Subscribe to RSS
DOI: 10.1055/s-2005-873170
Solanum chrysotrichum Hairy Root Cultures: Characterization, Scale-Up and Production of Five Antifungal Saponins for Human Use
Ma. Luisa Villarreal
Centro de Investigación en Biotecnología
Universidad Autónoma del Estado de Morelos
Cuernavaca 62210
Morelos
México
Phone: /Fax: +52-777-329-7057
Email: luisav@cib.uaem.mx
Publication History
Received: December 17, 2004
Accepted: July 13, 2005
Publication Date:
18 October 2005 (online)
Abstract
The production of five antifungal saponins (SC-2 - SC-6) recently extracted from Solanum chrysotrichum leaves was performed using hairy root cultures grown in 250-mL flasks and in 2-L modified draught-tube internal-loop airlift reactors (2-L MR). In both scales, first order growth kinetics were observed, reaching specific growth rates of 0.08 and 0.12 d-1, respectively. Root density at the end of the culture period was the same for the flasks and for the 2-L MR. In flasks the production of the most active saponin SC-2 was growth-associated, with a maximum yield of 0.04 % dry weight, while in the 2-L MR an SC-2 yield of 0.7 % dry weight was reached, representing a value six times greater than that observed for plant leaves. SC-3 was obtained from root biomasses grown in flasks, while SC-4 was recovered from biomasses at both levels under investigation. SC-5 and SC-6 were only detected in the culture medium of roots grown in 2-L MR. Solanum chrysotrichum hairy root cultures and their scale-up in reactors are feasible strategies for the production of these antifungal compounds for human use.
Solanum chrysotrichum has been most widely used by the indigenous people from the Mayan highlands of Chiapas, Mexico for the treatment of skin infections [1]. A spirostanol saponin designated as SC-1 was isolated and identified as the antifungal component of Solanum chrysotrichum [2]. Recently, five new active saponins (SC-2 - SC-6) were isolated and elucidated from the leaves, and the compound SC-2 was determined to be the most active saponin against T. mentagrophytes, T. rubrum and Candida albicans [3]. The production of the saponins from S. chrysotrichum leaves varies seasonally and according to the stage of development [3], [4]. It is therefore important to find a way of ensuring sustainable and controlled production of these saponins on a large-scale using tissue culture systems. A first attempt was the production of SC-1 using cell suspensions grown in 10-L airlift reactors [4]. Use of hairy root cultures is another alternative that has demonstrated genetic stability and increased levels of various phytochemicals [5], [6]. In a previous project we developed a novel fitting for growing the hairy roots of S. chrysotrichum in an airlift reactor [7]. In this work, we describe the growth and production of the antifungal saponins using hairy roots when conducted in 250-mL Erlenmeyer flasks and 2-L airlift bioreactors.[]

In flask cultures, the density of roots after 42 days was 4.3 g dry weight L-1 (DW L-1), 6 times higher than the inoculum. Conductivity of the culture medium (Δ) dropped in proportion to root tissue growth (Fig. [1] A). Root growth followed first order kinetics with a mean specific growth velocity (μ) of 0.08 d-1. Sucrose was hydrolyzed on day 20 when the exponential growth phase ended, even though glucose and fructose were poorly consumed, and at the end of the culture, 26 g L-1 of total sugar still remained in the culture medium (Fig. [1] B).
Production of the active extract (a mixture of saponins from chloroform extract) in the roots was growth-related. During the culture time, only three of the saponins (SC-2, SC-3 and SC-4) were recovered from root biomasses, which demonstrated discontinuous peaking patterns, but with differences in their accumulation profiles, so that maximum yields for saponins SC-2 (0.37 mg g DW-1) and SC4 (0.851 mg g DW-1) were registered at day 15 when saponin SC-3 was not present; while maximum yield for saponin SC-3 (0.189 mg g DW-1) was recovered at day 10, when the levels of SC-2 and SC-3 were diminished (Fig. [1] C). As reported earlier, compounds SC-1 - SC-5 belong to the same group of spirostan saponins which have identical aglycone portions but different saccharide chains, and significant variations in the levels of these compounds has also been observed in the leaves of wild plants harvested at different stages of development [3]. Even though it is difficult to explain the differential peaking fluctuations of the three saponins recovered from root biomasses, it may be due to conversion between compounds because of the different saccharides attached to them. Peaking fluctuations have been detected in various plant cell cultures producing secondary metabolites [8], [9].
Saponins were not released into the culture medium, and SC-5 and SC-6 were not observed either in the biomass or in the culture medium (Table [1]). As SC-2 is the most active saponin, its continuous production in the culture could represent an advantage when developing strategies for optimizing production. None of the saponin yields from hairy roots cultured in flasks was higher than those observed in plant leaves.
In order to evaluate the consumption of sugar by S. chrysotrichum hairy root cultures, the effects of sucrose concentration and light intensity on growth and saponin production were observed for four different concentrations of the carbohydrate: 3, 10, 20 and 30 g L-1, and under two light conditions: dark and continuous white light (8 - 10 μmol s-1 m-2). In Fig. [2] A, it can be observed that the highest biomass production was obtained at the maximum sucrose concentration, and that this biomass was significantly higher in continuous light than it was in the dark. It was also observed that when there was less sugar, growth diminished significantly in both dark and light conditions. Chlorophyll yields for roots grown in the light were greater than those observed in the dark (Fig. [2] B), Total sugar in the culture medium remained high in both dark and light conditions with maximum sucrose concentration of 30 g L-1 (Fig. [2] C); it was however the case that growth with this sucrose concentration was significantly higher in the light (Fig. [2] A). According to data reported in the literature concerning the cell suspensions and hairy roots cultures of some other species [10], [11], [12], these results may suggest that the S. chrysotrichum hairy root cultures exhibited some degree of photomixotrophism, as they contained substantial amounts of chlorophyll according to the range reported for photomixotrophic cultures, but still requiring exogenous sugar supply for their growth.
A low increment in biomass, both in light and dark, was registered when sucrose was not added into the culture medium, or added at a low dose (3 g L-1); this growth was probably accomplished by using the small contents of sugars carried by the roots at the time of subculturing.
Fig. [3] shows variations in the production of saponins with different sucrose concentrations and light conditions and indicates that, at 30 g L-1, production of SC-2, SC-3 and SC-4 was greater in the light (a) than in the dark (b) suggesting that as is the case with some other plant secondary metabolites [11], light may mediate effects on biosynthetic pathways.
Certain experiments were undertaken to evaluate growth caused by variations from the normal (1 × ) B5 concentrations of phosphates and/or nitrates. After 30 days in culture using high (3 × ) and low (0.3 × ) B5 concentrations of nitrates, roots did not grow in any of the phosphate tested concentrations (1 × , 1.5 × , 3 × and 0.3 × ); even though the cultures partially hydrolyzed sucrose. With a 50 % higher (1.5 × ) B5 nitrate concentration, roots demonstrated the same growth as under normal (1 × ) conditions in any of the four phosphate concentrations tested. These results demonstrate that significant variations in B5 nitrate levels may be critical for growth, while varying the phosphates did not alter this parameter. Similar results have been obtained for the hairy roots cultures of Catharanthus roseus [13].
In the 2-L reactor, root density calculated by conductivity measurements was of 4.4 g DW L-1 after 42 days, a value similar to that obtained in flasks. Root growth followed first order kinetics. The lag phase was 2 days longer than in the flask culture, although a μ of 0.11 d-1 (td = 6 d) with a maximum μI of 0.18 d-1 was observed. The doubling time was 2 - 3 days less than that observed in flask culture and similar to the one observed in cell cultures of S. chrysotrichum grown in the 10-L reactor [4]. Sucrose was totally hydrolyzed on day 20, and sugar concentration at the end of the culture was of 26.5 g L-1, a value similar to the one registered in the flask cultures.
Results observed in flasks and 2-L reactor cultures suggest that better physical growth conditions were achieved in the reactors where roots grew faster, due to physical modifications introduced with the novel design.
In root biomasses grown in the 2-L reactor, an SC-2 yield of 7.17 mg g DW-1 (0.7 % DW) was obtained, which was 19 and 6 times that of flask cultures or that obtained from plant leaves [3], SC-4 yield was lower than the one from flask cultures, and SC-3, SC-5 and SC-6 were not produced by biomasses. In the culture medium, small concentrations of SC-5 and SC-6 were recovered (Table [1]).

Fig. 1 S. chrysotrichum hairy root cultures in 250-mL flasks with 100 mL of B5 medium and an inoculum of 10 g FW L-1. (a) Biomass X () and conductivity Δ (✦). (b) Carbohydrate consumption: sucrose (▴), glucose (✦) and fructose (•). (c) Active extract (•), SC-2 (✦), SC-3 (), SC-4 (▴).

Fig. 2 S. chrysotrichum hairy root cultures in flasks (45 days) without sugar and with 4 sugar concentrations (3, 10, 20 and 30 g L-1) and 2 light conditions [light () and dark ()]. (a) Root biomass (X). (b) Total chlorophylls (Chl). (c) Total sugar.

Fig. 3 Saponin yields (Y) of SC-2 (), SC-3 (□), and SC-4 () in hairy root cultures of S. chrysotrichum in flasks without sugar and with 4 sugar concentrations (3, 10, 20, 30 g L-1) and 2 light conditions: (a) light and (b) dark.
Compound | Maximum yields (mg gDW-1) | ||||
Plant leaves |
Hairy root culture | ||||
Flasks (Biomass) |
2 - 1 reactor | ||||
Biomass | Medium | ||||
SC-2 | 1.237 | 0.370 ± 0.050 | 7.170 ± 1.4 | nd | |
SC-3 | 0.412 | 0.189 ± 0.03 | Nd | nd | |
SC-4 | 2.054 | 0.851 ± 0.12 | 0.137 ± 0.03 | nd | |
SC-5 | 3.186 | nd | nd | 0.028 ± 0.012 | |
SC-6 | 2.348 | nd | nd | 0.056 ± 0.014 | |
n = 3 ± SD. | |||||
nd = not detected. |
Materials and Methods
Hairy root propagation: The hairy root cell line, C58 - 431 which had been previously cultivated for more than two years [7], was used in this work. The roots were cultivated in Erlenmeyer flasks with 100-mL of B5 medium supplemented with sucrose, (30 g L-1) without hormones, and incubated at 26 - 27 °C under uniform white light conditions (8 - 10 μmol s-1 m-2), in a gyratory shaker (115 rpm). The roots were subcultured every three weeks by transferring 1 g FW into fresh medium.
Bioreactor cultures: The reactor used was a 2-L draught-tube internal-loop airlift reactor, previously described, in which a mesh draught with helixes replaced the glass draught in the original design, resulting in the homogeneous distribution of roots, so that a double biomass concentration was obtained, when compared with that produced in the basic design [7]. The scale-up was performed by maintaining the geometric relationships and with a constant Q/V (relation between gas flow and volume) throughout the time in culture. Twenty-day-old root cultures grown in a 1-L flask were used as inoculum (10 g FW L-1) for the reactor. During 6 weeks of cultivation, conditions for reactor culture were: 26 - 27 °C, 0.1 vvm (volume of air per liquid volume min-1), and uniform white light (8 - 10 μmol s-1 m-2).
Culture sampling: Three randomly chosen flasks were sacrificed every three days for growth and product analysis. In the reactors, 5-mL samples of the culture medium were taken every three days for growth and product analysis, and saponins concentrations in biomass were evaluated at the end of the culture period.
Sugar analysis: Sucrose, fructose and glucose analyses were carried out by HPLC with an IR detector. The column was of the NH2 type measuring 3.9 × 300 mm, with 125 Å pore size and 10 μm particle diameter, from Waters. Predetermined conditions were: mobile phase 20 : 80 acetonitrile:water with an operational flow of 1.5 mL min-1.
Chlorophyll determination: Chlorophyll was extracted from the root samples with 80 % acetone, analyzed in a Beckman DU 650 spectrophotometer and estimated by the procedure described by Flores et al. [8].
HPLC analysis: Saponins SC-2 - SC-6 for HPLC analysis were obtained from the methanol extract of wild plants of S. chrysotrichum, as described [3]. Extracts were analyzed on a Waters Delta prep 4000 modular HPLC system, consisting of a U 6K injector, a 600E pump system controller (Millenium 3.2 software), and a refractive index detector. The analysis was carried out on two ChromolithTM RP-18 (100 × 4.6 mm, 2μm) columns connected in series; the mobile phase was 35 : 65 acetonitrile:water at a flow rate of 1.5 mL min-1 for SC-2 and, 1.7 mL min-1 for SC-3 and SC-4. For SC-5 and SC-6, the mobile phase was 37 : 63 acetonitrile:water at a flow rate of 1.5 mL min-1.
Calibration curves were constructed separately for saponins SC-2 - SC-6 using solutions at the following dilutions: 100, 200, 400 and 800 μg mL-1 in MeOH. The identities of each peak were confirmed by co-injection of purified samples of SC-2, SC-3, SC-4, SC-5 and SC-6. The calibration curves were based on the peak areas of the HPLC chromatograms. The experiments were performed in five replicates. Values were expressed on the basis of the dry weight in grams.
Root tissue growth estimation: Root tissue growth during the reactor operation was indirectly monitored using conductivity on-line measurements as we previously reported [7].
#Acknowledgements
This work was supported in part by Consejo Nacional de Ciencia y Tecnología (CONACYT P 42 924-Q).
#References
- 1 Lozoya X, Navarro V, García M, Zurita M. Solanum chrysotrichum (Schdl) a plant used in México for treatment of skin mycosis. J Ethnopharmacol. 1992; 36 127-32
- 2 Alvarez L, Perez M C, González J L, Navarro V, Villarreal M L, Olson J O. SC-1 an antimycotic spirostan saponin from Solanum chrysotrichum . Planta Med. 2001; 67 372-74
- 3 Zamilpa A, Tortoriello J, Navarro V, Delgado G, Alvarez L. Five new steroidal saponins from Solanum chrysotrichum leaves and their antimycotic activity. J Nat Prod. 2002; 65 1815-9
- 4 Villarreal M L, Arias C, Vega J, Feria A V, Ramírez O T, Nicasio P. et al . Large-scale cultivation of Solanum crhysotrichum cells: production of the antifungal saponin SC-1 in 10-L airlift bioreactors. Plant Cell Rep. 1997; 16 653-6
- 5 Giri A M, Narasu L M. Transgenic hairy roots: recent trends and applications. Biotechnol Adv. 2000; 18 1-22
- 6 Doran P. Properties and applications of hairy roots cultures.
In: Plant Biotechnology and Transgenic Plants. Oskman-Caldentey KM, Barz W, editors New York; Marcel Dekker 2002: pp 143-62 - 7 Caspeta L, Quintero R, Villarreal M L. Novel airlift reactor fitting for hairy root cultures: developmental and performance studies. Biotechnol Prog. 2005; 21 735-40
- 8 Mori T, Sakurai M, Sakuta M. Changes in PAL, CHS, and DAHP synthase (DS-Co and Ds-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa . Plant Cell Tissue Organ Cult. 2000; 62 135-9
- 9 Bolta Z, Baricevic D, Bohanec B, Andrensek S. A preliminary investigation of ursolic acid in cell suspension culture of Salvia officinalis . Plant Cell Tissue Organ Cult. 2000; 62 57-63
- 10 Yen H E, Chen Y C, Yen S K, Lin J H. Sugar uptake by photomixotrophic soybean suspension cultures. Bot Bull Acad Sin. 1999; 40 147-52
- 11 Bhadra R, Morgan J A, Shanks J V. Transient studies of light-adapted cultures of hairy roots of Catharanthus roseus: growth and indole alkaloid accumulation. Biotechnol Bioeng. 1998; 60 670-8
- 12 Flores H E, Dai Y, Cuello J L, Maldonado M IE, Loyola V V. Green roots: photosynthesis and photoatutrophy in an underground plant organ. Plant Physiol. 1993; 101 363-71
- 13 Toivonen L, Balsevich J, Kurz W GW. Indole alkaloid production by hairy root cultures of Catharanthus roseus . Plant Cell Tissue Organ Cult. 1989; 18 79-93
Ma. Luisa Villarreal
Centro de Investigación en Biotecnología
Universidad Autónoma del Estado de Morelos
Cuernavaca 62210
Morelos
México
Phone: /Fax: +52-777-329-7057
Email: luisav@cib.uaem.mx
References
- 1 Lozoya X, Navarro V, García M, Zurita M. Solanum chrysotrichum (Schdl) a plant used in México for treatment of skin mycosis. J Ethnopharmacol. 1992; 36 127-32
- 2 Alvarez L, Perez M C, González J L, Navarro V, Villarreal M L, Olson J O. SC-1 an antimycotic spirostan saponin from Solanum chrysotrichum . Planta Med. 2001; 67 372-74
- 3 Zamilpa A, Tortoriello J, Navarro V, Delgado G, Alvarez L. Five new steroidal saponins from Solanum chrysotrichum leaves and their antimycotic activity. J Nat Prod. 2002; 65 1815-9
- 4 Villarreal M L, Arias C, Vega J, Feria A V, Ramírez O T, Nicasio P. et al . Large-scale cultivation of Solanum crhysotrichum cells: production of the antifungal saponin SC-1 in 10-L airlift bioreactors. Plant Cell Rep. 1997; 16 653-6
- 5 Giri A M, Narasu L M. Transgenic hairy roots: recent trends and applications. Biotechnol Adv. 2000; 18 1-22
- 6 Doran P. Properties and applications of hairy roots cultures.
In: Plant Biotechnology and Transgenic Plants. Oskman-Caldentey KM, Barz W, editors New York; Marcel Dekker 2002: pp 143-62 - 7 Caspeta L, Quintero R, Villarreal M L. Novel airlift reactor fitting for hairy root cultures: developmental and performance studies. Biotechnol Prog. 2005; 21 735-40
- 8 Mori T, Sakurai M, Sakuta M. Changes in PAL, CHS, and DAHP synthase (DS-Co and Ds-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa . Plant Cell Tissue Organ Cult. 2000; 62 135-9
- 9 Bolta Z, Baricevic D, Bohanec B, Andrensek S. A preliminary investigation of ursolic acid in cell suspension culture of Salvia officinalis . Plant Cell Tissue Organ Cult. 2000; 62 57-63
- 10 Yen H E, Chen Y C, Yen S K, Lin J H. Sugar uptake by photomixotrophic soybean suspension cultures. Bot Bull Acad Sin. 1999; 40 147-52
- 11 Bhadra R, Morgan J A, Shanks J V. Transient studies of light-adapted cultures of hairy roots of Catharanthus roseus: growth and indole alkaloid accumulation. Biotechnol Bioeng. 1998; 60 670-8
- 12 Flores H E, Dai Y, Cuello J L, Maldonado M IE, Loyola V V. Green roots: photosynthesis and photoatutrophy in an underground plant organ. Plant Physiol. 1993; 101 363-71
- 13 Toivonen L, Balsevich J, Kurz W GW. Indole alkaloid production by hairy root cultures of Catharanthus roseus . Plant Cell Tissue Organ Cult. 1989; 18 79-93
Ma. Luisa Villarreal
Centro de Investigación en Biotecnología
Universidad Autónoma del Estado de Morelos
Cuernavaca 62210
Morelos
México
Phone: /Fax: +52-777-329-7057
Email: luisav@cib.uaem.mx


Fig. 1 S. chrysotrichum hairy root cultures in 250-mL flasks with 100 mL of B5 medium and an inoculum of 10 g FW L-1. (a) Biomass X () and conductivity Δ (✦). (b) Carbohydrate consumption: sucrose (▴), glucose (✦) and fructose (•). (c) Active extract (•), SC-2 (✦), SC-3 (), SC-4 (▴).

Fig. 2 S. chrysotrichum hairy root cultures in flasks (45 days) without sugar and with 4 sugar concentrations (3, 10, 20 and 30 g L-1) and 2 light conditions [light () and dark ()]. (a) Root biomass (X). (b) Total chlorophylls (Chl). (c) Total sugar.

Fig. 3 Saponin yields (Y) of SC-2 (), SC-3 (□), and SC-4 () in hairy root cultures of S. chrysotrichum in flasks without sugar and with 4 sugar concentrations (3, 10, 20, 30 g L-1) and 2 light conditions: (a) light and (b) dark.