Duroia saccifera: in vitro germination, friable calli and identification of β-sitosterol and stigmasterol from the active extract against Mycobacterium tuberculosis

Duroia saccifera (Rubiaceae) occurs in the Amazon rainforest and their extracts showed antibacterial properties. To obtain greater quantities of active substances, leaf segments from in vitro D. saccifera seedlings were used as explants for calli induction; calli were multiplied via multiple subcultures, dried and extracted with hexane followed by ethyl acetate (EtOAc) and methanol (MeOH). As D. macrophylla had been reported to produce antimycobacterial substances, we assayed calli extracts against Mycobacterium tuberculosis (H37Rv strain). Calli EtOAc extract was active, with a minimal inhibitory concentration (MIC) of ≤ 25 mg mL, IC90 of 19.5 mg mL -1 and minimal bactericidal concentration (MBC) of 200 mg mL. EtOAc extract was analyzed by Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) to determine its chemical profile, and was found to be rich in terpenes. Chromatographic fractionation of the EtOAc extract yielded a mixture of two sterols, β-sitosterol and stigmasterol (in proportion of 2:1), which were identified by H and C NMR analysis. As far as we know, this is the first report of Duroia saccifera in vitro cell culture, antituberculosis activity of calli extract and β-sitosterol and stigmasterol isolation from in vitro plant cell culture.


Introduction
Tuberculosis (TB) is a chronic contagious disease caused by Mycobacterium tuberculosis (WHO 2017). TB is one of the 10 leading causes of mortality worldwide and more than 95% of TB deaths occur in low-and middleincome countries. In recent decades, the drugs clinically available for TB treatment (rifampin and isoniazid) have become ineffective, as the causative microorganism has developed resistance, so causing an increase in the number of patients who have the disease in a resistant form (WHO 2017). This serious public health problem has led to a renewed initiative to search for novel effective drugs, including new natural plant products, which might potentially fight TB.
As part of the search for new antituberculosis substances conducted by our bioprospection research group, we had previously isolated two triterpenes and eight indole alkaloids from Duroia macrophylla that were active against M. tuberculosis (H37Rv strain) Nunez et al. 2014).
To obtain a renewable source of D. saccifera material, we carried out an in vitro callus establishment protocol and phytochemical study of the ethyl acetate calli extract to isolate and identify calli-derived substances biologicallyactive against Mycobacterium tuberculosis.

Germination
A pre-disinfection of the fruit was made using a disinfectant solution of Mancozeb® (2 g L −1 ) plus streptomycin (100 mg L −1 ) for 30 minutes, followed by ethanol (70% v/v) immersion for 5 minutes. Seeds disinfection followed an ethanol (70% v/v) immersion for 1 minute in a laminar flow chamber, followed by an immersion in NaClO (2% v/v) for 5 minutes, with three washes in autoclaved distilled water between the disinfecting agents.
After disinfection, seeds were inoculated onto MS medium (Murashige & Skoog 1962) supplemented with sucrose (30 g L −1 ), agar (7 g L −1 ) and 21.54 μmol L −1 of gibberellic acid (GA 3 ), and cultivated for 90 days, without changing medium or subcultures. The seeds were maintained in a growth room at 26 ± 2 o C and 60% average humidity, under a 16:8 h photoperiod (light:dark), with a light intensity of 50 μmol −2 m −2 s −1 given by white fluorescent lamps. After germination, seedlings were subjected to successive subcultures every 30 days.

Callus induction
Complete leaves from in vitro seedlings of D. saccifera were used as explants for callogenesis induction. They were cut at the base of the leaf blade and inoculated into sterile test tubes containing 5 mL MS medium (Murashige & Skoog 1962), supplemented with sucrose (30 g L −1 ) and agar (7 g L −1 ) in a laminar flow chamber. We added 2,4-dichlorophenoxyacetic (2,4-D) (17.24 μmol L −1 ) and kinetin (8.62 μmol L −1 ) as growth regulators. Cultures were then maintained in a growth room at 26 ± 2 o C and 60% average humidity, under a 16:8 h photoperiod (light:dark), with a light intensity of 50 μmol −2 m −2 s −1 given by white fluorescent lamps.
After 60 days in culture, induced calli were subcultured in new tubes. After 30 days they were transferred from the test tubes to glass jars with 25 mL of medium, and subsequently subjected to successive subcultures every 30 days in order to increase mass.

Extraction, fractionation and isolation
Duroia saccifera calli were manually removed from the culture medium and combined achieving a fresh calli total weight of 1.238 kg. They were lyophilized (model ALPHA 1-2 LDplus, Christ) and powdered yielding 54.49 g (4.4% of dry material). The lyophilized powder was then extracted first with hexane (1 g:10 mL), using ultrasound for 20 min, filtered and re-extracted with hexane, repeating 4 times. Then the calli was extracted with ethyl acetate (EtOAc) by the same procedure 5 times and finally with methanol (MeOH), also 5 times by the same procedure. All extracts (hexane, EtOAc and MeOH) were concentrated in a rotary evaporator (model 802,Fisatom). Extracts and all fractions were analyzed by Thin Layer Chromatography (TLC) (Merck) of silica with UV 254 on aluminum support, eluted with suitable systems, and revealed under UV light (λ = 254 and 365 nm), sulfuric p-anisaldehyde, Ce(SO 4 ) 2 , FeCl 3 , and Dragendorff's reagent.

Structural characterization
In order to determine the chemical structure, the fraction was dissolved in CDCl 3 and analyzed by Nuclear Magnetic Resonance (NMR) obtained with a Fourier 300 spectrometer (Bruker), operating at 300 for 1 H and 75 MHz for 13 C nuclei, respectively. All NMR shifts were expressed in ppm related to TMS signal at 0.00 ppm as internal reference.

Antituberculosis activity
The resazurin microtiter assay (REMA) was performed to test extract activity against Mycobacterium tuberculosis, at the Medical Microbiology Research Nucleus (NUPEMM), Federal University of Rio Grande (FURG). Extracts were tested at the initial concentration of 400 μg mL −1 . In each experiment, 100 μL of extracts, serially diluted in Middlebrook 7H9 medium supplemented with 10% OADC (oleic acid, albumin, dextrose and catalase) and 100 μL of the inoculum, were incubated at 37 o C for 7 days in a 96 well plate. Following incubation, 30 μL of resazurin (0.02%) was added in each well and further incubated for two days at 37 o C. Biological activity was assayed based on the change of color from blue to pink, when oxidation of the reagent occurs due to bacterial growth. Rifampicin and isoniazid drugs were used as positive controls (Palomino et al. 2002).

Germination
Of the 108 initial seeds, 24% developed fungus contamination and 25% did not germinate. After 50 days of cultivation, the emergence of the radicle was observed in the remainder. The hypocotyl and radicle elongation occurred after 60 days. Secondary root formation occurred after 72 days and fully developed cotyledons after 82 days. After 90 days, 50% of the seedlings were fully formed (Fig. 1a-f).

Callus induction
Callus formation began after 15 days, when cell growth occurred in the central part of the leaf and at the end of leaf segments. After 60 days, the explants were completely transformed into callus and were then subcultured in new tubes, even though they still had zones of green coloration, which indicated cell masses with some degree of differentiation. After 90 days, they had a friable appearance, when the surface of the calli took on a smoothly nodulated appearance, and coloration ranged between whitish and yellowish ( Fig. 2a-f).

Extraction, fractionation and isolation
TLC analysis of EtOAc extract obtained from D. saccifera calli indicated a high concentration of terpenes when the plates were sprayed with Ce(SO 4 ) 2 and sulfuric anisaldehyde. When revealed with UV light (λ = 365 nm) fluorescence, the plates indicated the presence of molecules with conjugated double bonds. When the plates were sprayed with FeCl 3 and Dragendorff's reagent no spots were visible, which indicated the absence or very low concentrations of phenolic substances and alkaloids.

Structural characterization
1 H-NMR data for fraction 7-9 showed the presence of several signals in the shielded region between δ 0.6 (s) and 1.25 (s), characteristic of methyl hydrogens; and two signals at δ 5.35   It was also possible to observe steroid methyl singlets in the 1 H-RMN spectrum, which could be correlated with each carbon atom by HSQC experiment analysis. For β-sitosterol, the correlations of these methyl groups were: δ 0.68 with 11.85, δ 0.80 with 18.93, δ 0.82 with 19.73,   (Reis et al. 2016;Carrion et al. 2013). A second study reported the isolation of the triterpenes oleanoic and ursolic acid active against M. tuberculosis strain (MIC 200 μg mL −1 ) from D. macrophylla extracts .
Several other studies have shown terpenes to be responsible for antimycobacterial activity (Seidel & Taylor 2004;Aguiar et al. 2005;Higuchi et al. 2008;Subramaniam et al. 2014;Evina et al. 2017;Suja et al. 2017). Their high lipophilicity is probably the main factor that allows their penetration through the mycobacterial cell wall (Higuchi et al. 2008).
Reviews of the numerous plant species and active compounds showing antimycobacterial activity indicate β-sitosterol and stigmasterol to be important phytochemical components in this process (Chinsembu 2016;Tiwari et al. 2019). Suja (2017) reported growth inhibition activity of M. tuberculosis with a MIC of 25 and 100 μg mL −1 for β-sitosterol and stigmasterol, respectively. A study conducted by Saludes et al. (2002) reported the β-sitosterol and stigmasterol MICs to be 128 and 32 μg mL −1 , respectively. In the current study, we found a mixture of both substances which have similar structures, differing only in the position of an extra double bound (present in stigmasterol), which complicates their separation (Xu et al. 2005;Pierre & Moses 2015). This, and the small quantity of isolated mixture, meant it was not possible try to purify them further.

Conclusions
In this study, for the first time, we report in vitro germination, callus induction, and inhibitory activity, of EtOAc extract of D. saccifera calli against M. tuberculosis. β-sitosterol and stigmasterol were obtained for the first time from in vitro callus culture of this species. In vitro D. saccifera cultures have a broad potential for supply of active secondary metabolites, mainly terpenes. Fractionation of the EtOAc extract should be continued in order to isolate, identify and assay other possible active substances that could lead to the discovery of new anti-tuberculosis drugs.