New triterpenic acids produced in callus culture from fruit pulp of Acca sellowiana (O. Berg) Burret

A B S T R A C T
The aim of this work was the study of the best conditions for obtaining a callus culture from the pulp of Acca sellowiana, and to perform a quali-quantitative analysis of the secondary metabolites yielded by the in vitro callus culture. To this end, callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of growth regulators. Three previously undescribed ursane-type triterpenoids, 2α,3β,6α,23-tetrahydroXy-18α,19α-urs-20-en-28-oic acid, 2α,3β,23-trihydroXy-18α,19α-urs-20-en-28-oic acid and 2α,3β,6β,23-tetrahydroXy-18α,19α-urs-20-en-28-oic acid were isolated from the methanolic extract of A. sellowiana culture and characterized by 1D and 2D NMR experiments. Moreover, the quali-quantitative analysis (ESI-MSn and GC–MS) also showed the presence of β-sitosterol, phloridzin, oleanolic, ursolic, 3β-hydroXy- 18α,19α-urs-20-en-28-oic, maslinic, corosolic, 2α,3β-dihydroXy-18α,19α-urs-20-en-28-oic, and tormentic acid.

1.Introduction
It is known that fruits and vegetables contain many compounds, such as polyphenols, thiols, carotenoids, tocopherols, glucosinolates, and triterpenic acids, which have several beneficial health properties. In particular, triterpenic acids may protect against cardiovascular dis- ease, cancer, inflammation and cataracts (Ayeleso, Matumba, & Mukwevho, 2017; Giomaro et al., 2014; Laszczyk, 2009; Lee, Chen, Lin, & Chuang, 2018). Acca sellowiana (O. Berg) Burret (Myrtaceae), better known by its former name, Feijoa sellowiana (O. Berg) O. Berg, is a perennial evergreen shrub of the Myrtaceae family, originating from South America and growing in the subtropical zone. A food crop with good medicinal and nutritional value, A. sellowiana has many potential applications in the food, nutraceutical, pharmacological and cosmetic industries (Clerici & Carvalho-Silva, 2011). A. sellowiana essential oils from the skin and pulp show a broad spectrum of anti-microbial ac- tivity, and it can also be used as a food flavouring agent (Saj, Roy, & Savitha, 2008). Some authors reported that the A. sellowiana fruit acetonic extract exerted anticancer activities (Bontempo et al., 2007) and anti-inflammatory activity (Rossi et al., 2007).Today, many studies are being conducted using plant cell and organ cultures of different plants with the aim of producing health promoting secondary metabolites (Ahmad et al., 2014; Gawri, Singh, Shukla, & Upadhyay, 2013; Mihai, Mitoi, Brezeanu, & Cogalnicenu, 2010; Saradha, Ranjitham, & Paulsamy, 2014; Tabata et al., 1993; Zahedzadeh, Kakavand, & Mahna, 2015). The most commonly used plant materials for initiating in vitro cultures are mainly composed of the leaf, cotyledons, node, internode and petiole of the plants. Fruit pulp has rarely been used as a starting material for in vitro cultures for obtaining secondary metabolites, with only a few examples appearing in the literature (Nawa & Ohtani, 1992; Oota, Masuda, & Tamura, 1983). Even in the case of A. sellowiana, several authors have reported in vitro culture protocols starting from different plant organs (Cangahuala-Inocente, Dal Vesco, Steinmacher, Torres, & Guerra, 2007; Reis, Batista, & Canhoto, 2008; Stefanello, Dal Vesco, Ducroquet, Nodari, & Guerra, 2005), but none of these authors has used the fruit pulp.

Our research team recently published a work describing a method to obtain high biomass production from the pulp explants of apples. The results showed that apple pulps produced higher amounts of bioactive triterpenic acids in vitro than in vivo (Verardo, Gorassini, Ricci, & Fraternale, 2017). Considering these interesting results and the nutri- tional properties of A. sellowiana fruit, we have investigated the pro- duction of bioactive compounds of the in vitro callus cultures from the pulp of this fruit. Therefore, the scope of this study was to optimize the conditions for obtaining the highest biomass production of calluses from the pulp explants of A. sellowiana and subsequently, to analyze and identify the secondary metabolites eventually produced with particular attention to triterpenic acids. In fact, considering their biological ac- tivities (Ayeleso et al., 2017; Laszczyk, 2009), these compounds could have interesting nutritional value, functional properties and nu- traceutical applications.

2.Materials and methods
EXtraction and derivatization solvents were of analytical grade and were obtained from Sigma-Aldrich (Milan, Italy). LC-MS grade MeOH used for direct infusion experiments, cholesterol, phloridzin, β-sitos-terol, ursolic and oleanolic acids, used as standards, Sylon BFT [Bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% of trimethyl- chlorosilane (TMCS)], employed as silylating reagent, 6-benzylamino- purine (BA), 1-naphthaleneacetic acid (NAA), and agar were also ob- tained from Sigma-Aldrich. For solid phase extraction (SPE), ISOLUTE silica gel cartridges (1 g, 6 mL; StepBio, Bologna, Italy) were used.Electrospray ionization mass spectra (ESI-MS) and multi-stage mass spectrometry (MSn) experiments were performed with a Finnigan LXQ Linear Ion Trap (Thermo Scientific, San Jose, CA, USA) operating in the negative ion mode. A MeOH solution of each extract sample was properly diluted and infused into the ion source at a flow rate of 10 μL min−1 with the aid of a syringe pump. The typical source con-
ditions were transfer line capillary at 275 °C; ion spray voltage at 4.70 kV; sheath, auXiliary and sweep gas (N2) flow rates at 20, 5 and 0 arbitrary units, respectively. Helium was used as the collision damping gas in the ion trap set at a pressure of 1 mTorr. ESI-MSn spectra were obtained by collision induced dissociation (CID) experiments after isolation of the appropriate precursor ions in the ion trap (isolation width 1.2 m/z unit), and subjecting them to the following typical con- ditions: normalized collision energy between 20% and 30%, selected to preserve a signal of the precursor ion on the order of 5%; activation Q 0.25 and activation time 30 ms. Spectra were averages of 10 scans. GC–MS analyses were carried out using a Trace GC Ultra gas chro-matograph coupled to an ion-trap mass spectrometer (ITMS) detector Polaris Q (Thermo Scientific) and equipped with a split-splitless in- jector. The column was a 30 m × 0.25 mm i.d., 0.1 μm film thickness, fused silica SLB-5 ms (Supelco, Sigma-Aldrich). The initial oven tem- perature was 240 °C programmed to 280 °C at 2 °C min−1 and kept at 280 °C for 10 min, the temperature was then raised to 310 °C at a rate of 10 °C min−1 and maintained at this temperature for 20 min. Samples (1 μL) were injected in the split (1:10) mode. Injector, transfer line and ion source were set at 280, 280 and 200 °C, respectively. Helium was used as carrier gas at a flow of 1 mL min−1. The mass spectra were
recorded under electron impact at 70 eV electron energy with a mass range from m/z 50 to 1000 and a scan rate of 0.8 scan sec−1. Quantification of metabolites in full scan mode was carried out via the total ion current (TIC) peak areas according to the internal standard method. The data acquisition was under the control of Xcalibur soft- ware (Thermo Scientific).1D and 2D NMR spectra were recorded on a Bruker Avance III 600 MHz equipped with a TCI cryoprobe (Bruker Biospin, Milan, Italy) operating at 600 and 150 MHz for 1H and 13C, respectively, using acetone‑d6 as solvent. NMR peak locations were reported as δ values from TMS. NMR assignments were performed using HSQC and HMBC experiments. Optical rotations were determined on suitable solutions (g 100 mL−1) at 23 °C using an ATAGO AP-300 automatic polarimeter.Infrared (IR) spectra were obtained with a Bruker Vector 22 spec- trophotometer. Elemental analyses were performed with a FlashEA 1112 Series CHNS-O elemental analyzer (Thermo Scientific).

Mature pulp of A. sellowiana (O. Berg) Burret (Myrtaceae) fruit was used as the source of explants for callus induction. Mature fruits were randomly collected in October 2015 in autumn during a period of mild temperatures and little rain from plants growing in an open field near Pordenone (PN), Friuli-Venezia Giulia Region, Italy, global positioning system (GPS) coordinates: latitude 45.9669348, longitude 12.6408589, altitude 104 m at sea level.The fruits were first sterilized by washing their surface with ethanol. They were then flamed in the laminar flow cabinet and opened with a sterile blade. Dishes of 5 mm diameter and about 3 mm thick were excised from fruit pulp with a cork borer about 0.5 cm from the peel (Verardo et al., 2017).
Cultures were grown on Murashige and Skoog (MS; Murashige & Skoog, 1962) and Gamborg B5 (B5; DiXon, 1985) media, both con- taining sucrose (30.0 g L−1) and supplemented with 6-benzylamino- purine (BA, 0.89 μM) plus 1-naphthaleneacetic acid (NAA, 10.7 μM) or 0.89 μM BA alone or 10.7 μM NAA alone (Kondo, Tsukada, & Niimi,2001). Media were adjusted to pH 5.8 prior to adding agar (0.8%), autoclaved (120 °C, 104 KPa) for 20 min and dispensed into 90 mm Petri dishes (30 mL of medium per Petri dish). Three dishes containing five explants each were prepared per treatment. Each experiment was performed in triplicate. Cultures were incubated in the dark at 25 ± 2 °C. Subcultures were grown after 28 days in the same media.
Callus formation was recorded after two months of culture in ac- cordance with Simoes et al. (2009) on the basis of fresh weight (FW) and dry weight (DW) of the obtained callus in each experimental con- dition. Data concerning callus biomass production are reported as mean ± standard deviation of FW and DW in all culture conditions tested, and were analyzed using one-way analysis of variance (ANOVA). The Tukey test at 5% level of significance was used to test the difference among means and statistical software MSTAT-C was employed. The optimal conditions for obtaining the callus from the pulp of A. sell- owiana fruit were maintained using the same culture conditions de- scribed above with subcultures every 28 days. The obtained callus (28 days) was stored at −80 °C, freeze-dried, and ground in a pestle mortar prior to chemical analysis.

Powdered callus culture of A. sellowiana (1.0 g) was extracted in MeOH (80 mL) under magnetic stirring for 1 h at room temperature, followed by centrifugation at 5000 rpm for 8 min. The supernatant was collected in a flask and the residue was extracted once again in the same manner. All the supernatants were pooled and concentrated in vacuo at 40 °C. The residue was suspended in water (20 mL) and extracted with EtOAc (60 mL × 3). The collected organic phases were washed with H2O (25 mL × 3) and brine (15 mL) then dried (Na2SO4 anhydrous), filtered, and evaporated to dryness in vacuo. The residue (ca. 70 mg) was dissolved in CH2Cl2 (2 mL), subjected to SPE fractionation and each collected fraction (1.5 mL) underwent ESI-MS analysis after dilution in MeOH. Before sample loading, the cartridge was conditioned with CH2Cl2 (15 mL). The elution was carried out using the following step gradient of CH2Cl2-EtOAc (v/v): 100:0 (9 mL, discarded), 95:5 (9 mL, discarded), 70:30 (6 mL, discarded), 70:30 (3 mL) and 50:50 (3 mL)
containing compound 8, 50:50 (6 mL, discarded), 20:80 (4.5 mL, dis-carded), 20:80 (4.5 mL) containing compound 10, 0:100 (3 mL, discarded), 0:100 (4.5 mL) containing mainly compound 9, 0:100 (3.0 mL, discarded) containing both compounds 9 and 12, 0:100 (4.5 mL) con- taining mainly compound 12. The GC–MS analyses of the fractions containing compounds 9 and 12 allowed us to determine their relative amounts.This procedure was repeated four more times, always using 1.0 g of callus culture of A. sellowiana. At the end, the fractions containing the same compound were combined affording compound 8 (6.8 mg), 9(1.5 mg) containing ca. 38% of 12, 10 (4.1 mg) and 12 (1.2 mg) con- taining ca. 35% of 9. Due to the small amount of the sample, no further purifications of compounds 9 and 12 were attempted.

Quantification of the analytes in the callus culture was performed using the internal standard method based on the relative peak area of analyte to IS (cholesterol) from the average of two replicate measure- ments. A calibration curve was constructed for each corresponding standard compound in the extracts. For this purpose, 10.0 mg of each available standard (phloridzin, β-sitosterol, ursolic acid and oleanolic acid) was weighed and transferred to a 50 mL volumetric flask and diluted to volume with EtOAc in order to yield a standard solution at a concentration of 200 μg mL−1. Further dilutions were made, with the same solvent, to yield working solutions at 0.5, 1, 2, 4 and 8 μg mL−1 with a constant concentration of the IS (cholesterol, 40.0 μg mL−1). These solutions were analyzed in triplicate by GC–MS in order to cal- culate the calibration curve for each standard. The calibration curves were constructed by plotting the ratio of peak area of analyte/peak area of IS versus the corresponding concentration ratio at five points (0.5, 1, 2, 4 and 8 μg mL−1) using the least squares regression method. The calibration curves obtained had a high level of linearity with a corre- lation coefficient (r2) higher than 0.999 for all analytes.When standards were unavailable, the quantification of the target analyte was carried out using the calibration curve of available stan- dard of a similar chemical structure.

3.Results and discussion
A. sellowiana fruit pulp explants formed cream-coloured friable calluses on the upper face and along the cutting surface in both B5 and
MS culture media containing BA (0.89 μM) and NAA (10.7 μM) or NAA (10.7 μM) alone. Both the media containing BA (0.89 μM) alone do not induce the formation of calluses, and the explants turn brown and die after about 10 days of culture. Moreover, the combination of B5 medium with 0.89 μM BA and 10.7 μM NAA induced the highest bio- mass production of calluses from pulp explants of A. sellowiana (Table 1). These plant growth combinations were maintained, and subcultures were grown every 28 days to obtain in vitro plant material for extractions and analysis. The calluses obtained in MS and B5 media containing NAA alone grew with difficulty and could not be used for biomass production because after the first subculture, they turned brown and died. Indeed, we found that the presence of cytokinin BA is essential for the maintenance of callus culture from fruit pulp (Kondo et al., 2001; Verardo et al., 2017; Wang, Yan, & Wang, 2014).To our knowledge, this is the first time that callus culture has been obtained from the pulp of A. sellowiana fruit showed quasi-molecular ions at m/z 179 [M – H]−, 215 and 217 [M + Cl]− derived from glucose and/or fructose as confirmed by MSn experiments and comparison with authentic specimens. The second zone was characterized by the [M – H]− ions at m/z 455, 471, 487 and
503. A comparison of the fragmentation pattern (MSn).

Investigations of A. sellowiana fruit composition have revealed that, in addition to high amounts of vitamin C (Basile et al., 1997; Romero Rodriguez, Vazquez Oderiz, Lopez Hernandez, & Simal Lozano, 1992; Romero Rodriguez, Vazquez Oderiz, Lopez Hernandez, & Simal Lozano, 1994; Salvo, Toscano, & Dugo, 1987), iodine (Basile et al., 1997) and minerals (Romero et al., 1994; Salvo et al., 1987), A. sellowiana is rich in phenolic compounds such as catechins, proanthocyanidins, leu- coanthocyanins, flavonols, naphtoquinones, and tannins (Basile, Conte, Rigano, Senatore, & Sorbo, 2010; Foo & Porter, 1981; Ielpo et al., 2000; Weston, 2010).The extraction of secondary metabolites yielded by the A. sellowiana pulp cell culture was carried out in MeOH as it was in our previous study (Verardo et al., 2017). Unlike what was observed in the pulp, the preliminary ESI-MS analysis of the methanolic extract obtained from A. sellowiana pulp cell culture showed the presence of pentacyclic tri- terpene acids as the main compounds. The spectrum (Fig. 1) of the methanolic extract directly infused into the ESI source operating in the negative ion mode exhibited three zones: the first, from m/z 150 to 400,
suggesting the presence of a hydroXy-methylene group (Xia et al., 2015). As reported in our previous investigation (Verardo et al., 2017), the third zone of the spectrum showed the [M – H+ C6H12O6]− adduct ions formed between the triterpenic acids and a hexose at m/z 635, 651, 667, and 683. Finally, the ESI-MS spectrum showed a peak at m/z 435 [M – H]− identified as phloridzin by comparison with an authentic specimen.The identification of some of the secondary metabolites that were produced was performed by the GC–MS analysis of the corresponding trimethylsilyl derivatives. To this end, powdered culture material was extracted with MeOH under stirring at room temperature. To eliminate the coextracted sugars, MeOH was distilled off under reduced pressure, and the residue, suspended in H2O, was extracted with EtOAc, reduced to dryness and derivatized. The comparison of the mass spectra of the chromatographic peaks with those of standards and/or with those available in the literature (Budzikiewicz, Wilson, & Djerassi, 1963;Caligiani et al., 2013; Verardo et al., 2017) allowed the identification of the following compounds (Fig. 2): β-sitosterol (1), phloridzin (2),
oleanolic acid (3), ursolic acid (4), maslinic acid (6), corosolic acid (7) and tormentic acid (11). Compounds 8–10 and 12 were isolated by SPE (Silica-gel; mobile phase: CH2Cl2-EtOAc, gradient: from 100:0% to 0:100%) and their structures (Fig. 3) were established by a careful analysis of their 1D and 2D NMR (HSQC, HMBC, NOESY and 1H–1H-

Fig. 2. Typical total ion current (TIC) chromatogram obtained from the methanolic extract of A. sellowiana fruit pulp cell culture after elimination of sugars and trimethylsilyl derivatization. Peak numbers refer to the identified compounds.

Fig. 3. Structures of compounds 8–10 and 12. oCOSY) spectroscopic data.
The ESI-MS analysis of compound 8 showed a quasi-molecular ion at m/z 471 [M – H]−. The mass spectrum (GC–MS) of its trimethylsilyl derivative showed a molecular ion at m/z 688 and revealed the loss of two trimethylsilanol [(CH3)3SiOH] groups (2 × 90 u) and 118 u [HCOOSi(CH3)3], indicating the presence of two hydroXyls and a car-boXylic acid functionality. Moreover, the 1D and 2D NMR data of 8 matched well with those reported for Psiguanin A by Shao et al. (2012).On the basis of the above evidences, compound 8 was identified as 2α,3β-dihydroXy-18α,19α-urs-20-en-28-oic acid. A thorough discussion of the 2D NMR correlations observed for compound 8 can be found in the Supplementary material.The mass spectrum (GC–MS) of the trimethylsilyl derivative of compound 10 showed a molecular ion at m/z 776, which was 88 u [(CH3)3SiO – H] higher than the corresponding M+∙ of compound 8. This occurrence and the loss of 103 u [•CH2OSi(CH3)3] from the radical ion at m/z 686 [M – (CH3)3SiOH]+∙ observed in the mass spectrum of 10 point to the presence of three hydroXyl groups, one of which is linked to a methylene carbon (Fig. 3). Moreover, the similarity of the 1H and 13C NMR spectra of this compound (Table 2) to those of 8 (Sup- plementary material, Table 1S) show that compound 10 is a trihy- droXyurs-20-en-28-oic acid. Aided by HSQC experiment, the main dif- ferences were the disappearance of the methyl signal at δ 28.1 and the occurrence of a methylene group at δ 66.3. The methylene protons at δ 3.57 (d, J = 10.0 Hz) and 3.29 (d, J = 10.0 Hz) and the corresponding carbon at δ 66.3, together with the MS observations, indicated that this is a hydroXyl methylene group. In the HMBC spectrum of compound 10, these oXymethylene protons evidenced interactions with the carbon signals at δ 77.6 (C-3), 42.5 (C-4), 47.7 (C-5) and 12.7 (C-24). The NOESY spectrum of 10 showed the same interactions (Fig. 4) between H-3 (δ 3.40, d, J = 9.1 Hz) and H-5 (δ 1.31, m), as well as between H-25 (δ 0.99, s), H-2 (δ 3.69, m), H-24 (δ 0.73, s) and H-26 (δ 0.98, s), and between H-13 (δ, 2.50, ddd, J = 13.1, 9.8, 3.6), H-19 (δ 2.13, m) and H- 26 (δ 0.98, s) observed in compound 8 (Supplementary material, Fig. 2S). Therefore, the hydroXyl group was located at C-23 (δ 66.3) and the hydroXyl methylene moiety was α-equatorially oriented. The other correlations observed in the HMBC, 1He1H COSY and NOESY spectra of compound 10 were very similar (Table 2) to those of 8 (Supplementary material, Table 1S), suggesting the same rigid “all chair” structure with a trans junction between the five rings and with the C-26 and C-27 methyl groups, both axial, β- and α-oriented, respectively. Moreover, the C-29 methyl group at δ 22.9 and the carboXylic function C-28 (δ 175.9) were in equatorial and axial position, α- and β-oriented, re- spectively. Accordingly, the structure of 10 was identified as 2α,3β,23- trihydroXy-18α,19α-urs-20-en-28-oic acid. A quite similar compound was isolated by Yu, Duan, Gao, and Takaishi (2007) but, in this case, in the structure reported by these authors, the C-29 methyl group was β- oriented.

The molecular ion of the trimethylsilyl derivative of compound 9 appeared in the mass spectrum (GC–MS) at m/z 864, 88 u [(CH3)3SiO – H] higher than that of 10. Moreover, like compound 10, the mass spectrum of 9 also evidenced the loss of 103 u [•CH2OSi(CH3)3] from the radical ion at m/z 684 [M – 2 × (CH3)3SiOH]+∙, suggesting the presence of four hydroXyl groups, with one linked to a methylene carbon. The 1H, 13C and HSQC spectra of compound 9 showed that the methylene carbon present in 10 at δ 17.8 (C-6) disappeared and was replaced by a methine signal at δ 66.3 and the corresponding methylene protons H-6 at δ 1.48 (m) and 1.40 (m) were substituted by a methine signal at δ 4.00 (td, J = 10.6, 4.0 Hz), indicating the presence of a hydroXyl group at C-6 in compound 9 (Fig. 3). The two hydroXyl me- thine protons at δ 3.75 (m) (H-2) and 3.20 (d, J = 9.5 Hz) (H-3) showed the same HMBC and NOESY correlations observed in compounds 8 and 10 (Table 2, Fig. 4), suggesting that also in 9 the two hydroXyl groups
were located on C-2 (δ 68.4) and C-3 (δ 77.9), α- and β-equatorially oriented, respectively. The presence of a hydroXyl methylene group at δ
3.70 (d, J = 3.9 Hz), together with the corresponding carbon signal at δ 68.1 inferred from Phlorizin the HSQC experiment, confirmed the GC–MS results.