Stevia rebaudiana (Bert.) Bertoni is a small perennial shrub of the Compositae family. Estimates of total number of species in the genus ranges from 150 to 300, all distributed in the New World, from the southwestern United States to the northern Argentina. The native occurrence of Stevia rebaudiana is between 22-24° S and 53-56° W, in Paraguay and Brazil. This species was known by the Guarany Indians under several names (Caa-jhe-he, Caa-hee, Ca-a-yupe, Azuca-caa, Eira-caa) related to its sweet leaf taste and to its use in sweet beverages and remedies, especially the cooked “mate” (Ilex paraguariensis). Stevia rebaudiana became known by the Europeans due to its discovery by Moises Bertoni in 1899; the chemist Rebaudi was the first to study its chemical characteristics.
Stevia rebaudiana: Economic Importance and Uses
The economic importance of this species is the consequence of the presence, in the leaves, of substances with high sweetening power, whose physical, chemical, pharmacological and toxicological characteristics allow its use in human diet as natural and diethetic sweetening, with no colateral effects. Several reviews about this plant have been published over the past 40 years, including topics like its cultivation, commercialization, chemistry and biological effects. The first isolated sweetening principle was the stevioside, whose sweetening power ranges from 100-300 times higher than sucrose, depending on the sucrose concentration of the reference solution. Other sweetening principles were described after 1970: rebaudiosides A, B, C, D, E, dulcosides A and B, and the steviolbioside, whose sweetening power ranges from 100-400 times higher than sucrose at 0.4%.
All these substances are diterpenoid tetracyclic glycosides, having as common feature the aglycone steviol. The diethetic attributes are the result of their high sweetening power making the calorie intake negligible. Dubois (1982) substituted the glycosidic groups of the stevioside by non-glycosidic groups and concluded that the former were not necessary for the occurrence of the sweet taste. The stevioside is the major component but has an unpleasant aftertaste. This problem is solved by blending it with other compounds or by its conversion into rebaudioside A, which is normally present in the leaves in lower content (25% to 45% of the stevioside content), does not have an aftertaste, and has a sweetening power 1.2-1.6 times higher than stevioside. The other glycosides occur in low levels. These sweetening compounds are found mainly in the leaves, but small amounts were also detected in the flowers. Data on the yields of stevioside and other sweet glycosides are included in Table Sweet glycosides content of Stevia rebaudiana. The wide range in concentration values must be ascribed to both genetic.
Table Sweet glycosides content of Stevia rebaudiana
|Name||Plant part||Yield (%)|
Besides the sweetening compounds, other substances have been isolated: gibberellin A20 in roots, stigma-sterol and β-sitosterol (an anti-hyperlipoproteinemic) in leaves, jhanol, austroinulin, 6-0-acetyl austroinulin,β-amyrine acetate, lupeol esters in leaves and flowers, bactericide agents in leaves, gums and organic acids in leaves and various components of the volatile oil. Rutin (cardiotonic and restorative in cases of capillary fragility) and stigmasterol were also found in material cultivated in vitro.
In addition to the sweetening properties, some data indicating a hypoglycemic effect were obtained, but using only total leaf extracts. A slight inhibitory action of stevioside on enzymes that break sucrose and on the development of oral bacteria in vitro was observed by Yabu et al. (1977), indicating a possible anticariogenic effect.
A contraceptive effect of leaf extracts, reducing fertility in rat (female) described by Planas and Kuc (1968), was not confirmed. Tests for a possible anti-androgenic activity were negative for stevioside. For steviol and dihydrosteviol significant results were obtained only with high doses.
In the search to find possible side-effects some work was done, and the available data show that such substances are active on cell metabolism. Effects on the energy metabolism were observed in isolated mitochondria inhibiting several steps in the respiratory chain. In isolated cells and organs only the aglycones steviol and isosteviol were active. Regarding the carbohydrate metabolism, all stevioside derivatives showed inhibitory effects by altering the hexose carrier of the plasma membrane. Wingard et al. (1980) showed that stevioside and rebaudioside A are degraded in vitro to steviol by bacteria of the intestinal microflora of rats. Further, steviol is completely absorbed in the lower bowel and eliminated within 72 h. On the other hand, in toxicological tests in rats, crude extracts and stevioside revealed low toxicity. Other tests with crude extracts and stevioside showed neither induction of chromosomic aberration nor mutagenic and carcinogenic effects in bacterial and animal cells. Tests for antimitotic activity were also negative, nevertheless, metabolically activated steviol was found to be highly mutagenic. Some effects on the cardiovascular system and on the smooth muscle were also described.
Extracts with stevioside and rebaudioside showed effects similar to those of gibberellin in the activation of amylase in cereals and in the promotion of growth when applied to whole plants. The effects are probably related to the structural similarities of the aglyconic portion of the molecule to the gibberellins and to a possible role of the stevioside and its derivatives in the synthesis of that growth regulator. Several patents for use of the stevioside and its derived compounds as growth regulators are registered in Japan.
Commercial and Agronomic Aspects
Several industrial processes for extraction, purification, improvement of taste and use of the sweetening compounds in food were patented in Japan. In this country, Stevia sweetenings are used in a wide range, directly in the composition of liquid or solid foods, and as substitute for conventional sugars or artificial diethetics.
The demand for these chemicals generated by such uses is supplied through commercial cultures where the dry leaves for extraction of the sweetenings are produced. Besides the plantations in Japan (about 200 ha in 1982), others exist in several countries (Korea, Taiwan, Laos, Indonesia, China, Brazil and Paraguay), the production being mainly exported to Japan. Estimates of dried leaf annual consumption in Japan are variable, ranging from ca. 300tons in 1979 to 700-1000 tons in 1979 and 650-750 tons in 1981.
The culture of Stevia rebaudiana for economic purposes is influenced by climatic factors: cold and dry regions are not appropriate. The latitude is also important because this is a short-day plant with critical photoperiod between 13 —14h, the maximal leaf yield and stevioside content occurring at the end of the vegetative phase, the former being also a function of the length of this period.
Due to agronomic research carried out after 1970, when the species was introduced in Japan, several data became available, especially on the responses to nutrients, soil moisture, plant density and regrowth after reaping, growing behaviour, characteristics of propagation, time and frequency of harvest, hereditability and correlation amoung various characters, diseases and their control. This has improved the techniques of culture and increased the productivity.
Sexual propagation of the species is possible, and it is largely employed despite the low cultural value of seeds (10% -15%), owing to its low fertility index. This method does not allow the production of homogenous populations with pre-defined characteristics, resulting in great variability in important features like sweetening composition and level. Vegetative propagation through cuttings or division of the mature plants is also employed, but this method is limited by the lower number of individuals that can be obtained simultaneously from a single plant.
In crop plants, increase in productivity and decrease of production costs are fundamental goals. To reach them, tissue culture techniques have been used successfully, along with traditional agronomic methods. In Stevia rebaudiana the use of in vitro techniques as alternative or complement to conventional procedures of propagation and genetic improvement could facilitate obtaining disease-free homogenous populations, with higher levels of the sweetening compounds, larger foliar mass, resistance to drought, herbicides, breaking of stems and branches, etc. Another important goal is to break the negative correlation that exists between the contents of stevioside and rebaudioside A and of the former and the dried leaf yield (higher in early and late maturing plants, respectively).
Stevia rebaudiana: Concluding Remarks
Considering the present knowledge on Stevia rebaudiana and particularly the available data on the sweetening compounds which indicate, firstly, their low toxicity, and secondly the viability of production in industrial scale and utilization as non-caloric sweetenings, a significant increase in the economic and social importance of this species can be expected. Thus, with an increase in the demand for sweetening compounds, an increase in productivity will be needed. This objective could be reached through the improvement of the techniques of cultivation and of the genetic material. The use of techniques of tissue and cell culture could play a relevant role for these purposes.
In this species, several techniques for micropropagation are already available, and clones sensu stricto can be obtained from the mother plant. Thus, this technique will be useful to complement conventional vegetative propagation for rapid multiplication of individuals with desirable characteristics, produced by natural variability or induced in culture.
In relation to the production of sweet compounds in vitro, the few available results do not exclude the possibility of employing this technique as an alternative to the agronomic culture.
Nevertheless, much of the potential for tissue culture in Stevia rebaudiana remains unexplored. The use of the variability originating in in vitro cultures and selection techniques at the cellular level enhances the probability of obtaining better genotypes. Thus the establishment of techniques of protoplast fusion and culture, and further regeneration of plants are of fundamental importance. The data available for cell and protoplast culture represent only an initial step on the way to be explored. The establishment of effective techniques to regenerate plants via indirect organogenesis in callus derived from cell suspensions will undoubtedly provide the means for studies in view of obtaining new and improved genotypes.
Selections from the book: “Medicinal and Aromatic Plants II”, 1989.