the family as the basic unit is problematic, because the number of species varies greatly within families. In this study, 2677 species are reported from 154 plant families. However the 10 most speciesrich families (in decreasing order of number of species, Asteraceae, Poaceae, Brassicaceae, Rosaceae, Fabaceae, Cyperaceae, Caryophyllaceae, Ranunculaceae, Apiaceae, and Lamiaceae) make up more than half of all native species, while 43 (28%) of the families have just one, 18 (12%) have two, and 20 (13%) have three species. If only the percentages of species studied per family were to be shown, then small families in which 1 out of 1, 2/2, or 3/3 species had been studied would all be 100% investigated and overwhelm the species-rich families. A more differentiated approach was suggested by Moerman25 when studying the numbers of plants used medicinally in North America using regression analysis and residual values. This approach was adopted for the present study. Figure 2a shows the regression analysis and all plant families concerning the

Figure 2. Plot of the number of plant species per family versus the number of species of each family that have been investigated phytochemically (a) and for bioactivity (b), alongside the trend line for all families. The 13 plant families with more 50 species are labeled. 211

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Table 1. The 45 Most Species-Rich Plant Families with Their Numbers of Species Native to Switzerland and the Residual Values Determineda family

species number

residual value phytochemistry

residual value bioactivity

Asteraceae Poaceae Brassicaceae Rosaceae Fabaceae Cyperaceae Caryophyllaceae Ranunculaceae Apiaceae Lamiaceae Orchidaceae Orobanchaceae Plantaginaceae Boraginaceae Primulaceae Polygonaceae Rubiaceae Juncaceae Salicaceae Liliaceae Gentianaceae Campanulaceae Saxifragaceae

300 216 148 146 138 132 106 91 87 76 67 62 60 41 38 37 37 37 36 34 34 32 28

47.45 −49.24 −15.37 −9.31 15.91 −32.93 −8.21 6.7 20.81 31.61 −22.64 −6 8.05 4.08 −7.34 11.19 −0.81 −16.81 −6.29 0.77 2.77 −6.18 −10.07

14.31 −24.11 −2.25 16.22 5.08 −26.52 −8.45 3.04 17.98 32.54 −11.36 −11.19 −4.73 −0.3 1.4 9.64 −4.36 −8.36 −0.13 1.34 −3.66 −8.2 −7.26

family

species number

residual value phytochemistry

residual value bioactivity

Amaranthaceae Onagraceae Euphorbiaceae Violaceae Scrophulariaceae Crassulaceae Potamogetonaceae Valerianaceae Geraniaceae Ericaceae Caprifoliaceae Amaryllidaceae Papaveraceae Aspleniaceae Dipsacaceae Dryopteridaceae Clusiaceae Iridaceae Betulaceae Solanaceae Equisetaceae Polygalaceae

27 27 26 25 24 24 23 21 21 19 18 17 16 16 16 14 11 11 10 10 10 10

9.46 0.46 5.99 −9.48 6.04 0.04 −2.43 −4.37 1.63 3.68 5.21 2.74 4.26 −0.74 −3.74 −0.68 3.9 −2.1 4.43 3.43 3.43 0.43

3.97 −1.03 9.2 −2.56 0.67 −0.33 −2.1 −2.63 6.37 3.83 4.07 4.3 4.53 −2.47 −2.47 −1 4.7 −1.3 3.93 4.93 3.93 −2.07

a

Residual values result from subtracting the actual percentage of studied plants from the value predicted from averaging all families in a regression analysis.

Figure 4. Percentages of plants studied phytochemically (lighter columns) and for bioactivity (darker columns) according to the analysis of the full data set, as well as predicted when just 10%, 5%, and 2.5% of the data set were analyzed randomly. Percentages are shown with standard deviations from three replicates.

Figure 3. Plants categorized according to their commonness based on Aeschlimann and Burdet23 and percentages of each group that have been studied phytochemically (lighter columns) and for bioactivity (darker columns).

chemically or for their bioactivity have become more evident.9−13 In this study an attempt has been made to define the terms “phytochemically studied” and “studied for bioactivity”. To obtain a credible estimate of the true numbers of “studied” plants, a systematic search strategy was applied in a case study for the Swiss native flora. The analysis shows that a substantially larger proportion of Swiss plants has been studied than postulated for the global flora. These results should not be viewed as representative figures for other geographic areas, but as a case study for the application of this systematic approach. The compilation of a full data set for all species within this

shows the results of this analysis with the standard deviations obtained. The analysis of all plants showed that 55% and 28% of the native plants have been studied for phytochemistry and bioactivity, respectively. The representative analysis of 10% of the data showed the data to be 54% and 29%, respectively. The analysis of 5% of the species gave 55% and 29%, and an analysis of just 2.5% of the data revealed an average of 60% and 27%, respectively, as having been studied previously. Over the years, discrepancies in published estimates of the numbers of plants that are presumed to have been investigated 212

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elegantly. For a lively discussion on its suitability, the reader should refer to the contribution by Weckerle et al.32 Grasses, defined here as members of the Cyperaceae (132 species), Poaceae (216), and Juncaceae (37), are usually not toxic and only rarely used as medicinal plants. Furthermore, only few specialists are able to distinguish between grassy plant species. As a consequence, grassy plants are less than half as well studied as nongrassy plants. No compound has ever been reported from a native Carex species (Cyperaceae), even though this is the largest Swiss genus, with 91 species. Other poorly studied, species-rich families are usually taxonomically complex or rare families such as the Caryophyllaceae (106), the Orchidaceae (67), and the Orobanchaceae (62). The native Swiss species from 11 entire families, the Agavaceae, Elatinaceae, Hyacinthaceae, Isoetaceae, Juncaginaceae, Linderniaceae, Montiaceae, Najadaceae, Plumbaginaceae, Scheuchzeriaceae, and Theophrastaceae, have never been studied at all with respect to their phytochemistry and bioactivity. Medicinal plants have attracted particular attention. Most of the best-studied plants in this survey have medicinal uses, such as Cannabis sativa L., Digitalis purpurea L., Hippophaë rhamnoides L., Hypericum perforatum L., Petasites hybridus (L.) Gaertn., Rhodiola rosea L., Silybum marianum (L.) Gaertn., and Viscum album L. All official medicinal plants have been investigated phytochemically, and 91% of them examined for bioactivity. Many more Swiss native plants have been used medicinally than are listed in the Swiss Pharmacopoeia,26 but this criterion was chosen to create a clearly defined category. Plants considered toxic are also among the best studied as found in this survey. This probably reflects the interest historically paid to plant toxins as a source of drugs and as molecular tools for the exploration of biochemical processes. In this category it was decided to include plants as being toxic only when listed as such in Lauber and Wagner.20 Fragrant plants also have been extensively studied, and routine methods such as gas chromatography (GC) have made the characterization of the composition of essential oils relatively straightforward. To date, 15% of Swiss native plants have been studied for their essential oil composition, and plant families that typically accumulate essential oils, such as the Apiaceae and Lamiaceae, are among the best-studied families chemically. A plant may attract attention by being very common, large, or obvious, while rare, small, or inconspicuous species may be overlooked. Indeed, a clear correlation has been observed between commonness and the percent of species investigated. This, however, does not exlude that some very common species such as Phyteuma spicatum L. or Hieracium sylvaticum (L.) L. have never been studied. Even some common trees are yet unstudied, such as the field maple (Acer campestre L.) or the wild pear (Pyrus pyraster Burgsd.). The categories of commonness shown here should, however, be treated with some caution, because a plant rare in Switzerland may not necessarily be rare elsewhere. In addition, habitat may be a predictor of the likelihood of prior investigation. Plants from familiar and readily accessible habitats, such as fertilized meadows, or cultured plants, “weeds”, and ruderal plants are among the best-studied groups. The more remote and rare the habitat is, the less their plants have been studied, and plants from the highest mountain ranges were found to be the least studied in this survey. The analysis of plants according to their growth forms shows that trees are the best studied group, with 86% and 73%

defined region provided an absolute measure to compare with the predicted values provided by randomly sampled representative subsets. As shown in Figure 4, the numbers of species studied predicted by analyzing just 5% and 10% of the initial 2677 species delivered good approximations of the actual outcome with deviations of just a few percent. Thus, it can be reasonably anticipated that a representative analysis, following this approach, of perhaps 10% of the world’s approximately 300 0001,2 higher plant species would provide a more credible and differentiated estimate of how many plants have been studied chemically and for their bioactivity. This would not be an easy task, as it would be necessary to come to terms with all taxonomic synonyms used, but this could be done using plant databases.1 Ultimately, a scientifically more sound estimate on the study of the global flora could be obtained than has been proposed earlier.13−17 The requirement for a study to be included in the present literature search was that it had to be searchable by SciFinder Scholar, an extensive search engine accessing the Chemical Abstracts and PubMed databases.19 However, it is still unreasonable to expect to find every study ever done in these databases. No search strategy can access unpublished work done by companies, or all academic theses and dissertations done generations ago. Test samples showed that some relevant studies were missed. For example, according to SciFinder Scholar, the medicinal plant Verbascum phlomoides L. has been the subject of many phytochemical studies, but not on its biological activities, excluding one investigation on antioxidant effects. In Wichtl,27 however, there are references to reported expectorant activities that SciFinder Scholar had not included. To categorize plants as “studied” or “not studied”, as has been done here (and as has been used by various authorities9−15), is an oversimplification of a complex matter. These categories are surrogate parameters, which allow certain analyses. However, they do not always depict the underlying situation very well. Sometimes a single study can significantly change a statistic, as exemplified by Orobanche. The 20 relatively rare species of this genus undergo a subterranean parasitic life with sporadically emerging sprouts. Half of the Swiss Orobanche species have been studied phytochemically, which gives the impression of a relatively well-studied genus. However, nine of the species (four exclusively) were covered in a single chemotaxonomic study on fatty acids and tocochromanols present.28 The taxonomic particularities of a plant per se are probably rarely the driving force to spark scientific endeavor, but in some cases individual investigators have dedicated much effort to the study of a particular taxonomic group. A specific example is the Gentianaceae family, which appears among the best-investigated taxa in the present study. Detailed analysis of the retrieved literature has revealed that the Swiss phytochemists Kurt Hostettmann and André Jacot-Guillarmod studied more than half of the Swiss Gentianaceae.29,30 For a complete list of Gentianaceae species investigated by these authors with the corresponding references, see Supporting Information (Table S2). It has been shown repeatedly that medicinal plants are overrepresented in some families and under-represented in others.25,31 It is also these “medicinal plant families” such as the Apiaceae, Asteraceae, Fabaceae, and Lamiaceae that have been studied most intensely, as shown by a regression analysis with residual values (Figure 1, Table 1). This analysis method was chosen because it simplifies a complex phenomenon quite 213

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screening of all results, suitable terms to help identify phytochemical and bioactivity studies were defined. For phytochemical studies these terms were “isolation”, “essential oil”, “volatile oil”, “phytosterol”, “sterol”, “fatty acid”, and “identification”. The most suitable terms for bioactivity results were “antimicrobial”, “antifungal”, “in vivo pharmacology”, “mice”, “rats”, “in vivo”, “in vitro”, “cells”, “clinical studies”, “human”, “clinical”, and “activity”. The columns in Table S1 in the Supporting Information represent the search categories with the main categories “phytochemistry” and “bioactivity”, with each containing the data from subcategories. “Phytochemistry” was assigned the subcategories “isolation”, “fatty acid composition”, “essential oils”, and “phytosterols”. “Bioactivity” included the subcategories “in vitro pharmacological studies”, “in vivo studies”, “antimicrobial”, and “clinical”. If more than 10 studies were counted in one subcategory, then a value of >10 was entered. If a study contained data from more than one subcategory, then this was entered in several subcategories, but treated as just one study in the main categories “phytochemistry” and “bioactivity”. If there were no results on a plant species, the databank http://www.tela-botanica.org was searched for synonyms, and the search repeated for all of these. All these databank searches were done between January 16 and May 30, 2011.

investigated phytochemically and biologically, respectively. Trees are large and long-lived and represent only a small number of species (88) in Switzerland compared to other regions of the world. Less well studied are shrubs, dwarf shrubs, herbs, and aquatic plants. Aquatic plants contain a number of very complex genera, such as Potamogeton (Potamogetonaceae, 21 species), which are difficult to distinguish at a species level. It must be noted that in some taxa the number of accepted species varies greatly depending on which taxonomic authority one follows. The taxonomically complex genus Rubus (Rosaceae), for example, may contain as many as 20 native Swiss species20 or as few as three.23 Hence, the differences between herbs and shrubs would be less pronounced if fewer Rubus species were included for analysis.

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EXPERIMENTAL SECTION

List of Native Swiss Plants. Data supplied by the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL) were used.24 These are based on a long-term study from 1967 to 1979 by ca. 200 trained botanists, who systematically mapped the distribution of plant taxa in regions of Switzerland over this time period.20−23 The list consists of 3200 taxa of native plants, meaning truly indigenous or introduced, but necessarily established and reproducing over a minimum defined period in at least one of the mapping areas. The taxonomy of this study follows Flora Helvetica.20 The 3200 taxa were incorporated into a table (MS Excel, Microsoft, Redmond, WA, USA), and all subspecies were removed to give 2677 species. Apart from their botanical name and family, all plants were categorized as to whether they were listed as toxic or as medicinal plants, as were their growth forms and habitats according to the Flora Helvetica.23 Finally, the commonness of each plant was documented according to Aeschlimann et al.23 No data were available for a few species with regard to growth form, habitat, or commonness. These species were excluded from the corresponding statistics. Criteria for a Plant to be Studied. The criterion for a plant species to be considered phytochemically studied is that, in the literature searchable by SciFinder Scholar,19 there was at least one disclosed structure of a constituent of this plant species. Reports meeting this criterion were further subcategorized as follows: isolation of phytochemicals, essential oil analysis by gas chromatography, compounds identified by comparison with references using chromatographic methods (TLC, GC, or HPLC), fatty acid and lipid analysis by GC, and phytosterol analysis. The criterion for plant species to be considered studied for bioactivity was that literature was retrievable by SciFinder Scholar in which a defined bioactivity meeting the criteria below had been reported. It was considered irrelevant if activity was shown for purified compounds, chromatographic fractions, or extracts. The subcategories were as follows: in vitro studies such as results from assays on molecular targets (enzymes, receptors) or lower organisms (other than microbes), antimicrobial assays, in vivo studies, and clinical studies. In vitro antioxidant activity or radical scavenging was not considered as a form of bioactivity, but included as an additional category. If bioactivity had been reported for a compound from one plant, and the compound was known from another plant as well, then the activity was attributed only to the plant mentioned in the study. For example, if a common compound such as β-sitosterol has been reported from one plant and shown to be active in a bioassay, then other plants reported to contain β-sitosterol were not considered as “biologically studied”. Literature Search. All literature for the species in the list was searched systematically in the scientific database SciFinder Scholar. Phytochemical studies and studies on bioactivity were identified and categorized in the subcategories given above. If less than 250 publications were retrieved for a plant species, they were all visually screened, and if necessary, the full text was read. If more than 250 publications were found, the search was refined using the “refine” function in SciFinder Scholar. After a period of experimenting by comparing results from refined searches with those of systematic

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ASSOCIATED CONTENT

S Supporting Information *

Table with the results of the literature search for all the plants including their botanical authorities and the number of studies found for each category and subcategory, as well as a table with the Gentianaceae species investigated by Hostettmann and Jacot-Guillarmod and the corresponding references. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 (0) 61 267 15 34. Fax: +41 (0) 61 267 14 74. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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