Molecular networking of arachidin related compounds from elicited peanut hairy root culture and their potential activity against HCT116 colon cancer cells.

doi:10.21203/rs.3.rs-4706920/v1

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Molecular networking of arachidin related compounds from elicited peanut hairy root culture and their potential activity against HCT116 colon cancer cells.

https://doi.org/10.21203/rs.3.rs-4706920/v1

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Numerous stressors are known to trigger the synthesis of phytoalexins in plants. Specifically, stilbenoids, notably trans-arachidin compounds, are induced in elicited peanut hairy root cultures. This study employed two distinct elicitation strategies on peanut hairy root cultures and analyzed stilbenoid profiles alongside their respective biological effects. Results from HPLC-UV revealed that a combination of paraquat, methyl jasmonate, and cyclodextrin (PQ + MeJA + CD) over 72 hrs notably stimulated the production of trans-resveratrol (Res), surpassing levels of trans-arachidin-1 (Ara-1) and trans-arachidin-3 (Ara-3) in the culture medium. Conversely, elicitation with chitosan, methyl jasmonate, and cyclodextrin (CHT + MeJA + CD) led to higher concentrations of Ara-1 and Ara-3 compared to Res in the medium. LC-HRMS/MS and subsequent molecular networking revealed chemical diversity among trans-arachidins and related compounds in peanut hairy root samples. Differential comparison of elicitation mixtures using CHT instead of PQ in the elicitation mixture led to increased metabolite abundances in both roots and the culture medium, with higher levels observed in the medium samples. MTT assays on HCT116 human colon cancer cells demonstrated greater cytotoxicity of extracts from the culture medium compared to root tissue extracts. This study underscores that distinct stilbenes with comparable bioactivities can be induced through various elicitation methods in peanut hairy root cultures. However, the use of CHT as an elicitor holds particular interest due to its non-toxic and environmentally friendly nature compared to PQ.

Metabolomics

Molecular networking

Arachidin

Stilbenoid

Elicitation

Hairy root

Plants are sessile organisms that are constantly exposed to a wide range of environmental stresses, such as drought, salinity, extreme temperatures, and pathogen attacks. To cope with these stresses, plants have evolved a diverse array of defense mechanisms to perceive and respond to various external stimuli. One of the most fascinating aspects of plant defense mechanisms is their ability to produce specialized metabolites in response to stressors. Plant specialized metabolites are a diverse group of compounds that are not directly involved in the primary functions of growth and development. Instead, specialized metabolites have important roles, such as the interaction with the environment including defense against herbivores and pathogens. By applying elicitors, such as methyl jasmonate (MeJA) and salicylic acid (SA), it is possible to activate the plant immune system and induce the production of defense-related compounds, such as a variety of phenolics, including flavonoids, hydroxycinammic acid conjugates or stilbenes, which may act as phytoalexins (Jeyasri et al. 2023). For example, the abiotic stresses of salt and ozone treatments on two pomegranate cultivars showed that the cultivar Wonderful produced three times higher levels of cinnamic acid derivatives than the control group. On the other hand, the Parfianka cultivar displayed an adequate amount of constitutive polyphenolics to cope with environmental stresses (Calzone et al. 2023). The exogenous treatment of Pyrenean oak trees with MeJA resulted in increased levels of volatile terpenoid compounds, such as trans-β-ocimene and bourbonene, compared to the control group. As a result, the plants were protected from avian predators (Amo et al. 2022).

Peanuts (Arachis hypogaea) are a popular and widely consumed food that are known for their nutritional benefits, including high protein and healthy fat content. In recent years, researchers have been interested in the potential health benefits of certain compounds found in peanuts known as stilbenoids. Stilbenoids are well known as phytoalexins, representing a class of polyphenolic compounds that have also been studied for health-related properties like their antioxidant, anti-inflammatory, and anti-cancer activities (El Khawand et al. 2018; Lopes et al. 2011). Peanuts contain a unique set of naturally occurring stilbenoid compounds, including trans-resveratrol (Res), trans-arachidin-1 (Ara-1), and trans- arachidin-3 (Ara-3). Several studies have shown that trans-arachidin compounds may have a range of potential health benefits. The immunomodulatory properties of Res and Ara-1, extracted from peanut sprouts, were investigated for their effect on aging in ICR mice. The results indicated that Res and Ara-1 modulated the inhibitory functions of regulatory T cells by upregulating CTLA-4, TGF-β, and IL-10 expression, thereby facilitating the aging immune response and contributing to longevity (El Khawand et al. 2018). The Ara-1 alone and its combination with the anticancer drug paclitaxel (Pac) showed the highest cytotoxicity towards triple-negative breast cancer (TNBC) cells. The synergistic effect of Ara-1 and Pac resulted in the activation of p53 expression, the inhibition of cancer cell growth, and the induction of apoptosis via oxidative stress in mitochondria. The combination of Ara-1 also facilitated a reduction in Pac IC50 by approximately 2-fold in TNBC cells (Mohammadhosseinpour et al. 2023).

Peanut hairy root cultures have become an important tool in the production of stilbenoids. In our previous report, we demonstrated that peanut hairy roots were produced upon infection of peanut tissues with Agrobacterium rhizogenes (Pilaisangsuree et al. 2018). The hairy roots grow rapidly and have several advantages over other plant culture systems, including high growth rates, genetic stability, and the ability to produce large amounts of specialized metabolites. The elicitation approach was used to induce the production of specialized metabolites such as flavonoids, and alkaloids in hairy root culture. The elicitation process can be triggered by the use of various chemical substances and physical factors such as jasmonic acid, salicylic acid, chitosan, and UV irradiation, which stimulate the defense mechanisms in the plant tissue and induce the production of specialized metabolites (Halder et al. 2019). Exposure of pigeon pea hairy root cultures to UV-B radiation for example, resulted in a 1.5-fold increase in the production of flavonoids and a 2.3-fold increase in cajaninstilbene acid compared to the control. The increase in flavonoid production was consistent with the upregulation of genes associated with enzymes in the flavonoid biosynthesis pathway, chalcone synthase, isoflavone O-methyltransferase, isoflavone 3′-hydroxylase, and isoflavone reductase (Gai et al. 2022). The addition of 200 mg/L chitosan to Psammosilene tunicoides hairy root cultures resulted in a 4.5-fold increase in saponin content, including quillaic acid, gypsogenin, and gypsogenin-3-O-β-D-glucuronopyranoside. In the same study, transcriptomic data also indicated upregulation of genes that encode antioxidant enzymes, stress-responsive transcription factors, and terpenoid biosynthetic enzymes in response to the chitosan elicitation (Qiu et al. 2021). In our previous report, peanut hairy root cultures were utilized as a model system for producing stilbenoids such as Res, Ara-1, and Ara-3 through different strategies. The combination of paraquat (PQ), methyl jasmonate (MeJA), and methyl-β-cyclodextrin (CD); (PQ + MeJA + CD) induced high levels of stilbene compounds with antioxidant activity in uncut peanut hairy roots under dark condition (Somboon et al. 2019; Wongshaya et al. 2020). Additionally, the simultaneous treatment of chitosan (CHT) in combination with MeJA and CD (CHT + MeJA + CD) demonstrated substantial amounts of Ara-1 and Ara-3, which provides an advantage over the PQ + MeJA + CD induction strategy since the use of CHT is more environmentally friendly compared to the toxic substance PQ and also leads to higher amounts of Ara-1 and Ara-3 obtained through CHT + MeJA + CD treatment compared to PQ + MeJA + CD treatment (Chayjarung et al. 2022; Chayjarung et al. 2021).

In recent years, advances in high-throughput technologies have enabled the more comprehensive exploration of the molecular basis of plant metabolism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique used to simultaneously identify and quantify hundreds of low molecular weight biochemical constituents, in complex biological samples. This technique combines LC to first physically separate the compounds of interest prior to MS(/MS) to detect and identify the separated molecules based on their mass-to-charge ratio (m/z) and fragmentation patterns. The bioinformatic approach of molecular networking analysis enables the visualization and interpretation of metabolomic data by generating clusters of related metabolites, based on their fragmentation patterns and hence structural similarity (Quinn et al. 2017). This method has proven to be a potent tool for identifying metabolic changes in response of plants to environmental perturbations, including biotic and abiotic stress treatment (Katam et al. 2022). Metabolite profiling of 119 chemicals belonging to glucosinolates, flavonoids, phenolic acids, fatty acids, amides, peptides, sulfolipids, carotenoids, and a new class of sulfolipids was characterized and annotated in Moringa oleifera leaves using UPLC-HRMS-based molecular networking (Abdel Shakour et al. 2022). In addition, metabolite profiling of four Momordica species by LC-HRMS/MS in combination with molecular networking revealed various classes of metabolites, including terpenoids, lipids, and flavonoids. The putative structure annotation and their classification as known metabolites by molecular networking was confirmed by matching accurate mass and MS/MS spectra against libraries. The approach of molecular networking has great potential for understanding an improved plant phytochemical diversity (Ramabulana et al. 2021).

In this study, we applied different elicitation strategies, PQ + MeJA + CD and CHT + MeJA + CD, to peanut hairy root cultures and compared their respective profiles of different stilbenoids using LC-HRMS/MS and feature based molecular networking. The elicited compounds from each approach were then evaluated for bioactivity, allowing for comparison of the bioactive molecular substances produced by each elicitation method. By combining the metabolite profiles of stilbenoids with their bioactivity, we were able to attribute the observed bioactivity to the diverse phytochemical composition of the hairy root samples obtained with the different elicitation approaches.

2.1 Hairy root culture and elicitation approach

Peanut hairy roots (K2-K599 line) obtained from the peanut cultivar Kalasin 2 were transformed with A. rhizhogenes (K599), following the protocol previously described by Pilaisangsuree et al. (2018). After subculturing the hairy root tissues on Gamborg Medium (B5) (Gamborg et al. 1968) agar medium for 14 days, 1 g of hairy root tissue was transferred to 50 mL of ½ MS (Murashige and Skoog 1962) liquid medium containing 1.5% sucrose. Different elicitation strategies were employed as follows: 1) control group; 2) elicitation with a combination of 500 µM PQ, 100 µM MeJA, and 6.87 mM CD, (PQ + MeJA + CD) as described by Wongshaya et al. (2020); 3) elicitation with a combination of 200 mg/L CHT, 100 µM MeJA, and 6.87 mM CD, (CHT + MeJA + CD) as described by Chayjarung et al. (2021). All the hairy root liquid cultures were continuously shaken at 150 rpm and kept in the dark for 72 hrs. All experiments were performed using five biological replicates.

2.2 Extraction of hairy root tissue and culture medium

The hairy root tissues were separated from the culture medium. Hairy root tissues from each replication were lyophilized, macerated with 60 mL of methanol for 24 hrs prior to evaporation. The culture medium was extracted through partitioning with an equal volume (50 mL) of ethyl acetate, and the extraction process was repeated three times. The ethyl acetate fractions were then combined and dried using a Büchi rotary evaporator.

2.3 HPLC-UV analysis

The crude extract of the culture medium and the hairy root samples obtained from different elicitation strategies were examined using the HPLC method, as previously described by Limmongkon et al. (2018). A Luna 5 µm C18 (2) 100Å column (4.6 × 250 mm, Phenomenex, CA, USA) was employed for the separation of the crude extract. The separation was performed at a constant flow rate of 1 mL/min using a mobile phase consisting of acetonitrile and 2% formic acid in water (v/v) in a 30:70 (v/v) ratio. UV detection was carried out at different wavelengths, with OD 306 nm for the detection of trans-resveratrol and OD 340 nm for the detection of the trans-arachidin compounds.

2.4 LC-HRMS/MS and molecular networking analysis

2.4.1 LC-HRMS/MS analysis

LC-HRMS measurements were carried out as described earlier (Sauerschnig et al. 2017). In brief, hairy root sample extracts were analyzed on a QExactive HF Orbitrap instrument coupled to a Vanquish UHPLC system (both Thermo Fisher Scientific). Chromatographic separation was carried out on a reversed phase column (C18, Waters X-Bridge) with linear gradient elution in 45 min. For full scan measurements, the ESI source was operated in fast polarity switching mode.

LC-HRMS/MS fragment spectra were recorded in data dependent mode using an inclusion- as well as an exclusion list. The inclusion list consisted of m/z values of 36 arachidins and related compounds (exact mass of [M + H]⁺, [M-H₂O + H]⁺, [M + Na]⁺, [M + NH4]⁺ adduct ions). Re-occurring signals not related to the biological system were listed from earlier measurements of the same sample set and put on the exclusion list to avoid fragmentation of background ions or noise-related ions. Upon an initial full scan (mass range m/z 100–1000, mass resolving power setting of 120,000 (FWHM at m/z 200), five ions were selected to be fragmented with stepped collision energy (nce 20eV, 45eV, 70eV). If detected in the survey scan, ions from the inclusion list were preferentially selected as precursors for fragmentation. The remaining time interval for product ion fragmentation between successive survey scans was filled up with precursor ions according to their intensities starting with the most abundant. The fragmented ions were then set on a dynamic exclusion list for 3 seconds before they were re-considered for fragmentation. MS/MS fragment spectra were recorded in centroid mode with mass resolving power setting 15,000 (FWHM at m/z 200).

2.4.2 MS/MS data processing

The LC-HRMS/MS raw files were processed using MZmine3 (version 3.3.0). The workflow comprised of several modules: Mass detection, ADAP Chromatogram Builder (with parameters set at 5 ppm, minimum group size of 4 scans, and minimum highest intensity of 1E6), Local minimum feature resolver (with a 10% chromatographic threshold, minimum absolute height of 1E6, minimum ratio of peak top/edge set at 5, and a peak duration between 0.05–2 min), Feature list rows filter (with the "Validate ¹³C isotope pattern" parameter set to true, and removing if ¹³C is true), Join aligner (with a 10 ppm m/z tolerance, 0.2 min retention time tolerance, weight of 3 for m/z and 1 for retention time), Feature list blank subtraction (detected in at least 2 blank samples, minimum fold-increase in non-blank samples of 10), Feature list rows filter (keeping only MS2 features), local database search (with a 5 ppm mass deviation, adducts set to positive mode, and no retention time check), and finally exporting to GNPS-FBMN for molecular networking with the option to merge experimental MS/MS spectra (10 ppm deviation allowed across spectra of the same compound).

2.4.3 Molecular networking

The generated MGF and .csv file were then uploaded to GNPS and the feature based molecular networking workflow (release_28.2) was used (default parameters, data source MZmine2). The generated molecular network was downloaded and opened in Cytoscape (version 3.9.1). Annotated compounds were colored according to the most plausible compounds (i.e., lowest m/z deviation, isomeric annotations were discarded). All features that were not in a network with at least one annotated compound were also discarded. Illustrations of the remaining nodes were automatically generated with R (version 4.2.2) and the mzR

(https://www.bioconductor.org/packages/release/bioc/html/mzR.html, version 2.32.0) and MSnbase packages (https://www.bioconductor.org/packages/release/bioc/html/MSnbase.html, version 2.42.2).

2.5 In Vitro Assay for Cytotoxic Activity

2.5.1 Cell line

The human colorectal carcinoma (CRC) cell line HCT116 was purchased from the American Type Culture Collection (ATCC). HCT116 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/mL of penicillin and 100 g/mL of streptomycin and maintained at 37˚C in a humidified incubator with 5% CO₂ atmosphere.

2.5.2 Determination of cell viability by 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

HCT116 cells were seeded at density of 7x10³ cells/well in 96-well plates and cultured for 24 hrs. The cells were treated with a range of concentrations including 0, 200, 400, 600, 800 µg/mL of the extracts for 48 hrs. The final concentration of the DMSO in the treatment was maintained at less than 0.5%. After incubation, MTT solution at 0.5 mg/mL was added and incubated for 4 hrs at 37˚C. The formazan crystal was dissolved by DMSO and the absorbance at 540 nm was measured with a microplate reader. The experiments were repeated for at least three times under the same condition. The graphical representation of % cell viability and the half-maximal inhibitory concentration (IC₅₀) values were performed using GraphPad Prism 9.4.0 software.

2.6 Statistical analysis

IBM SPSS Statistics version 23 was used for statistical analysis, which included a one-way ANOVA followed by LSD post hoc analysis. The experiments were carried out in five biological replicates, and the results are presented as mean ± SD values with a significance level of p < 0.05.

R (version 4.2.2) was used for statistical analysis of the metabolic features detected in the LC-HRMS dataset. Metabolic features were subjected to auto-scaling (van den Berg et al. 2006) prior to principal component analysis. Moreover, univariate statistics was calculated on the relative peak abundances of the detected metabolic features without transformation. The p-value was calculated with a non-paired Welch-t-test and the log2-fold-change was derived from the group means. Statistical difference was tested with a critical p-value of 0.05 and an absolute log2-fold-change of at least 1 (i.e., a difference of at least 100% percent).

3.1 Characteristics of elicited hairy root tissues and culture medium

Five biological replicates were used in the elicitation experiments. Figure 1 shows the hairy root tissues and culture medium after being treated with the two different elicitor mixtures for 72 hrs. All samples of hairy root tissues and culture medium treated with a combination of paraquat, methyl jasmonate, and cyclodextrin (PQ + MeJA + CD) as well as the combination of chitosan, methyl jasmonate, and cyclodextrin (CHT + MeJA + CD) displayed a yellowish color after 72 hrs of elicitation compared to the control group, indicating that both of the tested elicitor mixtures induced the production and release of substances from hairy root tissues into the culture medium. Moreover, the more intense color in the samples treated with CHT-containing elicitor mixture compared to the PQ-containing mixture suggested a differential production of substances produced and/or secreted from hairy root tissues

3.2 HPLC-UV determination of stilbenoids

HPLC-UV was used to analyze the profile and quantities of known stilbenoids, including Res and the prenylated forms Ara-1 and Ara-3, in crude extracts from both hairy root tissues and culture medium, as shown in Fig. 2 and Table 1. The hairy root tissues of the control group (Fig. 2A) and the PQ + MeJA + CD treated group (Fig. 2C) accumulated small amounts of Res and unidentified compounds, but no obvious trans-arachidin variant was detected. In contrast, only a low amount of Res was detected in hairy root tissues of the CHT + MeJA + CD treated group, while Ara-1 and Ara-3 accumulated in the hairy root tissues (Fig. 2E). Interestingly, after elicitation with different elicitors, the stilbenene compounds, including Res, Ara-1, and Ara-3, were detected in the culture medium (Fig. 2D and 2F) in constrast to the un-elicited control (Fig. 2B), in which these compounds were missing. The PQ + MeJA + CD treatment for 72 hrs produced a substantial amount of Res (21.2 ± 11.7 µmol/g crude extract), which was higher than the levels of Ara-1 and Ara-3 in the culture medium (19.1 ± 11.5 and 3.5 ± 1.2 µmol/g crude extract, respectively) (Fig. 2D). Conversely, the CHT + MeJA + CD treatment resulted in higher amounts of Ara-1 (24.7 ± 22.0 µmol/g crude extract) and Ara-3 (18.7 ± 11.1 µmol/g crude extract) compared to Res (3.1 ± 1.3 µmol/g crude extract) in the culture medium (Fig. 2F). The unique chromatogram profile may be attributed to the varied response mechanisms of hairy root tissue to different elicitors.

Table 1

The contents of *trans*-resveratrol (Res), *trans*-arachidin-1 (Ara-1) and *trans*-arachidin-3 (Ara-3) from crude extract of hairy root tissues and culture medium crude extracts treated with PQ + MeJA + CD and CHT + MeJA + CD at 72 hrs. Data represents the mean ± standard deviation of five replicates per sample.
Source	Treatment	Trans-resveratrol (µmol/g crude extract)	Trans-arachidin-1 (µmol/g crude extract)	Trans-arachidin-3 (µmol/g crude extract)
Root tissue	control	2.2 ± 1.5	ND	ND
	PQ + MeJA + CD	5.0 ± 2.4	ND	ND
	CHT + MeJA + CD	0.4 ± 0.2	2.5 ± 0.9	4.0 ± 1.6
Culture Medium	control	ND	ND	ND
	PQ + MeJA + CD	21.2 ± 11.7	19.1 ± 11.5	3.5 ± 1.2
	CHT + MeJA + CD	3.1 ± 1.3	24.7 ± 22.0	18.7 ± 11.1
ND: not detect

3.3 LC-HRMS/MS analysis with a focus on trans-arachidins and related compounds

Manual inspection of extracted ion chromatograms (EICs) of LC-HRMS data for compounds with the chemical formulas of Ara-1 and Ara-3 revealed a large number of chromatographic peaks representing isomeric compounds, presumably positional isomers (Online Resource 1). Next, the dataset was processed with MZmine3 with the aim to detect all metabolites in an untargeted manner for subsequent molecular networking. A total of 3,104 monoisotopic features together with their corresponding MS/MS spectra were detected in the samples and subsequently classified into several molecular families using the feature based molecular networking workflow of the GNPS platform (Nothias et al. 2020; Wang et al. 2016). Of these, only molecular families with at least one feature having been annotated as a trans-arachidin or a related compound were kept. Features annotated as either Ara-1 (blue) or Ara-3 (purple) are mostly located within two well-connected clusters A and B (Fig. 3). The molecular structures of stilbene compounds, particularly those related to arachidin, are depicted in Fig. 4. The generated network confirms that the different isomers with the m/z values of Ara-1 and Ara-3 have highly similar MS/MS fragmentation spectra (Fig. 5).

The feature cluster depicted in Fig. 3A mostly represent different isomers directly connected and the annotated features using the principal ions of the compounds (i.e., [M + H]⁺, [M + Na]⁺, and [M + NH₄]⁺). Furthermore, the crudely purified trans-arachidin extracts also confirm these compounds to likely be the desired compounds. Figure 5A-5C shows the MS/MS spectra of three such compounds from the molecular network A with the principal fragments of the spectra either identical or having an m/z corresponding to one additional oxygen atom in the precursor ions’ sum formulas. The difference of one oxygen atom is retained for the precursor and also some of the fragments (e.g, 241 ◊ 257 or 107 ◊ 123). A closer inspection of the two subgroups of the compounds annotated as Ara-1 in the molecular family A also shows that these are either shifted by the oxygen atom (i.e., pink nodes) or not (i.e., blue nodes) (Fig. 3A). Moreover, there seem to be two tightly connected but non-overlapping subparts of the molecular family that are annotated as Ara-1, which is a strong indication that the respective compounds have a highly similar structure and that the position of the additional oxygen atom is at different positions of the stilbene core structure. Furthermore, the retention times of these compounds are different confirming them to be individual compounds and not in-source fragments or artifacts of a single compound. Interestingly, in the subgraph A there is another group of features with high similarity that are connected to mostly only one subgroup of the Ara-3 metabolites (Fig. 3). Their m/z value is 329.1373, which resembles the chemical formula of Ara-1 with an another oxygen atom (or Ara-3 with two additional oxygen atoms). This can also be seen in the MS/MS spectra of a respective example compound (Fig. 5D).

Several reports indicate that stilbenoids found in peanut extracts typically contain either a resveratrol (3,5,4’-trihydroxystilbene) or a piceatannol (3,5,3’,4’-tetrahydroxystilbene) structure, often with a prenyl group substitution (Aguamah et al. 1981; Keen and Ingham 1976; Sobolev et al. 2020). This prenyl group may consist of isopentenyl (3-methyl-but-1-enyl or 3-methyl-but-2-enyl) or isopentadienyl (3-methyl-but-1,3-dienyl) variants. In MS/MS negative mode, the identified derivatives include isoprenylated forms of Res with a nominal mass of 296 Da, such as Ara-2 and Ara-3, and isoprenylated forms of piceatannol with a nominal mass of 312 Da, including Ara-1 and Ara-5. Notably, two isomers of isopentenyl stilbenoids, named Ara-3 and Ara-2, exhibit different positions of the double bond in the prenyl chain, resulting in a longer conjugated system in Ara-3 compared to Ara-2 (Aisyah et al. 2015). Additionally, in peanuts, the isopentadienyl group attached to Res can produce trans-3′-isopentadienyl-3,5,4′-trihydroxystilbene (IPD) with a nominal mass of 294 Da, while the isopentenyl group attached to piceatannol can yield 4-isopentadienyl-3,5,3’,4’-tetrahydroxystilbene (IPP) with a molecular mass of 310 Da (Cooksey et al. 1988).

In summary, trans-arachidin compounds derived from peanut hairy root samples seem to be chemically quite diverse with highly similar, yet distinct, fragment spectra. Most notably, the hydroxy group (or groups) can be at different positions in the compound leading to this high number of isomers.

3.4 Differences among experimental groups

Statistical analysis of the stilbenoids annotated as trans-arachidins and their structurally related derivatives (i.e., all compounds depicted in the molecular network subgraphs A and B) revealed differing EIC profiles between the tested experimental conditions. Based on their stilbenoid profiles, the samples separated in a two-dimensional PCA scores plot according to the respective elicitor composition and sample type (i.e. medium or root extracts respecively) (Fig. 6A).

Univariate comparison of the concentration levels of the arachidin-releated metabolites were carried out pairwise for all experimental variants (Volcano plots, Fig. 6B). A comparison between the elicitor-treated samples and the control samples (Fig. 6B) showed that for both treatment variants, the elicitor (CHT + MeJA + CD and PQ + MeJA + CD) increased the concentration of most of the annotated compounds compared to the respective controls, regardless of whether they were root extracts or the culture medium. Complementary, when the two elicitors were compared, the presence of CHT instead of PQ in the mixture resulted in higher abundances in both the roots and the culture medium. Finally, higher abundances of the metabolites are generally observed in the medium samples than in the root samples.

3.5 Cytotoxic Activity

The study investigated the impact of crude extracts obtained from both hairy root tissues and culture medium with both of the tested mixtures of elicitors on HCT116 human colon cancer cells, employing the MTT assay. In Fig. 7A, the cell morphology and density were observed under a microscope after 48 hrs of treatment, using the highest concentration of 800 µg/mL extracts and a control group. Notably, the application of extracts from hairy root tissues did not result in any cytotoxic effects on HCT116 cells. In contrast, the treatment with extracts from the culture medium led to cell detachment and a reduction in cell number.

As demonstrated in Fig. 7B, the MTT results indicated that treating HCT116 cells with root tissues, whether elicited by PQ + MeJA + CD or CHT + MeJA + CD, did not exhibit any toxicity compared to untreated control cells. Interestingly, treatment with root extracts, obtained after elicitation with CHT + MeJA + CD at concentrations of 400 and 600 µg/mL, significantly increased cell viability, while at a concentration of 800 µg/mL, it showed similar viability to the control. Conversely, treating HCT116 cells with culture medium elicited by PQ + MeJA + CD and CHT + MeJA + CD resulted in a dose-dependent inhibition of cell viability, as seen in Figs. 7A and 7C. The cytotoxic effects of crude extracts from the culture medium after PQ + MeJA + CD and CHT + MeJA + CD treatment exhibited similar effects at concentrations of 200, 400, and 800 µg/mL. However, the treatment with 600 ug/mL of PQ + MeJA + CD showed a more pronounced cytotoxic effect than CHT + MeJA + CD (Fig. 7C).

Furthermore, IC50 values were employed to assess the cytotoxic activity of these extracts on HCT116 cells, as shown in Table 2. The IC50 values for root tissues elicited by both PQ + MeJA + CD and CHT + MeJA + CD were above 800 µg/mL. In contrast, the IC50 values for culture medium extracts elicited by PQ + MeJA + CD and CHT + MeJA + CD were 418 ± 1 and 471 ± 1 µg/mL, respectively. These results indicate that extracts from the culture medium exhibited greater cytotoxicity towards HCT116 cells compared to extracts from hairy root tissues. Furthermore, extracts from hairy roots elicited by different elicitors displayed similar cytotoxic activity against HCT116 human colon cancer cells.

Table 2

IC50 values of extracts in HCT116 human colon cancer cells. The p-values represent comparisons between treatments with PQ + MeJA + CD and CHT + MeJA + CD.
Extract	IC50 (µg/mL) at 48 hrs		p-value
Extract	PQ + MeJA + CD	CHT + MeJA + CD	p-value
Root tissue	> 800	> 800	N/A
Culture Medium	418 ± 1	471 ± 1	0.1199

Specialized metabolites are natural compounds that are not essential for the growth and development, but play an important role in various environmental interactions including plant defense. In addition to their role in the direct interaction with biotic and abiotic factors, many specialized metabolites also provide valuable health-related properties, such as anti-inflammatory, antimicrobial, and anticancer activities, and are therefore of interest for pharmaceutical, and industrial applications. Stilbenoids, including Res, Ara-1, Ara-3, IPP, IPD, chiricanine A, and arahypins, are major phytoalexins that can be induced by filamentous fungi in germinated peanut seeds (Aisyah et al. 2015; Wu et al. 2011). Several reports have demonstrated that prenylated stilbenoids, such as Ara-1 and Ara-3, exhibit promising bioactivity compared to Res. The purified Ara-1 from germinated peanut kernels has demonstrated anti-cancer properties by inducing programmed cell death in human leukemia HL-60 cell lines via the intrinsic apoptotic pathway (Huang et al. 2010). In another study, prenylated stilbenoid Ara-1 from elicited peanut hairy root culture was reported to have the potential to inhibit cell proliferation and induce apoptosis in Triple-negative breast cancer (TNBC) cells when used in combination with chemotherapy drug paclitaxel (Mohammadhosseinpour et al. 2023).

Several strategies such as generating a hairy root culture as an experimental material and elicitation using diverse elicitors, have been investigated to obtain a high amount of prenylated stilbenes for bioactivity assays. Various chemical compounds, including PQ, MeJA, CDs, and CHT, examined within this research, have been recognized as elicitors for the production of secondary metabolites in plants. PQ acts as a herbicide, inducing plant stress by generating reactive oxygen species (ROS) and thereby triggering the production of secondary metabolites as part of the plant's defense against oxidative stress (Sipari et al. 2022). MeJA, derived from linolenic acid via jasmonic acid, is an endogenous plant hormone, well known to be involved in plant defense responses, including the formation of specialized metabolites (Jeyasri et al. 2023; Wasternack and Strnad 2016). Although CDs themselves do not directly induce stress, their ability to form complexes facilitates the delivery of molecules that influence plant physiology and secondary metabolite production, potentially augmenting the effects of elicitors (Almagro and Pedreño 2020). Lastly, CHT, obtained from chitin, prompts stress responses in plants by activating defense signaling pathways, upregulating genes involved in secondary metabolite biosynthesis, enhancing plant immunity, and modulating phytohormone signaling (Malerba and Cerana 2016). The optimization of elicitor treatment is a crucial step to obtain sufficient amounts of the desired prenylated stilbene products compared to the untreated control. Our previous reports demonstrated the use of a combination of PQ + MeJA + CD (Wongshaya et al. 2020) and CHT + MeJA + CD (Chayjarung et al. 2021) as elicitors of peanut hairy roots. Due to the available standards and purified reference compounds, only the major compounds of Res, Ara-1, and Ara-3, were determined and reported in each elicitor treatment. However, the complexity of stilbene compounds, based on their structural similarities, have not been investigated with an unbiased approach so far. This is the first study to investigate the complex interactions between elicited stilbene compounds, where a feature based molecular networking has been carried out to find stilbene compounds and structurally related derivatives after elicitation with PQ + MeJA + CD and CHT + MeJA + CD.

The molecular networking analysis is a powerful computational tool used in metabolomics research to analyze metabolic pathways and structural relationship between metabolites in biological systems. The present results demonstrated that the trans-arachidin compounds found in peanut hairy root samples exhibit significant chemical diversity with distinct highly similar structures. Particularly, the position of the hydroxy groups in the compound can vary, resulting in a large number of isomers. This corresponds to the report on peanut hairy roots elicited by MeJA and CD, which can be detected in the culture medium as prenylated forms of Ara-2 and Ara-5 with a nominal m/z of 297 and 313, for the [M + H]⁺ ions respectively. However, the yields of Ara-1 and Ara-3 were significantly higher than those of the dimethylallyl analogs of Ara-2 and Ara-5 (Yang et al. 2016). Statistical analysis revealed distinctions between the PQ + MeJA + CD and CHT + MeJA + CD treatment conditions concerning trans-arachidins and associated compounds. The abundance of these compounds notably increased in the culture medium after elicitation, affirming the production and release of these compounds into the medium. This was also evident from the distinctly intense yellowish colour observed in the culture medium following elicitation with both PQ + MeJA + CD and CHT + MeJA + CD.

Bioactivity assays conducted on crude extracts obtained from each elicitation approach demonstrated cytotoxic effects against HCT116 human colon cancer cells were exclusively observed in the extracts derived from the culture medium. In contrast, extracts obtained from hairy root tissues, whether elicited with CHT + MeJA + CD or PQ + MeJA + CD, did not exhibit such effects. These findings strongly suggest that the anti-proliferative impact on HCT116 cells originated from biochemical constituents released into the culture medium. Additionally, HPLC-UV analysis revealed elevated levels of stilbenoids, specifically Res, Ara-1, and Ara-3, within the culture medium relative to root tissues. This observation aligns with the volcano plots depicted in Fig. 6B, highlighting a notable prevalence of arachidin-related compounds detected in the medium in comparison to their abundance in root tissue. Consequently, the secretion of stilbenoid compounds resulting from various elicitation processes in hairy root culture also resulted in the demonstrated anti-colon cancer activity with the tested cell culture model. Upon comparing the impact of extracts from hairy root tissues elicited using different methods, it became evident that at the highest concentration, which was 800 µg/mL, both extracts did not exhibit any cytotoxic effects on HCT116 cells. There was no noticeable increase in cell numbers observed under the microscope. However, intriguingly, at concentrations of 400 and 600 µg/mL, extracts from hairy root tissues, which had been elicited with CHT + MeJA + CD demonstrated a statistically significant promotion of cell growth when compared to extracts from root tissues elicited with PQ + MeJA + CD. This outcome suggests that the extracts might contain elicitor-type specific substances with the potential to stimulate cell growth and could have other beneficial bioactivities, such as anti-aging effects, among others. Recent research has shown that ethanolic extracts derived from Ipomoea pes-caprae (IPC) possess properties that can combat skin aging by stimulating cell growth and collagen production in human fibroblasts (CCD-986sk cells). Additionally, the primary chemical constituents found in the leaf extracts of IPC include phenolic compounds, flavonoids, and tannins. Among these, the predominant phenolic compound identified in IPC extract is 3,5-di-caffeoylquinic acid (Panichakul et al. 2022). Several studies have indicated that a substance at high concentrations may be toxic to cells, but at subtoxic concentrations, it can enhance cell viability (Hecker et al. 2022). For instance, Alsaleh et al. (2023) found that subtoxic levels (6.25 µg/mL) of zinc oxide and nickel oxide nanoparticles increased mitochondrial activity in Raw 264.7 cells, potentially triggering detoxification pathways, whereas higher concentrations (12 µg/mL) were toxic and reduced cell viability. In the present study, this adverse effect was observed only in extracts elicited with CHT + MeJA + CD, and not in those elicited with PQ + MeJA + CD.

When we analyzed the extracts originating from the culture medium induced by the tested elicitors, it was evident that both types of extracts effectively suppressed the growth of colon cancer cells in a dose-dependent manner. The inhibitory impact was notably pronounced in extracts stimulated with PQ + MeJA + CD at precise concentrations, notably 600 µg/mL. However, no significant difference in IC50 values was observed between the two elicitation approaches (IC50 for the PQ + MeJA + CD approach: 418 ± 1 µg/mL; IC50 for the CHT + MeJA + CD approach: 471 ± 1 µg/mL). An analysis of the composition of stilbene compounds in each extract revealed varying groups of stilbenoids. In extracts elicited with PQ + MeJA + CD, high levels of Res and low levels of arachidin were detected. Conversely, in extracts elicited with CHT + MeJA + CD, arachidin levels were significantly higher than those of Res, suggesting that both groups of stilbenes possess similar abilities to inhibit the growth of colon cancer cells. Alternatively, other compounds apart from stilbenes may contribute to the inhibitory effect on the growth of HCT116 cells. For being able to deepen the current understanding, it is imperative to undertake further experiments for comprehensive investigation. A previous report indicated that Ara-1 and Ara-3 are prenylated analogs of Res and piceatannol, which exhibit slow metabolism and enhanced bioavailability. They also retain biological activity through cannabinoid receptor binding when compared to their non-prenylated parent compounds (Brents et al. 2012). Recent studies have shown that Ara-1 has higher cytotoxicity against TNBC cell lines compared to Res (Mohammadhosseinpour et al. 2022). Therefore, Ara-1 and Ara-3 may hold greater promise for therapeutic applications than Res. This study demonstrates that peanut hairy root culture elicitation using different methods can induce the production of distinct stilbenes with similar bioactivities, such as anti-colon cancer properties. Further studies are needed to elucidate the underlying mechanisms by which each extract inhibits the growth of HCT116 cells.

In conclusion, LC-HRMS/MS and molecular networking analysis revealed intricate relationships among stilbene compounds in induced hairy root cultures, highlighting their structural resemblances with remarkably similar MS/MS fragmentation spectra. Elicitation methods, such as (PQ + MeJA + CD) and (CHT + MeJA + CD), significantly boosted metabolite release into the culture medium, exerting distinct impacts on Res, Ara-1, and Ara-3 levels. Bioactivity assays demonstrated heightened cytotoxicity of culture medium extracts, suggesting promising therapeutic potentials. Further exploration could concentrate on elucidating the precise mechanisms behind the varied effects of elicitors on metabolite production and release, as well as investigating options for therapeutic applications of identified compounds in cancer or other diseases in the future. Integrating multi-omics approaches would provide a comprehensive understanding of the regulatory networks governing stilbene metabolism in elicited hairy root cultures, thus enhancing the potential for targeted metabolic engineering or optimization of elicitation strategies for enhanced metabolite production.

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Author contribution statement

Apinun Limmongkon: Project administration, Conceptualization, Writing-Review & Editing manuscript. Christoph Büschl: Methodology, Investigation, Statistical analysis, Writing & Editing manuscript. Maria Doppler: Methodology, Validation, Investigation, Data collection, Editing manuscript. Rainer Schuhmacher: Supervision, Conceptualization, Visualization, Editing manuscript. Suchada Phimsen: Conceptualization, Visualization, Writing manuscript. Chonnikan Tothong: Methodology, Validation, Data collection. Chanyanut Pankaew: Methodology, Validation, Data collection. Piyathida Wongkham: Methodology, Validation, Data collection. Warissara Wongkham: Methodology, Validation, Data collection.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgement

We gratefully acknowledge the Core Facility Bioactive Molecules: Screening and Analysis (BMoSA) at the University of Natural Resources and Life Sciences, Tulln, Austria, for providing the high-throughput mass spectrometry analysis.

Funding

This work was supported by Naresuan University (NU) and the National Science, Research and Innovation Fund (NSRF) [grant number R2567B051].

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Molecular networking of arachidin related compounds from elicited peanut hairy root culture and their potential activity against HCT116 colon cancer cells.

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Hairy root culture and elicitation approach

2.2 Extraction of hairy root tissue and culture medium

2.3 HPLC-UV analysis

2.4 LC-HRMS/MS and molecular networking analysis

2.4.1 LC-HRMS/MS analysis

2.4.2 MS/MS data processing

2.4.3 Molecular networking

2.5 In Vitro Assay for Cytotoxic Activity

2.5.1 Cell line

2.5.2 Determination of cell viability by 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

2.6 Statistical analysis

3. Results

3.1 Characteristics of elicited hairy root tissues and culture medium

3.2 HPLC-UV determination of stilbenoids

3.3 LC-HRMS/MS analysis with a focus on trans-arachidins and related compounds

3.4 Differences among experimental groups

3.5 Cytotoxic Activity

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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