Saponaria officinalis L.: In Vitro Culture and the Production of Triterpenoidal Saponins
There are very few studies on the production of triterpenoids and their saponins by in vitro plant culture. These products now enjoy growing interest since their chemical extraction and purification have become easier and their structural identity has been made possible by methods like RMN-13C or Fab-MS. Among the plants producing triterpenoidal saponins, some contain great amounts of very polar saponins, essentially in the rhizome and the roots (Saponaria officinalis L., Gypsophila sp., Caryophyllaceae) or in the bark (Quillaja saponaria Mol., Quillaja smegmadermos D.C., Rosaceae). These saponins are among the biggest with nine to ten oses bound to a pentacyclic triterpenoid acid. Their amphiphilic structure confers to them some well-known properties such as detergent, emulsive, hemolytic and toxic substances. Some of them are still largely used as shampoo (Quillaja saponins) or to make photographic emulsion (saponins of S. officinalis, fuller’s herb or of Gypsophila sp., soapwort). First results showed us the presence of these compounds in plant cell culture in vitro, so we have tried to investigate their production and metabolism using S. officinalis cells to elucidate their role in the cell.
Distribution and Importance of the Plant
Saponaria officinalis L. Caryophyllacease usually named fuller’s herb, is encountered in most of Europe, in France, Spain and Italy, for example, and also in North Africa and Syria, up to 1600 m altitude. This plant is ubiquitous, but prefers moisture and light, for example along the rivers on raised banks. Its delicate colours of pale pink and its graceful bearing make it an ornamental plant with simple or double flowers. It needs little attention and can be propagated easily by rhizomes.
Historically, S. officinalis was known from antiquity as a detersive plant. It was used by the Romans as soap. It was also used as medicine, in external application, against leprosy, scrofula, skin ulcers, psoriasis. Orally, it was employed to enhance milk secretion and as purgative, emmenagogue and diuretic. Actually, either the roots and the rhizomes or the shoots are used in the preparation of different medicines (21 medicaments are sold in France containing S. officinalis). Triterpenoidal saponins are the principles known to be active in the plant. It is usually used as hepatic depurative or expectorant (Vigneau 1985) in infusions and other herbal drinks (P. Hatinguais, Ets P.F. Medicament, France, pers. commun.). In 1970, 25 tons of dry plant material were used in Europe. In France, 250 kg of roots and rhizomes and 700 kg of shoots are employed by different laboratories to make medicines. The actual prices are 8 to 12 F/kg for the shoots and 20 to 30 kg for the roots and rhizomes (J. Elphege, Ets Gifrer France, pers. commun.), which are richer in saponins than the shoots.
Conclusion and Prospects
S. officinalis belongs to the group of plants used in soft medicine, essentially as infusions for hepatic depurative or expectorant treatment. Cultured in vitro, this plant produces calli and suspensions easily and the growth parameters (cell doubling time 50 h, and maximum at the stationary phase 12 to 15 g/1 dry wt) are average for cell suspension cultures.
In contrast to different plants we studied, Glycyrrhiza glabra (Leguminoseae), Centella asiatica (Umbelliferease), for example (unpubl.) S. officinalis tissues and cell suspension cultures can produce triterpenoidal saponins easily at about the same level as the shoot does. This saponin production has remained more or less constant over the past 6 years. Thus, for us, it constitutes essentially a biological model for studies of the metabolism regulation of these compounds to explain why these substances are produced in vitro by S. officinalis cells and not by other plants with great medicinal interest.
Plant Material Origin. S. officinalis shoots and seeds were kindly provided by the Museum National d’Histoire Naturelle of Paris, France. The shoots and the seeds were surface sterilized in 5% sodium hypochlorite (10 min for the shoots and 30 min for the seeds). The seeds were placed to germinate on Knop medium in sterile flasks. Seedlings were used when they were about 2 months old.
Saponin Extraction and Purification. Saponins were extracted with methanol 20% for 4 h and more and precipitated at -5 to 0°C in absolute ethanol. The precipitate was purified in the first experiments on silica gel column chromatography eluted with n-butanol/ethanol/ammonia 25% (70/20/50) and more recently on TSK Fractogel HW 40S column chromatography eluted with a gradient of methanol water (10% to 30%).
Determination of Saponins Containing Quillaic Acid. Saponins of S. officinalis were quantified as their genin, quillaic acid. Determination of quillaic acid was performed as its y-lactone after hydrolysis with refluxing HC1 for 12 h. The quillaic lactone was then extracted with ethyl ether and derivatized with BSTFA (N,O-bis-[trimethyl-silyl]-trifluoroacetamide) at room temperature. Quantification was then performed on GL chromatography on glass column filled with SE-30 2% on Chromosorb WHP80-100 mesh at 300°C, by comparison with a standard, 18/?-glycyrrhetinic acid.
M. Henry, Medicinal and Aromatic Plants II (1989)