Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

Acanthopanax trifoliatus (L.) Merr. (A. trifoliatus) belongs to the family Araliaceae, which is called "Le Cai", and is an indigenous plant to Guangdong Province that has been prevalently planted for years. A. trifoliatus is used in folk medicine and has ginseng-like activity. Kaurenoic acid ((−)-kaur-16-en-19-oic acid, KA) is a kaurane-type diterpenoid that is regarded as a major compound in A. trifoliatus. early studies have reported the determination of KA by HPLC capillary electrophoresis. However, KA could not be completely separated from other components in the plant extract by HpLc because of their similar molecular structures and physical and chemical properties. UHpLc-MS/MS could be a useful tool to identify and quantify KA. In the present work, a UHPLC-ESI-MS/MS method for determining KA in A. trifoliatus was developed and validated. KA was extracted from lyophilized A. trifoliatus leaves by ultrasound-assisted extraction and further purified by solid phase extraction (SPE). KA was quantified and separated on an Accucore c 18 Lc column. Mass spectrometry with multi-reaction monitoring (MRM) and quantitative fragment ion/product ion (m/z: 301.3/301.3) in ESI negative mode was used for quantification. The intra-assay and inter-assay relative standard deviation (R.S.D.) were 2.8% and 3.2%, respectively. The inter-person R.S.D. on the same day was 3.6%. The inter-instrument R.S.D. with the same model on the same day was 2.9%. The recoveries evaluated upon spiking three different concentrations of KA were above 97%. A minor matrix effect of 94% was observed. This method has been applied successfully for the determination of KA in A. trifoliatus leaves. Acanthopanax is a plant genus that embraces 18 plant species that are mainly distributed in Asia and the far-eastern region of Russia 1,2. One of the Acanthopanax species is A. trifoliatus, which is called "Le Cai" in Chinese, and is an indigenous plant in Enping, Guangdong Province that has been prevalently planted for years. The planting area has reached 420 hectares, and the total value is worth approximately 120 million. A. trifoliatus, with ginseng-like activity, has been used in folk medicine in southern China since old time to treat sinew, bone pains, rheumatism, bruises, neuralgia, impotence, gout, hepatitis and diabetes 3-5. In Cambodia, Laos, and Vietnam, A. trifoliatus has been used to treat nervous affections and improve memory 6,7. It has also been shown to have a good curative effect on the common cold, jaundice, gastric pain, diarrhea and ulcers 8. A decoction of leaves, young shoots or the bark of A. trifoliatus has been used to treat tuberculosis and lung hemorrhages 9. Some studies have indicated that extracts of A. trifoliatus possess several beneficial biological effects, including anti-inflammatory, immunostimulatory, and antioxidant properties, protein tyrosine phosphatase inhibitory activity and cytotoxic activities towards some types of cancer cells 10-12 .

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“…The LOD was determined by S/N ratio of 3 and found LOD was 1.3 pg. These results were lower than those from an early report 26 .…”

Section: Acontrasting

confidence: 81%

“…Therefore, we chose the MRM transition as 301.3 > 301.3. These unique characteristics were also observed by another study 26 . Different collision energies were tested on KA from 0 eV to 40 eV and there was no unique fragments observed.…”

Section: Asupporting

confidence: 78%

Determination of Kaurenoic Acid in Acanthopanax trifoliatus by Ultra-High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry (UHPLC-MS/MS)

Peng

Chen

Duan

et al. 2020

Sci Rep

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“…The recovery ratio of Ppcps/ks cell differentiation was set to 100% using 1 mM KA, and cell differentiation activities were detected in fractions 15 and 18 ( Figure 1B and Supplemental Figure 1C). Possible KA metabolites in these fractions were determined using liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), as previously reported (Miyazaki et al, 2015). Considering KA (molecular weight 302) and the GA-catabolite GA 34 (molecular weight 348), negatively charged ions were monitored from m/z 299 > 299 to 350 > 350 ( Figure 1C), which revealed that fraction 18 contained unmetabolized KA with an m/z 301 > 301 [M À H]ion ( Figure 1C).…”

mentioning

confidence: 99%

An Ancestral Gibberellin in a Moss Physcomitrella patens

et al. 2018

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Most of the plant hormones found in flowering plants contribute to growth regulation in the moss Physcomitrella patens. This moss does not produce gibberellin (GA) ( Figure 1A). An entkaurene synthase (PpCPS/KS) and an ent-kaurene oxidase (PpKO) have been identified in P. patens, but in contrast to flowering plants, P. patens has no ent-kaurenoic acid oxidase (KAO) homologs (Hayashi et al., 2010; Miyazaki et al., 2011, Figure 1A). Nevertheless, several 2-oxoglutarate-dependent dioxygenase (2ODD) genes, which might be related to the GA synthesis pathway, have been annotated (Hirano et al., 2007), and ent-kaurene-deficient P. patens mutants (Ppcps/ks) showed limited protonemal cell differentiation of chloronemata to caulonemata, which are fast-growing cells that produce gametophores. The application of ent-kaurenoic acid (KA), but not GA, recovered the phenotype. These results support that P. patens lacks a GA biosynthetic pathway downstream of KA, indicating that KA metabolite(s) instead of GA may play physiological roles in P. patens.In this letter, we report the identification, biosynthesis, and characterization of a hormonal diterpenoid compound biosynthesized from KA in P. patens. For detecting KA metabolites, we established a complementation bioassay system using Ppcps/ks mutants that quantifies KA within the 0.0001-1 mM range (Supplemental Figure 1A and 1B). After applying KA to the mutant, metabolites were extracted, fractionated by reversedphase high-performance liquid chromatography (HPLC), and subjected to the assay system. The recovery ratio of Ppcps/ks cell differentiation was set to 100% using 1 mM KA, and cell differentiation activities were detected in fractions 15 and 18 ( Figure 1B and Supplemental Figure 1C). Possible KA metabolites in these fractions were determined using liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), as previously reported (Miyazaki et al., 2015). Considering KA (molecular weight 302) and the GA-catabolite GA 34 (molecular weight 348), negatively charged ions were monitored from m/z 299 > 299 to 350 > 350 ( Figure 1C), which revealed that fraction 18 contained unmetabolized KA with an m/z 301 > 301 [M À H]ion ( Figure 1C). On the other hand, a possible candidate was found in fraction 15: an m/z 317 > 317 [M À H]ion at t R 7.8 min ( Figure 1C). The 16 m/z ions larger than that from KA were assumed to compose the KA skeleton with an oxygen without any desaturation.A further analysis with gas chromatography-EI-MS was performed. The methyl ester-trimethylsilyl ether derivatization of fraction 15 revealed the possible parental ion (m/z 318 + 86 = 404) of OH-KA-Me-TMS at t R 11.16 min (KRI 2508)

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“…1 Quantitative Analysis of Anthocyanin. The frozen plant tissues treated with or without compounds were homogenized, pretreated, and then applied to cation-exchange SPE (Oasis MCX) using the same procedure as described by Miyazaki et al 23 The SPE column was ■ RESULTS PAMD Inhibits the Growth of A. thaliana. PAMD was isolated from a chemical library of 9600 randomly synthesized compounds using SA-induced β-glucuronidase (GUS)-expressing plants (PR1::GUS line) and was determined to be an effective inhibitor of SA signaling.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Development of Inhibitors of Salicylic Acid Signaling

Jiang

Kurimoto

Seo

et al. 2015

J. Agric. Food Chem.

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Salicylic acid (SA) plays important roles in the induction of systemic acquired resistance (SAR) in plants. Determining the mechanism of SAR will extend our understanding of plant defenses against pathogens. We recently reported that PAMD is an inhibitor of SA signaling, which suppresses the expression of the pathogenesis-related PR genes and is expected to facilitate the understanding of SA signaling. However, PAMD strongly inhibits plant growth. To minimize the side effects of PAMD, we synthesized a number of PAMD derivatives, and identified compound 4 that strongly suppresses the expression of the PR genes with fewer adverse effects on plant growth than PAMD. We further showed that the adverse effects on plant growth were partially caused the stabilization of DELLA, which is also related to the pathogen responses. These results indicate that compound 4 would facilitate our understanding of SA signaling and its cross talk with other plant hormones.

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Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

Cited by 6 publications

References 19 publications

Determination of Kaurenoic Acid in Acanthopanax trifoliatus by Ultra-High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry (UHPLC-MS/MS)

Determination of Kaurenoic Acid in Acanthopanax trifoliatus by Ultra-High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry (UHPLC-MS/MS)

An Ancestral Gibberellin in a Moss Physcomitrella patens

Development of Inhibitors of Salicylic Acid Signaling

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