Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Cydonia oblonga Mill. Pulp Callus Inhibits Oxidative Stress and Inflammation in Injured Cells.

The pharmacological activity of a callus extract from the pulp of Cydonia oblonga Mill., also known as quince, was investigated in murine macrophage (RAW 264.7) and human keratinocyte (HaCaT) cell lines. In particular, the anti-inflammatory activity of C. oblonga Mill. pulp callus extract was assessed in lipopolysaccharides (LPS)-treated RAW 264.7 by the Griess test and in LPS-treated HaCaT human keratinocytes by examining the expression of genes involved in the inflammatory process, including nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), nuclear factor-kappa-B inhibitor alfa (ikBα), and intercellular adhesion molecule (ICAM). The antioxidant activity was evaluated by quantizing the reactive oxygen species (ROS) production in the hydrogen peroxide and tert-butyl hydroperoxide-injured HaCaT cell line. The obtained results indicate that C. oblonga callus from fruit pulp extract has anti-inflammatory and antioxidant activities, suggesting its possible application in delaying and preventing acute or chronic diseases associated with aging or in the treatment of wound dressing.

Chemical composition, antioxidant and anti-inflammatory properties of Monarda didyma L. essential oil.

In the present study, Monarda didyma L. essential oil (isolated from the flowering aerial parts of the plant) was examined to characterize its chemotype and to evaluate, in addition to the quali-quantitative chemical analysis, the associated antioxidant and anti-inflammatory activities. The plants were grown in central Italy, Urbino (PU), Marche region. Different analyses (TLC, GC-FID, GC-MS and 1H-NMR) allowed the identification of twenty compounds among which carvacrol, p-cymene and thymol were the most abundant. On this basis, the chemotype examined in the present study was indicated as Monarda didyma ct. carvacrol. The antioxidant effect was assessed by DPPH assay. Moreover, this chemotype was investigated for the anti-inflammatory effect in an in vitro setting (i.e., LPS-stimulated U937 cells). The decreased expression of pro-inflammatory cytokine IL-6 and the increased expression of miR-146a are suggestive of the involvement of the Toll-like receptor-4 signaling pathway. Although further studies are needed to better investigate the action mechanism/s underlying the results observed in the experimental setting, our findings show that M. didyma essential oil is rich in bioactive compounds (mainly aromatic monoterpenes and phenolic monoterpenes) which are most likely responsible for its beneficial effect.

Characterization of the Biological Activity of the Ethanolic Extract from the Roots of Cannabis sativa L. Grown in Aeroponics.

Cannabis sativa var. Kompolti, a variety routinely used for food production purposes, is characterized by a low concentration of psychoactive molecules, although containing many other biologically attractive metabolites in all parts of the plant, including the roots. In the present work, we evaluate the specific biological activities of the roots' extract from plants cultivated through aeroponics, an affordable and reliable method facilitating the isolation and processing of roots, with the advantage of being suitable for industrial scale-up. Furthermore, aeroponics results in an increased net accumulation of the most biologically attractive constituents (ß-sitosterol, friedelin and epi-friedelanol) found in the roots. The ethanolic extract of the aeroponic roots of C. sativa (APEX) and its separate components are studied to evaluate their anti-inflammatory (modulation of the expression level of specific markers upon LPS stimulation in U937 cells, such as IL-6, IL-8, TNF-α, IkB-α, iNOS, IRAK-1 and miR-146a) and antioxidant (in either acellular or cellular settings) activities. The APEX anti-inflammatory and antioxidant capacities are also functionally benchmarked using the wound-healing assay. On the whole, the data obtained show that APEX and its main components showed significant anti-inflammatory and antioxidant activities, which may render the exploitation of roots as a source of natural antioxidants and anti-inflammatory agents highly attractive, with the additional technical and economic advantages of aeroponics compared to soil cultivation.

Extracellular pH, osmolarity, temperature and humidity could discourage SARS-CoV-2 cell docking and propagation via intercellular signaling pathways.

The COVID-19 pandemic and its virus variants continue to pose a serious and long-lasting threat worldwide. To combat the pandemic, the world's largest COVID-19 vaccination campaign is currently ongoing. As of July 19th 2021, 26.2% of the world population has received at least one dose of a COVID-19 vaccine (1.04 billion), and one billion has been fully vaccinated, with very high vaccination rates in countries like Israel, Malta, and the UEA. Conversely, only 1% of people in low-income countries have received at least one dose with examples of vaccination frequency as low as 0.07% in the Democratic Republic of Congo. It is thus of paramount importance that more research on alternate methods to counter cell infection and propagation is undertaken that could be implemented in low-income countries. Moreover, an adjunctive therapeutic intervention would help to avoid disease exacerbation in high-rate vaccinated countries too. Based on experimental biochemical evidence on viral cell fusion and propagation, herein we identify (i) extracellular pH (epH), (ii) temperature, and (iii) humidity and osmolarity as critical factors. These factors are here in discussed along with their implications on mucus thick layer, proteases, abundance of sialic acid, vascular permeability and exudate/edema. Heated, humidified air containing sodium bicarbonate has long been used in the treatment of certain diseases, and here we argue that warm inhalation of sodium bicarbonate might successfully target these endpoints. Although we highlight the molecular/cellular basis and the signalling pathways to support this intervention, we underscore the need for clinical investigations to encourage further research and clinical trials. In addition, we think that such an approach is also important in light of the high mutation rate of this virus originating from a rapid increase.

Yield, Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation.

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C.sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, ß-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth-particularly of the roots-and a significant increase (up to 20-fold in the case of ß-sitosterol) in the total content of the aforementioned roots' bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy.

Prunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms' lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and anti-inflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.

Bioconversion of Callus-Produced Precursors to Silymarin Derivatives in Silybum marianum Leaves for the Production of Bioactive Compounds.

The present study aimed to investigate the enzymatic potential of Silybum marianum leaves to bioconvert phenolic acids produced in S. marianum callus into silymarin derivatives as chemopreventive agent. Here we demonstrate that despite the fact that leaves of S. marianum did not accumulate silymarin themselves, expanding leaves had the full capacity to convert di-caffeoylquinic acid to silymarin complex. This was proven by HPLC separations coupled with electrospray ionization mass spectrometry (ESI-MS) analysis. Soaking the leaf discs with S. marianum callus extract for different times revealed that silymarin derivatives had been formed at high yield after 16 h. Bioconverted products displayed the same retention time and the same mass spectra (MS or MS/MS) as standard silymarin. Bioconversion was achieved only when using leaves of a specific age, as both very young and old leaves failed to produce silymarin from callus extract. Only medium leaves had the metabolic capacity to convert callus components into silymarin. The results revealed higher activities of enzymes of the phenylpropanoid pathway in medium leaves than in young and old leaves. It is concluded that cotyledon-derived callus efficiently produces compounds that can be bio-converted to flavonolignans in leaves tissue of S. marianum.

Prunus spinosa Extract Loaded in Biomimetic Nanoparticles Evokes In Vitro Anti-Inflammatory and Wound Healing Activities.

Prunus spinosa fruits (PSF) contain different phenolic compounds showing antioxidant and anti-inflammatory activities. Innovative drug delivery systems such as biomimetic nanoparticles could improve the activity of PSF extract by promoting (i) the protection of payload into the lipidic bilayer, (ii) increased accumulation to the diseased tissue due to specific targeting properties, (iii) improved biocompatibility, (iv) low toxicity and increased bioavailability. Using membrane proteins extracted from human monocyte cell line THP-1 cells and a mixture of phospholipids, we formulated two types of PSF-extract-loaded biomimetic vesicles differing from each other for the presence of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG). The biological activity of free extract (PSF), compared to both types of extract-loaded vesicles (PSF-DOPCs and PSF-DOPGs) and empty vesicles (DOPCs and DOPGs), was evaluated in vitro on HUVEC cells. PSF-DOPCs showed preferential incorporation of the extract. When enriched into the nanovesicles, the extract showed a significantly increased anti-inflammatory activity, and a pronounced wound-healing effect (with PSF-DOPCs more efficient than PSF-DOPG) compared to free PSF. This innovative drug delivery system, combining nutraceutical active ingredients into a biomimetic formulation, represents a possible adjuvant therapy for the treatment of wound healing. This nanoplatform could be useful for the encapsulation/enrichment of other nutraceutical products with short stability and low bioavailability.

MicroRNAs Bioinformatics Analyses Identifying HDAC Pathway as a Putative Target for Existing Anti-COVID-19 Therapeutics.

Over 313,000 SARS-CoV-2 positive cases have been confirmed in Italy as of 30 September 2020, and the number of deaths exceeding thirty-five thousand makes Italy among the list of most significantly affected countries in the world. Such an enormous occurrence of infections and death raises the urgent demand for effective available treatments. Discovering the cellular/molecular mechanisms of SARS-CoV-2 pathogenicity is of paramount importance to understand how the infection becomes a disease and how to plan any therapeutic approach. In this regard, we performed an in silico analysis to predict the putative virus targets and evidence the already available therapeutics. Literature experimental results identified angiotensin-converting enzyme ACE and Spike proteins particularly involved in COVID-19. Consequently, we investigated the signalling pathways modulated by the two proteins through query miRNet, the platform linking miRNAs, targets, and functions. Our bioinformatics analysis predicted microRNAs (miRs), miR-335-5p and miR-26b-5p, as being modulated by Spike and ACE together with histone deacetylate (HDAC) pathway. Notably, our results identified ACE/ACE2-ATR1-Cholesterol-HDAC axis signals that also matched with some available clinical data. We hypothesize that the current and EMA-approved, SARS-CoV-2 off-label HDAC inhibitors (HDACis) drugs may be repurposed to limit or block host-virus interactions. Moreover, a ranked list of compounds is provided for further evaluation for safety, efficacy, and effectiveness.

A New Strategy to Increase Production of Genoprotective Bioactive Molecules from Cotyledon-Derived Silybum marianum L. Callus.

There is a need to enhance the production of bioactive secondary metabolites and to establish new production systems, e.g., for liver-protective compounds of Silybum marianum seeds. Quantifying and identifying the produced phytochemicals, and examining their protective effects against genotoxic agents, is of great interest. This study established a protocol for the qualitative and quantitative production of hepatoprotective compounds in cotyledon-derived Silybum marianum callus through optimized supplementation of the MS medium with the growth regulators 2,4-D, benzylaminopurine, myoinositol, and asparagine. High-performance liquid chromatography (HPLC) coupled with electrospray ionisation mass spectrometry (ESI-MS) allowed for identification and quantification of the produced compounds. None of the growth medium combinations supported a detectable production of silymarin. Instead, the generated calli accumulated phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acid, as revealed by HPLC and mass spectrometric analysis. 4-Nitro-o-phenylenediamine (NPD) was employed in the AMES-test with Salmonella typhimurium strain TA98 because it is a potent mutagen for this strain. Results revealed that callus extract had a high anti-genotoxic activity with respect to standard silymarin but more evident with respect seed extract. The callus produced chlorogenic acid and dicaffeoylquinic acid, which revealed higher bioactivity than silymarin. Both compounds were not formed or could not be detected in the seeds of Silybum marianum Egyptian ecotype.

In Vitro Anticollagenase and Antielastase Activities of Essential Oil of Helichrysum italicum subsp. italicum (Roth) G. Don.

The chemical composition of the essential oil of flowering aerial parts of Helichrysum italicum subsp. italicum cultivated in central Italy, Marche region, was analyzed by means of gas chromatography-mass spectrometry. Seventy-eight components, accounting for 98.71% of the whole essential oil composition, were identified and quantified. Neryl acetate showed the largest relative abundance in the composition, accounting for 15.75% of the oil, followed by α-pinene (8.21%); 4,6,9-trimethyl-8-decene-3,5-dione, (Italidione I), (7.34%); ar-curcumene and ß-selinene (5.37%); γ-curcumene (4.83%); nerol (4.75%); α-selinene (4.68%); limonene (4.55%); linalool (4.42%), and 2,4,6,9-tetramethyl-8-decene-3,5-dione (Italidione II), (4.26%). The oil inhibited in vitro collagenase and elastase activities, with IC50 values of 36.99 ± 1.52 and 135.43 ± 6.32 µg/mL, respectively. Neryl acetate, nerol, and linalool, distinctive compounds of the oil obtained from this plant, tested alone or in mixture, at the same percentages shown in the essential oil, exhibited no activity against the two enzymes. On the contrary, α-pinene and limonene, tested alone and in mixture, showed inhibitory activity on both collagenase and elastase.

Chemopreventive Potential of Apple Pulp Callus Against Colorectal Cancer Cell Proliferation and Tumorigenesis.

This study focused on the evaluation of the chemopreventive potential of tissue in vitro culture of the "Mela Rosa Marchigiana" apple (MRM callus) that allows the amplification of secondary metabolites. The MRM pulp and MRM callus chemopreventive potential was evaluated in terms of antiproliferative activity, inhibition of tumorigenesis in soft agar cultures, cell cycle and western blotting analyses in CaCo2 and LoVo colon cancer cell lines and in JB6 promotion-sensitive (JB6 P+) cells. MRM callus induced a strong concentration-dependent inhibition of colon cancer cell proliferation and suppressed 12-o-tetra-decanoyl-phorbol-13-acetate-induced tumorigenesis of JB6 P+ cells in soft agar cultures. MRM callus inhibited the phosphorylation of JNK, p38, and eIF2alpha. Our data indicate that the MRM callus exerts a good antiproliferative and antitumorigenic potential through the MAP kinase inhibition and could provide natural compounds with chemopreventive properties.

Bioeffects of Prunus spinosa L. fruit ethanol extract on reproduction and phenotypic plasticity of Trichoplax adhaerens Schulze, 1883 (Placozoa).

The aim of this work was to test and analyse the bioeffects of Prunus spinosa L. (Rosacaee) fruit ethanol extract on Trichoplax adhaerens Schulze, 1883 (Placozoa) laboratory cultures which-for the first time-were employed as in vivo biological model to assess the bioactivity of a natural extract. The ethanol extract of P. spinosa was administrated during a 46 day experimental period; ultrastructural (by optical, confocal, TEM and SEM microscopy) and morphometric analyses indicated that treated Trichoplax adhaerens showed significant differences in viability, reproductive modalities, body shape and colour with respect to the control group. Finally, P. spinosa bioactive compounds seem to exert profound protective effects on T. adhaerens reproduction and phenotype. Our results may support additional investigations related to other bioactive compounds properties useful for nutraceutical preparations to be used as food supplements.

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

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,3ß,6α,23-tetrahydroxy-18α,19α-urs-20-en-28-oic acid, 2α,3ß,23-trihydroxy-18α,19α-urs-20-en-28-oic acid and 2α,3ß,6ß,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 ß-sitosterol, phloridzin, oleanolic, ursolic, 3ß-hydroxy-18α,19α-urs-20-en-28-oic, maslinic, corosolic, 2α,3ß-dihydroxy-18α,19α-urs-20-en-28-oic, and tormentic acid.

The In Vitro Activity of Angelica archangelica L. Essential Oil on Inflammation.

The use of herbs with medicinal value and biomedical effects has increased tremendously in the last years. However, inadequate basic knowledge of their mode of action is the main issue related to phytotherapy, although they have shown promising potential. To provide insights into these important issues, we tested here on appropriate in vitro models the efficacy of Angelica archangelica essential oil (Aa-EO) for anti-inflammatory properties. The results demonstrated that Aa-EO induced significant apoptosis and necrosis at high doses in U937 cells. We used nontoxic concentrations to treat for anti-inflammatory capacity. The results also demonstrated a decreased proinflammatory cytokine interleukin-6 level in human umbilical vein endothelial cells, as senescence in vitro model, when cells are challenged with lipopolysaccharide (LPS), one of the most powerful proinflammatory inducer in the presence of Aa-EO. In addition, down expression of miR-126 and miR-146a (inflammamirs) produced by LPS stimulation was reverted by Aa-EO simultaneous treatment. These results provide noteworthy basis for the development/formulation of new drugs for future clinical uses and new food products or dietary supplements for contrasting inflammation.

Photochemical response of parsley (Petroselinum crispum (Mill.) Fuss) grown under red light: The effect on the essential oil composition and yield.

The effect of different wavelengths on plants morphological characters has been widely described, but also the chemical composition of the essential oil is influenced by the lighting conditions in which they are grown. In the present study, the effect of both the enrichment (reverse Emerson effect) and the monochromatic lighting treatments with red light has been evaluated on the essential oil compositions of parsley (Petroselinum crispum (Mill.) Fuss). Multivariate statistical analysis was performed on the results, with both the hierarchical cluster and principal component analyses. Whilst the red-enrichment of the light spectrum did not induce major changes in the essential oil composition, the end of the day monochromatic red (660 nm) treatment caused a chemotype switch in the essential oil and relevant differences in the overall composition, with an increment of the relative abundance of oxygenated compounds, coupled with a relevant decrement in the abundance of phenylpropanoids. The extraction yields remained unchanged in all the three tested conditions of light (control, red-enriched and monochromatic red). Different lighting conditions could be used as a tool to modulate the compounds present in the essential oil, but further studies would be advisable to assess the effects on different species and chemical classes of compounds.

The Essential Oil of Monarda didyma L. (Lamiaceae) Exerts Phytotoxic Activity in Vitro against Various Weed Seed.

The chemical composition of the essential oil of the flowering aerial parts of Monarda didyma L. cultivated in central Italy was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The major compounds of the oil were thymol (59.3%), p-cymene (10.3%), terpinolene (9.2%), δ-3-carene (4.4%), myrcene (3.7%), and camphene (3.4%). The essential oil was tested in vitro for its anti-germination activity against Papaver rhoeas L., Taraxacum officinale F. H. Wigg., Avena fatua L., Raphanus sativus L. and Lepidium sativum L. seeds, demonstrating good inhibitory activity in a dose-dependent way. The exposure of the employed weed seeds to M. didyma essential oil and thymol solution (59.3%) increased the level of hydrogen peroxide (H2O2) and malondialdehyde (MDA), markers of oxidative stress, in emerging 5-day-old rootlets.

High Triterpenic Acids Production in Callus Cultures from Fruit Pulp of Two Apple Varieties.

INTRODUCTION: Very rarely fruit pulp has been used in in vitro culture to produce secondary metabolites useful in promoting health. OBJECTIVES: The aims of this work were the study of the best conditions to obtain the callus cultures from the pulp of two varieties of apples, Golden Delicious (GD) and "Mela Rosa Marchigiana" (MRM), and the quali-quantitative analysis of secondary metabolites produced by the two in vitro callus cultures. METHODOLOGY: Callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of supplements. To achieve the maximum recovery of secondary metabolites produced, preliminary extraction tests were carried out on GD apple culture using two different organic solvents (MeOH and EtOAc). The quali-quantitative analysis of the methanolic extract of both cultures was carried out by ESI-MSn and GC-MS techniques. RESULTS: The GC-MS analysis revealed the presence of triterpenic acids, in particular, oleanolic, ursolic, maslinic, pomolic, tormentic, corosolic and annurcoic acid along with a phytosterol, ß-sitosterol. In addition, GD callus culture produced phloridzin, absent in the MRM culture. In this last culture, however, the total amount of secondary metabolites was markedly higher. The in vivo production of these bioactive compounds were also quantified in the GD and MRM apple pulps. CONCLUSION: Apple pulps produced higher amounts of triterpenic acids in vitro than in vivo. The present work can be considered a method to amplify the production of important secondary metabolites which exert beneficial effects on human health. Copyright © 2016 John Wiley & Sons, Ltd.