Production of Two Isomers of Sphaeralcic Acid in Hairy Roots from Sphaeralcea angustifolia

The Sphaeralcea angustifolia plant is used as an anti-inflammatory and gastrointestinal protector in Mexican traditional medicine. The immunomodulatory and anti-inflammatory effects have been attributed to scopoletin (1), tomentin (2), and sphaeralcic acid (3) isolated from cells in suspension cultures and identified in the aerial tissues of the wild plant. The hairy roots from S. angustifolia established by infecting internodes with Agrobacterium rhizogenes were explored to produce active compounds based on biosynthetic stability and their capacity to produce new compounds. Chemical analysis was resumed after 3 years in these transformed roots, SaTRN12.2 (line 1) produced scopoletin (0.0022 mg g−1) and sphaeralcic acid (0.22 mg g−1); instead, the SaTRN7.1 (line 2) only produced sphaeralcic acid (3.07 mg g−1). The sphaeralcic acid content was 85-fold higher than that reported for the cells in the suspension cultivated into flakes, and it was similar when the cells in suspension were cultivated in a stirring tank under nitrate restriction. Moreover, both hairy root lines produced stigmasterol (4) and β-sitosterol (5), as well as two new naphthoic derivates: iso-sphaeralcic acid (6) and 8-methyl-iso-sphaeralcic acid (7), which turned out to be isomers of sphaeralcic acid (3) and have not been reported. The dichloromethane–methanol extract from SaTRN7.1 hairy root line had a gastroprotective effect on an ulcer model in mice induced with ethanol.


Introduction
Sphaeralcea angustifolia (Cavanilles) G. Don (Malvaceae) is used as an anti-inflammatory and to treat gastrointestinal diseases in Mexican traditional medicine [1][2][3]. The extracts from the aerial tissues of this plant exhibited an anti-inflammatory activity in acute (auricular edema induced with phorbol ester-TPA) and polyarthritis inflammation (induced with Freund's complete adjuvant (FCA)) mouse models, with β-sitosterol, αand β-amyrin, trans-cinnamic acid, and scopoletin identified as the main active compounds. Subsequently, using an FCA model, the extract decreased the serum levels of pro-inflammatory interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) and increased the anti-inflammatory IL-10 levels in the synovial fluid of mice with untreated polyarthritis [4][5][6]. With these backgrounds, a gel formulation containing 1% S. angustifolia dichloromethane extract standardized in the scopoletin content was evaluated in patients with osteoarthritis of the hands. The therapeutic effectiveness and tolerability of this phytomedicine were approximately 90% [7]. Scopoletin has been isolated from many plants [8] and important pharmacological activities have been reported, namely: anti-inflammatory [9,10]; antioxidant [11,12]; inhibition of nuclear transcription factor-kappa β (NF-κβ) and inflammatory cytokine production; inhibition of pro-inflammatory mediators [13,14]; anti-angiogenic in an arthritis mouse model [15]; anti-proliferative [16]; and gastroprotective as a potential preventive and therapeutic agent for gastro-esophageal inflammation, mainly through its anti-secretory and prokinetic activities [17].
Cell suspension culture was employed as a feasible methodology to produce scopoletin, tomentin, and sphaeralcic acid ( Figure 1); scopoletin and sphaeralcic acid production was improved by the total nitrate reduction in the culture medium [18][19][20]. The production of sphaeralcic acid in the cell suspension culture was scaled up using a bioreactor in a stirred tank and nitrate restriction [21]. Tomentin and sphaeralcic acid were identified as potent anti-inflammatories in acute (edema induced with λ-carrageenan footpad and with TPA) and chronic (kaolin/λ-carrageenan-induced arthritis) mice models [18,22,23]. Similarly, both compounds modulated the production of pro-(IL-1β) and anti-inflammatory (IL-10 and IL-4) cytokines [22,23].
Cell suspension culture was employed as a feasible methodology to produce scopoletin, tomentin, and sphaeralcic acid ( Figure 1); scopoletin and sphaeralcic acid production was improved by the total nitrate reduction in the culture medium [18][19][20]. The production of sphaeralcic acid in the cell suspension culture was scaled up using a bioreactor in a stirred tank and nitrate restriction [21]. Tomentin and sphaeralcic acid were identified as potent anti-inflammatories in acute (edema induced with λ-carrageenan footpad and with TPA) and chronic (kaolin/λ-carrageenan-induced arthritis) mice models [18,22,23]. Similarly, both compounds modulated the production of pro-(IL-1β) and anti-inflammatory (IL-10 and IL-4) cytokines [22,23].
Scopoletin (1) Tomentin (2) Sphaeralcic acid (3) Stigmasterol (4) β-Sitosterol (5) R = H, Iso-sphaeralcic acid (6) R = CH3, 8-methyl-iso-sphaeralcic acid (7) Hairy root cultures from S. angustifolia were also established, mediated by node infection with Agrobacterium rhizogenes ATCC15834, to produce scopoletin and sphaeralcic acid [24]. The aim of this study was to characterize scopoletin and sphaeralcic acid production in the SaTRN12.2 and SaTRN7.1 hairy root lines from S. angustifolia after 3 years Hairy root cultures from S. angustifolia were also established, mediated by node infection with Agrobacterium rhizogenes ATCC15834, to produce scopoletin and sphaeralcic acid [24]. The aim of this study was to characterize scopoletin and sphaeralcic acid production in the SaTRN12.2 and SaTRN7.1 hairy root lines from S. angustifolia after 3 years in culture. In addition, it aimed to determine the chemical profiles of dichloromethane-methanol extracts via the purification and identification of other sec-Plants 2023, 12, 1090 3 of 12 ondary metabolites biosynthesized in these cultures; the sterols and sphaeralcic acid isomers were categorized as new. Moreover, we aimed to evaluate the anti-ulcerogenic effect of the dichloromethane-methanol extract of SaTRN7.1 hairy root line in the ulcer model in mice induced with ethanol; this extract comprised sphaeralcic acid and its two isomers, as well as scopoletin and β-sitosterol, which reported a gastro-protective effect.

S. angustifolia Hairy Root Cultures Scopoletin and Sphaeralcic Acid Production
Hairy root culture is a useful biotechnological system for producing scopoletin and sphaeralcic acid in S. angustifolia [24]. The two hair root lines previously established and chemically analyzed in this project were classified according to the production of active compounds (Table 1): one, SaTRN12.2 (line 1), as a producer of two active compounds, (Figure 1) scopoletin (0.0022 mg g −1 ) and sphaeralcic acid (0.22 mg g −1 ), this hairy root line had been reported to be a not producer by Reyes-Pérez et al. 2022 [24]; a second one, SaTRN7.1 (line 2), as a strong producer of sphaeralcic acid (3.07 mg g −1 ) at 10-fold higher levels according to the analysis previously reported (scopoletin, 0.011 mg g −1 and sphaeralcic acid,1.22 mg g −1 ) for this hairy root line [24]. The sphaeralcic acid yield in the hairy root line 2 was 85-fold higher than that reported for the cells in the suspension (0.0359 mg g −1 ) of S. angustifolia cultivated in the MS medium with nitrate restriction [19], and similar to that reported in the same biotechnological system (3.47 mg g −1 ) cultivated in a stirring tank bioreactor [21]. From the dichloromethane-methanol extract of SaTRN12.2 (line 1) of S. angustifolia, the thin-layer chromatography (TLC) analysis of sub-fraction SaL1F3R6 (12 mg) revealed the presence of sterols. The compounds stigmasterol (4) and β-sitosterol (5) were detected using ultra-performance liquid chromatography (UPLC, tuning) coupled to a mass spectrum (fast atom bombardment mass spectrometry (FAB-MS)); the molecular structure (C 29 Table 1). Stigmasterol (4) and β-sitosterol (5) were identified by a comparison of the 1 H and 13 C nuclear magnetic resonance (NMR) spectra (Table S1) with those reported in the literature [25]. Stigmasterol was identified in the dichloromethane extract from the aerial tissues of the S. angustifolia wild plant, and it was reported to have anti-inflammatory and immunomodulatory activities [6].
From the dichloromethane-methanol extract of the SaTRN7.1 hairy root (line 2), the compounds stigmasterol (4) and β-sitosterol (5) were detected (Table 1)  It was confirmed that one compound was stigmasterol C 29 H 48 O from its molecular weight of 412 g/mol, and another one was β-sitosterol C 29 H 50 O from its molecular weight of 414 g/mol. sub-fraction SaL2F2R4 (4 mg). It was confirmed that one compound was stigmasterol C29H48O from its molecular weight of 412 g/mol, and another one was β-sitosterol C29H50O from its molecular weight of 414 g/mol.
The development of a methodology to generate hairy root cultures represents a technological approach to produce metabolites of pharmacological importance, consid ering that the transformed roots have a higher growth rate and accumulate high levels o active compounds compared with the wild plants [26]. Oncogenes are essential modu lators of plant growth and cell differentiation, and their ability to improve secondary metabolite production in transformed cells has been described [27]. Four T-DNA role oncogenes (ORFs), called rolA, rolB, rolC, and rolD, are critical for the induction, growth and morphology of hairy roots in infected plants. The hairy root transformation of S angustifolia (SaTRN12.2, line 1, and SaTRN7.1, line 2) was previously confirmed by the presence of the rolC gene [24]. The rolC gene has an important role as a modulator of secondary metabolite production among medicinal plants [27].
Hairy root culture has many advantages for producing high-value metabolites because of their fast growth and biosynthetic stability for many successive generations [28][29][30]. In addition, this culture could produce new compounds not produced by the plant for example, from Lopezia racemose, the new compound identified was (23R)-2α,3β,23,28-tetrahydroxy-14,15-dehydrocampesterol [31] or cadaverin, an amine detected for the first time in the hairy roots of Brugmansia candida [32]. The hairy roo culture of S. angustifolia produced two new compounds (6 and 7) derived from naphthoic acid and sphaeralcic acid [18], not yet identified in the wild plant or in the cells in sus pension.
A relationship was reported between the base chemical structure of the plant compound groups and the pharmacological activity. It has been suggested tha Based on an analysis of the spectroscopic data of single and two-dimensional NMR spectroscopy (Table 2) and a comparison with the data described in the literature [18], this compound was determined to be 2-(4-hydroxy,3-isopropyl,6-methyl, 7,8-dimethoxy) naphthoic acid (Figure 1), named 8-methyl-iso-sphaeralcic acid (7). In addition, the 1 H and 13 C NMR spectra (Table 2) showed the same signals as those described for compound 6, except for the presence of a methoxyl group. This work is the first report of compounds 6 and 7 isolated from the hairy root cultures of S. angustifolia.
The development of a methodology to generate hairy root cultures represents a technological approach to produce metabolites of pharmacological importance, considering that the transformed roots have a higher growth rate and accumulate high levels of active compounds compared with the wild plants [26]. Oncogenes are essential modulators of plant growth and cell differentiation, and their ability to improve secondary metabolite production in transformed cells has been described [27]. Four T-DNA role oncogenes (ORFs), called rolA, rolB, rolC, and rolD, are critical for the induction, growth, and morphology of hairy roots in infected plants. The hairy root transformation of S. angustifolia (SaTRN12.2, line 1, and SaTRN7.1, line 2) was previously confirmed by the presence of the rolC gene [24]. The rolC gene has an important role as a modulator of secondary metabolite production among medicinal plants [27].
Hairy root culture has many advantages for producing high-value metabolites because of their fast growth and biosynthetic stability for many successive generations [28][29][30]. In addition, this culture could produce new compounds not produced by the plant, for example, from Lopezia racemose, the new compound identified was (23R)-2α,3β,23,28-tetrahydroxy-14,15-dehydrocampesterol [31] or cadaverin, an amine detected for the first time in the hairy roots of Brugmansia candida [32]. The hairy root culture of S. angustifolia produced two new compounds (6 and 7) derived from naphthoic acid and sphaeralcic acid [18], not yet identified in the wild plant or in the cells in suspension.
A relationship was reported between the base chemical structure of the plant compound groups and the pharmacological activity. It has been suggested that isosphaeralcic acid (6) and 8-methyl-iso-sphaeralcic acid (7) could have anti-inflammatory and immunomodulatory effects similar to the activities shown for sphaeralcic acid in acute and chronic inflammation models in mice [20,23].
The galphimines A and B (nor-seco-triterpenes) isolated from Ghalphimia glauca, and their synthetic derivatives, have presented anxiolytic effects in mice, considering that the determining factor of this activity was attributed to the presence of hydroxyl groups in C-4, C-6, and C-7 and the presence of a double bonding in the ring A [33]. In the same way, the anti-inflammatory effect of galphimine-A and galphimine-E from G. glauca is related to the presence of an oxygenated function group in C-6 [34].
Reports of the structure-activity relationship of flavonoids as antibacterial agents have suggested that hydroxyl groups at special sites on aromatic rings improve the activity. Instead, methylation of the active hydroxyl groups decreases the activity. The phenyl groups, alkyl-amino chains, alkyl chains, and nitrogen or oxygen also enhance the activity of flavonoids [35]. Similarly, the relationship between the structural characteristics of flavonoids shows that the substitution pattern of free hydroxyl groups on the flavonoid skeleton helps determine the free radical scavenger potential [36].

Gastroprotector Effect of Dichloromethane-Methanol Extracts from SaTRN7.1 Hairy Roots Line of S. angustifolia
In this study, the effects of dichloromethane-methanol extract from the SaTRN7.1 hairy root (line 2) of S. angustifolia on ethanol-induced gastric ulcers in mice was examined. The mice administered with the vehicle (1% Tween-20) presented stomachs with an average ulcerated index of 0.664 ( Table 3). The mice treated with omeprazole and dichloromethane-methanol extract (100 mg/kg) of the hairy root presented stomach areas with less damage and smaller ulcer indices according to the ANOVA and Dunnette's test. According to Student's T test (p < 0.05), the extract showed a protective activity against gastric ulcers induced with ethanol, superior to the effect of omeprazole (1 mg/kg). The oral administration (100 mg/kg) of the dichloromethane-methanol extract inhibited 92% ulcer development. The extract contained mainly sphaeralcic acid, a compound with anti-inflammatory and immunomodulatory properties, sphaeralcic acid isomers (iso-sphaeralcic acid, 6 and 8-methyl-iso-sphaeralcic acid, 7), and stigmasterol and β-sitosterol. Stigmasterol is a vegetal sterol with anti-osteoarthritic, anti-hypercholesterolemia, cytotoxic, anti-tumoral, hypoglycemic, anti-mutagenic, antioxidant, and anti-inflammatory effects [37]. Stigmasterol acts as an anti-inflammatory that reduces the pro-inflammatory production of pro-inflammatory molecules, which contributes to osteoarthritis-induced cartilage degradation [38,39]. In addition, it had an analgesic effect in synergy with 9-hexacosane [40]. β-sitosterol was reported to have immunomodulatory [41] and anti-inflammatory properties on mouse models of edema auricular induced with TPA [42] and paw edema induced with λ-carrageenan [43]. The hairy root culture of S. angustifolia is proposed as a relevant source of new molecules with possible anti-inflammatory and gastroprotective activities. It will be important to evaluate the iso-sphaeralcic acid (6) and 8-methyl-iso-sphaeralcic acid (7) in acute and chronic inflammation models in mice, as well as sphaeralcic acid and its isomers in ethanol-induced gastric ulcers in mice.

S. angustifolia Hairy Root Cultures
The SaTRN12.2 (1) and SaTRN7.1 (2) hairy root lines of S. angustifolia were previously established by infecting internodes with Agrobaterium rhizogenes [24] and were provided by the research group of Dr. Nicasio-Torres.
The hairy roots (10 g) were grown in 1 L Erlenmeyer flasks with 400 mL of phytoregulator-free MS culture medium [24,44] and 3% sucrose. The cultures were incubated at . The hairy roots were transferred to fresh medium every three weeks using a 6% inoculum. At this time, each hairy root line was vacuum filtered separately using a Buchner funnel (Whatman filter paper No. 1, 9-cm diameter), and the retaining roots were weighed and transferred to flasks with fresh medium, preserving the same inoculum for its growth and proliferation.

Extract Preparation of Hairy Root Lines
Flasks of each hairy root line were vacuum filtered using a Buchner funnel (Whatman filter paper No. 1, 9-cm diameter). First, the retained roots were washed with sterile distilled water, and the harvested roots were then dried in an oven (Thelco 160 DM) at 65 • C for 48 h. Next, the dry and ground hairy root lines (173 g SaTRN12.2 and 100 g SaTRN7.1) were extracted thrice by maceration (24 h for each procedure) at room temperature, with a mixture of reactive grade solvents (dichloromethane-methanol 9:1; Merck) at a ratio of 1:50 (w/v). Finally, the extracts were filtered, pooled, and concentrated to dryness under reduced pressure in a rotary evaporator [24].

HPLC Conditions
The HPLC analyses were performed using a Waters system (2695 Separation Module) coupled to a diode array detector (2996) with a 190-600-nm detection range and were operated through the Manager Millennium software system (Empower 1; Waters Corp.). The separations were performed on a Spherisorb RP-18 column (250 × 4.6 mm, 5.0 µm; Waters Corporation) using a constant temperature of 25 • C during the analysis. Samples (20 µL) were eluted at a flow rate of 1.0 mL/min with a mobile phase gradient of high purity: (A) water with trifluoroacetic acid (0.5%, Sigma-Aldrich, St. Louis, MO, USA) and (B) acetonitrile (Merck, Darmstadt, Germany). The mobile phase was started with water (100%) and was maintained for 1 min. The concentration of solvent B was increased gradually to 5% (at 2 min), 30% (at 12 min), 50% (at 4 min), and 80% (at 1 min). During the next 2 min, solvent B was increased to 100%. Finally, the next 3 min were used to return the mobile phase to the initial condition. The chromatographic method had a 25 min run time [18][19][20][21][22][23][24] The extract from line 1 (3 g) was fractionated by chromatography on an open glass column (2.5 × 44 cm) packed with silica gel (25 g, Merck) and a dichloromethane-methanol gradient elution system with 5% polarity increments of methanol and the collection of 10 mL aliquots, yielding 30 fractions. The analysis by TLC allowed for the pooling of aliquots in 10 fractions (SaL1F1 to SaL1F10). Fraction SaL1F3 (0.089 g) was purified by column chromatography (1.5 × 5 cm) on silica gel (1 g, RP-18, Merck) using a water-acetonitrile gradient system with polarity changes of 10% and volumes of 10 mL, to give 40 fractions. Analysis by TLC allowed for pooling into seven sub-fractions (SaL1F3R1 to SaL1F3R7). In the SaL1F3R6 sub-fraction (12 mg), a single spot was observed by TLC, indicating a mixture of two compounds (4 and 5) identified by 1 H and 13 C NMR as stigmasterol and β-sitosterol, respectively.
The extract from line 2 (3.6 g) was fractionated by glass open column chromatography (2.5 × 44 cm) packed with silica gel (25 g, 70-230 mesh, Merck) and an n-hexane-ethyl acetate gradient elution system with polarity increments of 10%. Aliquots of 10 mL were collected (78), and 16 fractions (SaL2F1 to SaL2F16) were pooled according to their chemical profile observed by TLC.
The SaL2F2 fraction (0.03 g) was purified by column chromatography (1.5 × 21 cm) of silica gel (5 g) using a dichloromethane-acetone system with polarity changes of 5%, to provide 22 sub-fractions of 10 mL. After the TLC analysis, the samples were grouped into eight sub-fractions (SaL2F2R1 to SaL2F2R8). In sub-fraction SaL2F2R4, a mixture of stigmasterol (4) and β-sitosterol (5) was identified.

NMR Equipment and Masses
All NMR spectra were recorded on Agilent DD2-600 at 600 MHz, for 1 H NMR, 1 H-1 H COSY, HMQC, and HMBC, and 150 MHz for 13 C NMR and 13 C DEPT, using CDCl 3 and CD 3 OD as solvents. Chemical shifts were reported in ppm relative to TMS. FAB-MS and HRFAB-MS were performed using a JEOL MS-700 mass spectrometer.

Gastric Ulcers Induced with Absolute Ethanol in Mice
Female ICR mice (35)(36)(37)(38)(39)(40)Envigo) were integrated with 10 animals per cage and were preserved in the bioterium at 25 • C, with 12 h light/12 h dark cycles. Water and food (pellets from Harlan Rodent Lab Diet) were provided ad libitum. The ethical use of animals was conducted following with the Mexican Official Regulation dating from 1999 (NOM-062-ZOO1999). The research protocol was approved by the Local Committee for Research in Health (CLIS-1702) and Ethical Committee (CONBIOETICA 17 CEI 00120190121) of Instituto Mexicano del Seguro Social (IMSS) on 13 July 2021, with registration number 2021-1702-06.
Mice weighing between 35 and 40 g were divided into groups of six mice each. The mice were deprived of food with free access to drinking water 24 h prior to experimentation. After fasting, the mice were pre-treated with a single dose of vehicle (1% Tween-20; 0.1 mL/10g), omeprazole (20 mg/kg), and dichloromethane-methanol extract (100 mg/kg) of the SaTRN7.1 hairy root (line 2). Gastric lesions were induced with absolute ethanol (0.10 mL/10g body weight) 1 h after administering the treatments [45]. The mice were anesthetized with Sedalphorte (pentobarbital sodium 25 mg/kg) administered intraperitoneally after 1 h of ulcer induction, and then sacrificed by cervical dislocation. The stomachs were removed, opened along the greater curvature, and rinsed gently with water to remove the gastric contents. Ethanol-induced ulcers appeared as elongated bands of hemorrhagic lesions in the glandular region. After identifying the ulcers (damaged area), photographs were taken of each stomach, and the damaged area was measured from the photographs by planimetry using ImageJ software. The area of each ulcer lesion was measured by counting the number of small squares, 0.1-1 mm, covering the length and width of each ulcer. In each group, the area sum of all lesions for each stomach was used for the ulcerated area (UA) calculation (mm 2 ). The ulcer index (UI) was estimated by dividing the UA by the stomach total area (mm 2 ). The level of protection (%) was determined by the UI of the control minus the UI of the treated group divided by the UI of the control per 100. UI = UA stomach total area % Protection = UI control − UI treated group UI control × 100

Conclusions
The hairy root lines (SaTRN12.2 and SaTRN7.1) of the S. angustifolia plant are producers of the anti-inflammatory and immunomodulatory compounds scopoletin, sphaeralcic acid, stigmasterol, and β-sitosterol, and two isomers of sphaeralcic acid have been described for the first time: iso-sphaeralcic and 8-methyl-iso sphaeralcic. Furthermore, the protective activity against gastric ulcers induced with ethanol of the dichloromethanemethanol extract of SaTRN7.1 confirmed the traditional use of the S. angustifolia plant to treat gastrointestinal illness; this extract, similar to the plant, contains mainly compounds with an anti-inflammatory activity and gastroprotective effect similar to scopoletin and β-sitosterol. Biotechnologically, stable and enhanced active compound production of the SaTRN7.1 hairy root culture could be scaled up using a bioreactor.