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. 2022 Jul 17;151(1):215–219. doi: 10.1007/s11240-022-02342-0

Biosynthetic studies through feeding experiments in Eclipta prostrata (L.) L. hairy roots

Adriana A Lopes 1,✉, Giuliana R S Souza 1, Suzelei de Castro França 1, Miriam V Lourenço 1,✉
PMCID: PMC9288585  PMID: 35875188

Abstract

Eclipta prostrata (L.) L. is a medicinal plant of the Asteraceae family, and several extracts and isolated compounds of E. prostrata (L.) L. showed a wide range of biological activities such as antimicrobial, anticancer, hepatoprotective, neuroprotective, hair growth promoting activities, and more recently against covid-19. Eclipta prostrata (L.) L. hairy roots produce wedelolactone (WL), demethylwedelolactone (DWL) and 3,5-di-O-caffeoylquinic acid (3,5-diCQA), and there is no data in literature regarding biosynthetic pathways are involved. To verify the biosynthetic route, feeding experiments were carried out using sodium [2-13C]acetate, [3-13C]dl-phenylalanine, and 13C-labeled compounds (WL, DWL and 3,5-diCQA) were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-QTOF-MS). Analysis showed that the metabolic pathways operative of coumestans (WL and DWL) are derived from acetate and shikimate pathways, while that the phenylpropanoid (3,5-diCQA) biosynthesis is exclusively from shikimate pathway.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11240-022-02342-0.

Keywords: Asteraceae, Coumestans, Phenylpropanoid, Acetate pathway, Shikimate pathway

Key message

This work showed the feeding experiments using 13C-precursors in order to clarify the biosynthetic route of bioactive compounds from Eclipta prostata (L.) L. hairy roots.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11240-022-02342-0.

Introduction

Eclipta prostrata (L.) L., native to Brazil, is a medicinal species from the Asteraceae family (Souza et al. 2003). The biological efficacy of E. prostrata (L.) L. has been correlated to chemical constituents present in its extracts such as wedelolactone (WL) and demethylwedelolactone (DWL). More recent activity data showed that WL displays anti-glycation and anti-diabetic activities and could be promisor therapeutic agent against obesity and related metabolic diseases (Shahab et al. 2018; Yao et al. 2022). WL reduces pulmonary fibrosis (Yang et al. 2019), attenuates doxorubicin-induced inflammation (Zhu et al. 2019), is promising agent in the mantle cell lymphoma treatment (Romanchikova and Trapencieris 2019), exhibits strong antioxidant activity (Li et al. 2020), may be a promising anti-inflammatory candidate to combat Pseudomonas aeruginosa keratitis (Xu et al. 2021) and reduces acute pancreatitis and associated lung injury (Fan et al. 2021). In previous work was investigated the suitability of E. prostrata (L.) L. hairy root cultures biosynthesize the 3,5-di-O-caffeoylquinic acid (3,5-diCQA) (Maciel et al. 2021), a promising biological compound against SARS-CoV-2, the virus that causes covid-19 (Joshi et al. 2020; Shah et al. 2021; Sumon et al. 2021; Kadioglu et al. 2021). Although WL, DWL and 3,5-diCQA are known, their biosynthesis of compounds from E. prostrata (L.) L. are not related in the literature. Coumestans, such as coumestrol, are derived from shikimate and acetate pathways (Dewick 2009), while the phenylpropanoid, such as chlorogenic acid, are synthesized by shikimate pathway (Dewick 2009; Mahesh et al. 2007). Hence, the main aim of this article was to fed E. prostrata (L.) L. hairy roots with sodium [2-13C]acetate and [3-13C]dl-phenylalanine. After time incubation the crude extracts were prepared and analyzed by HPLC-QTOF-MS in order to find 13C-labeled WL, DWL and 3,5-diCQA.

Materials and methods

Chemicals

The sodium [2-13C]acetate and [3-13C]dl-phenylalanine (13C-precursors) were purchased from Sigma-Aldrich®.

Maintenance of E. prostrata (L.) L. hairy roots

Eclipta prostrata (L.) L. hairy roots C19 clone (Diogo et al. 2009; Maciel et al. 2021) have been cultivated in MS liquid culture medium under agitation (100 rpm) at 25 ± 1 ºC in the dark.

13C-tracer experiments with sodium [2-13C]acetate and [3-13C]dl-phenylalanine in E. prostrata (L.) L. hairy roots

Eclipta prostrata (L.) L. hairy roots C19 clone (1 month old; 2 g each; n = 6) were inoculated in liquid MS medium with 30 g.L− 1 of sucrose, pH 6.0 (± 0,05) supplemented with sodium [2-13C]acetate (0.25%) or [3-13C]dl-phenylalanine (10 mM). Additionally, a control group of roots that did not receive 13C-precursors was also prepared (1 month old; 2 g each; n = 6). Hairy root cultures were kept at 25 °C ± 1 °C, in the dark and under agitation at 100 rpm for 10 days (55–60% relative humidity). After 10 days of cultivation, the roots were harvested and subjected to extraction using methanol.

Equipment and HPLC-QTOF-MS conditions from feeding experiments

Methanolic extracts were performed in a Shimadzu HPLC System (pump LC-20AD, auto-injector SIL-20 A HT, column oven CTO-20 A, system controller CBM-20 A and degasser DGU-20A3), coupled to diode array detector (SPD-M20A) and a micrOTOF-QII (Bruker) mass spectrometer, operating with an electrospray ionization source (ESI-MS) in negative analysis mode. A volume of 5 µL at a concentration of 50 µg.mL− 1 of the samples was injected into a Luna C18 column (250 mm × 4.6 mm, 5 µ, Phenomenex). The chromatographic condition used was: (A:B; A-H2O ultrapure + 0.1% formic acid; B-MeOH + 0.1% formic acid), in an exploratory gradient elution; from 0 to 40 min starting at (95:5) until (0: 100). The flow of the mobile phase was 1.0 mL min− 1 and the compounds were monitored at λ = 210–600 nm. The operating parameters used in the Z-spray source were: capillary voltage = 3000 V, end plate offset 500 V, Z-spray source temperature = 200 °C, nebulizer gas pressure 4 bar and desolvation gas flow = 8–10 L.min− 1. The mass range used in the full-scan analysis mode was 100 to 600 Da.

Results and discussion

Isotopic labeling studies were carried out in order to elucidate the WL, DWL and 3,5-diCQA biosynthesis. Eclipta prostrata (L.) L. hairy roots were inoculated in liquid medium supplemented with sodium [2-13C]acetate or [3-13C]dl-phenylalanine, and after 10 days of culture, a methanolic extract from fresh hairy roots was prepared and analyzed by HPLC-QTOF-MS (see supplementary data). Table 1 shows 13C-incorporation data from WL, DWL and 3,5-diCQA compounds after isotopic labeling experiments using sodium [2-13C]acetate and [3-13C]dl-phenylalanine. During incorporation, for each 13C added, one mass unit was expected to be added. The calculation is based on the ratio between the molecular ion peak area, and its isotopes peak area, for both the control and 13C-precursor experiments. The smaller value of the ratio (between M-H peak area and its corresponding isotope [M-H + 1]−, [M-H + 2]− or [M-H + 3]− peak area), means 13C incorporation during biosynthetic process, thus confirming the incorporation of the 13C-labeled precursor (Musquiari et al. 2021). WL (m/z = 314.246) and DWL (m/z = 300.219) coumestans incorporated three acetate units from sodium [2-13C]acetate and, therefore, the M + 3 ion was selected, showing that the acetate pathway is one of the metabolic pathway in the biosynthesis of these compounds (Fig. 1). In the experiment using [3-13C]dl-phenylalanine only one 13C unit was incorporated and, therefore, the M + 1 ion was selected (see supplementary data). Thus, WL and DWL are biosynthesized by both the acetate and shikimate pathways. It is likely that DWL is the precursor of WL, because the chemical structures differ by just one methyl group. The S-Adenosyl methionine enzyme (SAM) probably mediates an addition of one methyl group into WL. The isotopic labeling of 3,5-diCQA (m/z = 515.443) led to the incorporation of two units of [3-13C]dl-phenylalanine confirming the shikimate pathway is the exclusive route for the phenylpropanoid biosynthesis (Fig. 1). WL, DWL and 3,5-diCQA are present in E. prostrata (L.) L. hairy roots derived from a common precursor, the shikimic acid, and whose pathway is formed from the regenerative process of the carbohydrate erythrose-4-phosphate, synthesized during the photosynthesis, further of the phosphoenolpyruvate, an intermediate from glucose metabolism. Thereby, the isotopic experiments allowed us to establish the biosynthetic pathway of bioactive compounds from E. prostrata (L.) L. hairy roots.

Table 1.

Mass spectrometric data of isotopic experiments using sodium [2-13C]acetate and [3-13C]dl-phenylalanine in E. prostrata (L.) L. hairy roots

Compound Retention time Ratio Control Sodium [2-13C]acetate [3-13C]dl-phenylalanine
3,5-diCQA 23’ RM + 2 12.5 – 1.22
DWL 25’ RM + 1 5.2 – 3.6
RM + 3 219.6 4.19 –
WL 30’ RM + 1 5.2 – 4.7
RM + 3 113.2 39.4 –
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(RM + 1: value of the ratio between [M-H]- peak area and its corresponding isotope [M-H + 1]- peak area; RM + 2: value of the ratio between [M-H]- peak area and its corresponding isotope [M-H + 2]- peak area; RM + 3: value of the ratio between [M-H]- peak area and its corresponding isotope [M-H + 3]- peak area)

Fig. 1.

Fig. 1

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Biosynthesis of WL, DWL and 3,5-diCQA after incorporation of sodium [2-13C]acetate (in red dots) and [3-13C]dl-phenylalanine (in blue dots) by E. prostrata (L.) L. hairy roots. (Color figure online)

Conclusions

Our isotopic experiments allowed us to establish the biosynthetic pathway of bioactive compounds from E. prostrata (L.) L. hairy roots; WL and DWL are from acetate and shikimate pathways while 3,5-diCQA is from shikimate pathway exclusively.

Supplementary Information

Below is the link to the electronic supplementary material.

11240_2022_2342_MOESM1_ESM.docx (417KB, docx)

Supplementary material 1 (DOCX 417.0 kb)

Acknowledgements

The authors are grateful to the Biotechnology Unit, Universidade de Ribeirão Preto (UNAERP) for funding applied in research and experimental development. G.R.S.S. also thanks CAPES for the award of scholarships. The authors would like to thank Prof. Dr. Norberto P. Lopes for HPLC-QTOF-MS analyses.

Author contributions

AAL and MVL designed research; GRSS and AAL performed research; AAL, MVL and SCF analyzed data; AAL wrote the manuscript and all authors reviewed the manuscript.

Funding

The authors are grateful to the Biotechnology Unit, Universidade de Ribeirão Preto (UNAERP) for funding applied research and experimental development.

Declarations

Conflict of  interest

The authors declare no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Adriana A. Lopes, Email: alopes@unaerp.br

Miriam V. Lourenço, Email: mvlouren@gmail.com

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Supplementary Materials

11240_2022_2342_MOESM1_ESM.docx (417KB, docx)

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