Establishment of a Cell Suspension Culture of Eysenhardtia platycarpa : Phytochemical Screening of extracts and Evaluation of Antifungal Activity.

: Eysenhardtia platycarpa (Fabaceae) is a medicinal plant used in México and it lacks biotechnological studies for its use. The aim of this work was to establish a cell suspension cultures (CSC) of E. platycarpa , determine the phytochemical profile, and evaluate its antifungal activity. Friable callus and CSC were established with 2 mg/L 1-naphthaleneacetic acid plus 0.1 mg/L kinetin. The highest total phenolics of CSC was 15.6 mg GAE/g dry weight and the total flavonoids content ranged from 56.2 to 104.1 µg QE/g dry weight. CG‒MS analysis showed that the dichloromethane extracts of CSC, sapwood and heartwood have a high amount of hexadecanoic acid (22.3 ‒ 35.3 %) and steroids (13.5 ‒ 14.7%). Heartwood and sapwood defatted hexane extracts have the highest amount of stigmasterol (≈ 23.4%) and β -sitosterol (≈ 43%), and leaf extracts presented β -amyrin (16.3%). Methanolic leaves extracts showed mostly sugars and some polyols, mainly D-pinitol (74.3%). Dichloromethane and fatty hexane extracts of CSC exhibited the percentages inhibition higher for Sclerotium cepivorum with 71.5 and 62.0%, respectively. The maximum inhibition for Rhizoctonia solani was with fatty hexane extracts of the sapwood (51.4%). Our study suggest that CSC extracts could be used as a possible complementary alternative to synthetic fungicides. 60 calculated based on the calibration curve of gallic acid at concentrations of 0 to 50 mg/L. Samples of methanolic extracts The results were expressed in terms of gallic acid equivalents (GAE) in mg/g of dry biomass (DW). at 510 nm. The calibration curve was performed with standard using concentrations of 100 to 1600 µg/mL. The results were expressed in terms quercetin equivalents (QE) in µg/g of dry biomass (DW).

cell culture provides a renewable source of natural products, since plant cell culture can be produced and harvested at all times of the year [3,4]. In fact, some plants extract or compounds such as ajmalicine, anthraquinones, berberine, caffeic acid, ginsenoside, nicotine, rosmarinic acid, shikonin have been produced using the plant cell, tissues, and organ culture [5].
In this regard, Eysenhardtia platycarpa (Fabaceae), is a plant with a wide variety of uses such as manufacture of utensils, furniture, as well in traditional Mexican medicine [7,8,9]. Among the few existing investigations for E. platycarpa, all of them have used the wild plant as a source of pharmacological studies [10]. For instance, flavonoids with cytotoxic and antibacterial activity were isolated from methanolic extracts of branches and leaves of E. platycarpa [11]. The in vivo antiinflammatory activity of flavones isolated from the leaves has also been reported [12]. However, no effort has been made to carry out studies aimed at sustainable use of this species. In a biotechnological study developed for Eysenhardtia polystachya (a closely related species), the antifungal activity of cell suspension culture extracts was reported against R. solani and S. cepivorum [13]. To our knowledge, however, there are no reports using biotechnological studies of E. platycarpa. Therefore, it is necessary to look for biotechnological techniques that allow to obtain bioactive extracts while preserving natural diversity and the environment.
The main aim of this study was to establish a cell suspension culture from E. platycarpa internodal segments and characterize their growth. We also characterized the CSC and intact plant extracts by gas chromatography-mass spectrometry. Further, the biological activity of the extracts against the phytopathogenic fungi Rhizoctonia solani and Sclorotium cepivorum was explored.

Plantlets Obtainment and Callus Induction
In vitro culture of Eysenhardtia platycarpa are shown in Figure 1. Seeds had 98% germination under in vitro conditions after 10 days of culture. The in vitro plants grew easily without plant growth regulators after being transferred to 1 L jars. (Figure 1a). In another Fabaceae species such as Prosopis laevigata, the in vitro germination can occur from 3 to 7 days in mechanically scarified seeds [14,15]. Regarding callus induction, a suitable response of callus formation was exhibited on internodal segments at 15 days of culture ( Figure 1b). All treatments had percentages of callus induction greater than 50.0%, regardless of the type of auxins NAA and 2,4-D or cytokinin KIN (Table 1).  The control (PGRs-free) showed callus formation in 50% of segments, but it exhibited scarce growth, showed a brown color, and later died. It is possible that E. platycarpa contains amounts of auxin in their leaves and stems, which, may have caused that even in the control treatment the segments formed callus. In fact, it has been reported that indoleacetic acid (IAA) occurs naturally in plants, mainly in young leaves or seeds [16,17]. Moreover, it is known that levels of naturally occurring auxin in explant tissues depend on the mother plant from which the explants were taken [18].
In internodal segments of E. platycarpa, all evaluated treatments showed a positive effect on callus formation; 62.0% of the treatments with NAA and KIN formed callus on 100% of the segments, mainly with NAA (1.0 or 2.0 mg/L), regardless KIN concentration. Callus of this treatment were clear greenish and more friable in appearance than the other treatments; moreover, callus had a homogeneous growth during all subcultures. The other treatments exhibited smaller and semicompact callus, and most of them did not show growth. In the case of treatments with 2,4-D plus KIN, it was observed that as the concentration of 2,4-D increased, there was a trend in increasing the percentage of callus induction and this occurred generally in presence of KIN (Table 1). Similarly, treatments with 2,4-D (1.0 or 2.0 mg/L) combined with KIN (0.1 or 2.0 mg/L) had 100% of explants with callus. The remaining treatments, including control, showed calluses with percentages between 50.0 and 94.0%. Calluses of the best treatments with both auxins, i.e., NAA (2.0 mg/L) with KIN (0.1 mg/L) or 2,4-D (2.0 mg/L) with KIN (0.1 mg/L) were subcultured periodically for 6 months to increase callus production.
In a study reported for E. polystachya was found that callus induction was variable, according to the PGRs, i.e., percentages of callus between 65.6 and 98.4% in leaf explants were obtained with picloram (PIC) plus KIN, and from 64.1 to 100% with NAA plus KIN [13]. Studies carried out in P. laevigata, callus from cotyledons, hypocotyls and roots explant were obtained with 2,4-D plus 6-benzylaminopurine (BAP) or KIN, in percentages of 28.0 to 100% [15]. This indicates that combining both, auxins and cytokinins play an indispensable role in inducing and increasing the percentage of calluses [19].

Growth Kinetics and Sucrose Consumption
Callus induced with NAA (2.0 mg L) and KIN (0.1 mg/L) or 2,4-D (2.0 mg L) and KIN (0.1 mg L) were the best treatments showing friable characteristics and they were used to initiate the establishment of the cell suspension cultures (CSC) of E. platycarpa with the same plant growth regulators; however, during one month of culture, cells cultured in MS liquid medium with 2,4-D (2.0 mg/L) and KIN (0.1 mg/L) had poor growth; therefore, this treatment was discarded from the experiment. In contrast, cell cultured with NAA (2.0 mg/L) and KIN (0.1 mg L) exhibited growth and an abundant accumulation of biomass was observed from day 10 of growth kinetics (Figures 1c-e,2). Another species such as P. laevigata, the treatment with 2,4-D (1.5 mg/L) and KIN (1.0 mg/L) was the most suitable for establishing the CSC [15]. It is known that response to callus induction treatments on semisolid culture medium, are not always the ones that best adapt to the liquid culture medium. For instance, P. laevigata leaf explants presented 100% of callus with trichlorophenoxyacetic acid (2,4,5-T, 1.28 mg/L) with BAP (1.13 mg/L) or 2,4,5-T (1.28 mg/L) with KIN (1.08 mg/L); however, only callus containing KIN were the most suitable for establishing the CSC [14]. This indicates that even among closely related species, the genotype is an influential factor in the response of cell cultures; moreover, the PGRs activity varies depending on the presence of transporter or receptor biosynthesized proteins in the explants, affecting in vitro culture development [20].
The growth kinetics of E. platycarpa was maintained for 18 days, during which it exhibited typical growth ( Figure 2). The lag phase lasted 4 days, the exponential phase was 6 days. The stationary phase was observed between 10 and 12 days; then, the senescence phase was gradually observed. In addition, the maximum accumulation of biomass dry weight (18.62 g/L DW) occurred at 10 days. The specific growth rate (µ) was 0.206, the doubling time (td) was 3.36 days, and the growth rate was 5.35, obtaining a yield of 0.621 g dry biomass/g sucrose.
In another study conducted in E. polystachya, the maximum accumulation of dry biomass was 14 g/L at 10 days of culture [13]. Biomass yield was lower than that reported in the present study for E platycarpa at the same time. Studies conducted by Maldonado-Magaña et al. [15] in P. laevigata also found a dry biomass yield of 15.6 g/L at 21 days of culture. During the adaptation phase of E. Biomass Sucrose platycarpa, the amount of sugars remained unchanged, however, in the exponential growth phase there was a greater demand for sugars and, consequently, a higher production of biomass. From day 12, the sugars in the culture medium almost completely consumed and coincided with the stationary phase and the senescence phase. This behavior is also reported in other species such as C. brasiliense cell suspension cultures [21].

Total Phenolics and Flavonoids Content
It has been reported that phenolics (TPH), flavonoids (TFL) and other compounds of plant extracts are effective against phytopathogenic fungi [22,23]; therefore, it is important to quantify these groups of compounds in the extracts. The TPH and TFL content from E. platycarpa CSC had low variation during culture growth (Figure 3). During the adaptation phase of the culture, there was a notable increase in TPH (15.6 mg GAE/g DW) and then it remained constant, showing amounts between 5.2±0.63 and 6.2 mg GAE/g DW. Similar studies reported by Giri et al. [24] found a concentration of 10.17 mg GAE/g DW at 28 days of culture of Habenaria edgeworthii. In this work, the TPH content of E. platycarpa leaves (11.77 mg GAE/g DW), was lower than cell suspension cultures on day 2; however, during this stage biomass production is low.
Regarding the TFL content, this also remained constant between day 2 and 14, except the day zero was 104.1 µg QE/g DW. Near to the end of the senescence phase (day 16), the TFL decreased at 56.2 µg QE/g DW (Figure 3). On the other hand, low concentrations of TFL were also found in wild plant leaves (88.2 µg QE/g DW). Studies conducted in Saussurea medusa (Maxim) cell suspension cultures the total flavonoid production was 607.8 mg/L after 15 days of culture [25]. In other species such as Clinacanthus nutans, large amount of total polyphenols, phenolic acids and flavonoids contents were found on in vitro cultures compared with a conventional cultures [6].

Yield of Extracts of Cell Suspension Culture and Intact Plant
In general, the methanolic and dichloromethane extracts exhibited the highest dry weight yields (Table 2). This coincides with that reported in related species such as E. polystachya, in which the highest yields were obtained with methanol and dichloromethane [26]. In Tectona grandis yields of 2.9, 2.3 and 3.6% were reported, through a sequential extraction with hexane, ethyl acetate and , also report the best yields of extracts using these solvents [28]. For E. platycarpa, the yield of the methanolic extract of the heartwood was 36.9% and 30.08% for leaf, being the highest yields (Table 2). In another study conducted for C. sappan L., methanolic extracts of heartwood and leaf showed higher yields with 17.60 and 17.05%, respectively [29].

Compounds Identified by GC-MS
Fabaceae family produce a high diversity of bioactive compounds as defense against bacterial and fungi [30]. In the literature, few studies have been reported on determination of intact plant compounds of E. platycarpa; however, there are no studies on the phytochemical profile of its cell culture extracts.
By using the gas chromatography-mass spectrometry (GC-MS) analysis, the phytochemical profile of the hexane, dichloromethane and methanolic extracts of the cell suspension cultures, sapwood, heartwood and leaves of the intact plant of E. platycarpa were determined.
Chromatograms with main compounds identified by GC-MS are in Figures S1-S16 and their mass spectra are in Figures S17-S29.

Antifungal Activity 45
As an assay of biological activity, we evaluate the antifungal potential of the extracts on 46 phytopathogenic fungi available in our laboratory (Rhizoctonia solani and Sclerotium cepivorum), which 47 have the broad host range and cause significant losses on crop quantity and quality of many crop 48 species. In a previous study, we reported the antifungal activity of extracts of Eysenhardtia polystachya 49 [13], a species close to E. platycarpa. In the current work, it was found that the antifungal activity of E. 50 platycarpa extracts were statistically significant (p = 0.05) on the mycelial growth of R. solani and S. 51 cepivorum (Figure 6,7). 52  showed 62.0% inhibition for S. cepivorum (Figure 6a). The other fatty hexane extracts had values lower 61 than 25.0% inhibition. In the case of defatted hexane extracts, the inhibition percentages for R. solani 62 were low (less than 22%), while the values for S. cepivorum ranged from 22 to 35% (Figure 6b). The 63 Cercobin fungicide did not affect the growth of R. solani but was efficient in inhibiting the growth of 64 S. cepivorum. In a previous study, we reported the antifungal activity of E. polystachya extracts, in 65 which, the defatted hexane extract of CSC showed 66.0% inhibition for R. solani and it was also higher 66 than the Cercobin, while the fatty hexane extracts had low inhibition [13]. 67 Regarding the dichloromethane extracts, only the cell suspension cultures extract showed 68 moderate inhibition on R. solani (36.0%) compared with Cercobin (9.0%) (Figure 7a). 69

74
In contrast, the dichloromethane extract from cell suspension culture showed the most effective 75 inhibition against S. cepivorum (71.5%), followed by heartwood (55.2%) and leaves extracts (45.9%) 76 (Figure 7a). In a study carried out for E. polystachya we also report that dichloromethane extracts of 77 sapwood and heartwood inhibited the growth of S. cepivorum by 73.0 and 80.0%, respectively [13].

78
On the other hand, the methanolic extracts from E. platycarpa had low inhibition compared with 79 Cercobin. Despite, CSC extracts showed the maximum percentage of inhibition for S. cepivorum with 80 44.2% (Figure 7b).

81
Many plants have been reported to inhibit the in vitro growth of phytopathogenic fungi, which 82 promise to be better than commercial fungicides [32,33]. It is possible that the growth inhibition of R. solani and S. cepivorum with the fatty hexane extract of the sapwood and CSC (Figure 6a) is due to a 84 synergism between saturated fatty acids, saturated diacids and steroids, since these are in greater 85 amount in this extract (Figure 4a, Table 3). In fact, the defatted hexanic extracts also contain saturated 86 fatty acids and high amount of steroids, but scarce amount of saturated diacids compared with fatty 87 hexane extract. Therefore, there was also a decrease in the inhibition of fungal growth (Figure 6b). 88 Several free fatty acids (lauric acid, myristic acid, palmitic acid, oleic acid, and linoleic acid) are 89 known to have an inhibitory effect on fungal germination, mycelial growth, and sporulation [34,35]. 90 The possible mechanisms of antifungal activity have been previously studied and focused on fungal 91 membrane disruption causing an increase in membrane fluidity, causing leakage of the intracellular 92 components and cell death [36]   After disinfection, the seeds were rinsed three times with sterile distilled water in a horizontal 126 laminar flow cabinet (CFLH-90E, Novatech). 127 The disinfected seeds were sown in MS culture medium [46]) supplemented with 3% sucrose 128 (w/v) (Sigma-Aldrich, St. Louis, MO, USA) and only half of the macronutrients were used. Culture 129 medium was adjusted to pH 5.8 and then gelled with 2 g/L of Phytagel ® (Sigma-Aldrich). The culture 130 medium was transferred to Gerber flasks of 100 mL capacity and were sterilized in a manual 131 autoclave (CV300-A, AESA) at 121 °C, 15 psi, for 18 min. Four disinfected seeds were placed in jars 132 type Gerber containing 25 mL of MS medium. 133 All cultures were incubated at 25 ± 2 °C under 16-h photoperiod of white fluorescent light with 134 a light intensity of 60 µmol/m 2 /s. Eight days after germination, the plantlets were transferred to flask 135 of 1 L volume capacity, containing 80 mL of MS culture medium. Plantlets were used for 136 subsequently experiments of callus induction. biomass contained in three flasks were harvested every two days, filtered, and washed with distilled 157 water to remove excess of the culture medium. The biomass was dried in an oven at 50 °C until 158 constant weight. The experiment was repeated twice, and the biomass dry weight (DW) data were 159 used to plot the growth curve. 160

Sucrose Consumption Determination 161
In addition, the culture medium filtered from the CSC of the growth kinetics were used to 162 determine the total sugars. For each sampling, three flasks were harvested, an aliquot of 5 mL was 163 taken for each flask (n = 3) and were stored in freezing until analysis by the phenol-sulfuric method 164 [47]. Aliquot of 250 µL culture medium was diluted in distilled water (1:400); then, 500 µL aliquot 165 was taken and 500 µL of phenol (5%) was added; subsequently, 2.5 mL of concentrated sulfuric acid 166 were added. The sample was vigorously mixed for 3 s and allowed to react at room temperature for 167 30 min. Samples were read in a spectrophotometer at 490 nm using distilled water as a blank. To 168 carry out the calibration curve, sucrose was used as a standard at concentrations of 1-40 g/L. of 500 µL methanolic extract was mixed with 125 µL of FC and then 125 µL Na2CO3 (20% w/v) was 177 added. The mixture was supplemented with distillated water up to 2 mL total volume. The reaction 178 was maintained at room temperature for 60 min under dark conditions. TPH were calculated based 179 on the calibration curve of gallic acid at concentrations of 0 to 50 mg/L. Samples of methanolic extracts 180 from leaves and CSC were analyzed in a Varian Cary® 50 UV-Vis spectrophotometer at 765 nm. The 181 results were expressed in terms of gallic acid equivalents (GAE) in mg/g of dry biomass (DW). 182 Total flavonoids content (TFL) were quantified with the aluminum chloride colorimetric method 183 [49]. An aliquot of methanolic extracts (240 µL) was mixed with 1.50 mL of distilled water, then 90 184 µL NaNO2 (5%) was added and allowed to react for 6 min under dark conditions. After the reaction, 185 180 µL AlCl3 (10%) was added to the mixture and stirred vigorously. After 5 min, 600 µL NaOH (1 186 M) was added and brought to 3 mL final volume with distilled water. Samples were analyzed in a 187 Varian Cary® 50 UV-Vis spectrophotometer at 510 nm. The calibration curve was performed with 188 quercetin as standard using concentrations of 100 to 1600 µg/mL. The results were expressed in terms 189 of quercetin equivalents (QE) in µg/g of dry biomass (DW). temperature was set at 100 °C (1 min), rate of 10 °C/min up to 150 °C, rate of 3 °C/min up to 300 °C (4 209 min). Helium was used as the carrier gas with a flow rate of 1.0 mL/min and the injector temperature 210 was set at 250 °C. Compounds were identified as TMS derivatives by comparing their mass spectra 211 with the NIST library version 1.7a and by comparing their fragmentation patterns with published 212 data [50,51]). For semiquantitative analysis, peaks were integrated using a GC ChemStation software 213 version C.00.01. The composition was reported as a percentage of the total peak area. 214

In Vitro Antifungal Evaluation of Extracts 215
The phytopathogenic fungi R. solani and S. cepivorum were provided from Colección del 216 Laboratorio de Patología, Departamento de Producción Agrícola (Universidad de Guadalajara). 217 Seven-day-old strains grown on potato dextrose agar (PDA, BD Bioxon) culture medium was used. 218 The antifungal evaluation was carried out according to the gar disk-diffusion method [52]. The 16 219 extracts were dissolved in 96% ethanol (1 mg/mL) and the 5 mm diameter filter paper discs (Whatman 220 No. 1) superposed on PDA culture medium were impregnated with 10 µL of each solution.

221
Cercobin® and ethanol were used as positive and negative controls, respectively. After applying 222 solutions extracts or controls on the discs, the solvent was allowed to evaporate for 1 h in a laminar 223 flow cabinet. Mycelium propagules (5 mm 3 ) were inoculated on discs treated into sterile polystyrene 224 Petri dishes (Interlux ® 90 x 15 mm) containing 10 mL of PDA medium and then incubated at 28 ± 2°C. 225 The results are reported as percentage inhibition (%) of mycelial growth at 72 h of cultures. 226

Statistical Analysis 227
The data corresponding to the percentage of callus induction, dry biomass of cell suspension 228 cultures, total phenolics and flavonoid content, and the inhibition percentage of the mycelial growth 229 were subjected to a normality test and then an analysis of variance (ANOVA) followed by Tukey's 230 multiple range test (p = 0.05). SAS 9.0 software (SAS Institute Inc.) was used for the statistical analysis. 231 All the experiments were conducted in triplicate. 232

Conclusions 233
The obtaining of a biotechnological culture of E. platycarpa, the phytochemical profile of cell 234 cultures and intact plant, and their antifungal activity are reported for the first time. The NAA auxin 235 was the most suitable to establish callus and CSC, producing 18.62 g dry biomass/L. Hexane extracts 236 exhibited high amounts of saturated fatty acids, mainly hexadecanoic acid in CSC. Although the 237 methanolic extracts had the best yields, they did not show the best antifungal activity. 238 Dichloromethane extract of CSC showed greater effectiveness to inhibit the in vitro growth for S. 239 cepivorum (62.0%). The fatty hexane extract of the sapwood had better inhibition of R. solani (51%), 240 but in the dichloromethane extracts, CCS was the only one that inhibited R. solani. In this paper we 241 demonstrated that CSC extracts could be used as an alternative to synthetic fungicides to control 242 phytopathogenic fungi as R. solani and S. cepivorum, however, further studies are needed.

273
Conflicts of Interest: The authors declare no conflict of interest.