#METABOLOMICS WORKBENCH Claude19_20250121_233924 DATATRACK_ID:5555 STUDY_ID:ST003707 ANALYSIS_ID:AN006082 PROJECT_ID:PR002301 VERSION 1 CREATED_ON February 5, 2025, 6:17 pm #PROJECT PR:PROJECT_TITLE Reprogramming the metabolome of Centella asiatica (L.) Urban callus: Profiling PR:PROJECT_TITLE of newly synthesized cryptic anthocyanins triggered by LED light exposure PR:PROJECT_TYPE Plant metabolomics PR:PROJECT_SUMMARY In addition to the pentacyclic triterpenoid centelloids, Centella asiatica also PR:PROJECT_SUMMARY synthesizes hydroxycinnamic acid conjugates as well as flavonoids. The latter is PR:PROJECT_SUMMARY the major class of secondary plant metabolites and comprises of various PR:PROJECT_SUMMARY subclasses, including anthocyanidins. Anthocyanins are rarely reported in PR:PROJECT_SUMMARY extracts from C. asiatica and differ structurally due to a flavylium PR:PROJECT_SUMMARY (2-phenylchromenylium) ion that carries a positive charge at the oxygen atom of PR:PROJECT_SUMMARY the C-ring of the basic flavonoid structure. Callus of C. asiatica was initiated PR:PROJECT_SUMMARY and propagated on synthetic media and subjected to different light regimes. PR:PROJECT_SUMMARY White callus resulted from white fluorescent illumination, while purple callus PR:PROJECT_SUMMARY developed in response to white light emitting diode (LED) illumination. In order PR:PROJECT_SUMMARY to profile the metabolites responsible for the intense purple colouration, PR:PROJECT_SUMMARY methanolic extracts were prepared from the two cell lines. Total phenolic, PR:PROJECT_SUMMARY flavonoid and anthocyanin content were determined and indicated (i) very low PR:PROJECT_SUMMARY levels of flavonoids and anthocyanins in white callus and (ii) that anthocyanins PR:PROJECT_SUMMARY dominate the flavonoid content of the purple callus. Extracts were subjected to PR:PROJECT_SUMMARY untargeted ultra high-performance liquid chromatography coupled to PR:PROJECT_SUMMARY high-definition mass spectrometry to profile newly synthesised anthocyanins. PR:PROJECT_SUMMARY Metabolite annotation was based on accurate mass determination and PR:PROJECT_SUMMARY characteristic fragmentation patterns. We report on the reprogramming of the PR:PROJECT_SUMMARY metabolome of white C. asiatica callus due to LED illumination and profile the PR:PROJECT_SUMMARY cryptic anthocyanins as well as putative flavonoid and caffeoylquinic acid PR:PROJECT_SUMMARY co-pigments in purple callus. PR:INSTITUTE University of Johannesburg PR:DEPARTMENT Biochemistry PR:LABORATORY Prof Dubery Lab PR:LAST_NAME Hamany Djande PR:FIRST_NAME Claude Yasmine PR:ADDRESS 81A Fourth Avenue Westdene PR:EMAIL claudeh@uj.ac.za PR:PHONE 0814415123 #STUDY ST:STUDY_TITLE Reprogramming the metabolome of Centella asiatica (L.) Urban callus: Profiling ST:STUDY_TITLE of newly synthesized cryptic anthocyanins triggered by LED light exposure ST:STUDY_TYPE Plant metabolomics ST:STUDY_SUMMARY In addition to the pentacyclic triterpenoid centelloids, Centella asiatica also ST:STUDY_SUMMARY synthesizes hydroxycinnamic acid conjugates as well as flavonoids. The latter is ST:STUDY_SUMMARY the major class of secondary plant metabolites and comprises of various ST:STUDY_SUMMARY subclasses, including anthocyanidins. Anthocyanins are rarely reported in ST:STUDY_SUMMARY extracts from C. asiatica and differ structurally due to a flavylium ST:STUDY_SUMMARY (2-phenylchromenylium) ion that carries a positive charge at the oxygen atom of ST:STUDY_SUMMARY the C-ring of the basic flavonoid structure. Callus of C. asiatica was initiated ST:STUDY_SUMMARY and propagated on synthetic media and subjected to different light regimes. ST:STUDY_SUMMARY White callus resulted from white fluorescent illumination, while purple callus ST:STUDY_SUMMARY developed in response to white light emitting diode (LED) illumination. In order ST:STUDY_SUMMARY to profile the metabolites responsible for the intense purple colouration, ST:STUDY_SUMMARY methanolic extracts were prepared from the two cell lines. Total phenolic, ST:STUDY_SUMMARY flavonoid and anthocyanin content were determined and indicated (i) very low ST:STUDY_SUMMARY levels of flavonoids and anthocyanins in white callus and (ii) that anthocyanins ST:STUDY_SUMMARY dominate the flavonoid content of the purple callus. Extracts were subjected to ST:STUDY_SUMMARY untargeted ultra high-performance liquid chromatography coupled to ST:STUDY_SUMMARY high-definition mass spectrometry to profile newly synthesised anthocyanins. ST:STUDY_SUMMARY Metabolite annotation was based on accurate mass determination and ST:STUDY_SUMMARY characteristic fragmentation patterns. We report on the reprogramming of the ST:STUDY_SUMMARY metabolome of white C. asiatica callus due to LED illumination and profile the ST:STUDY_SUMMARY cryptic anthocyanins as well as putative flavonoid and caffeoylquinic acid ST:STUDY_SUMMARY co-pigments in purple callus. This study will provide contribute to the ST:STUDY_SUMMARY knowledge this understudied class of metabolites and will bring light into the ST:STUDY_SUMMARY role of LED in the activation their production in vitro. ST:INSTITUTE University of Johannesburg ST:DEPARTMENT Biochemistry ST:LABORATORY Prof Dubery Lab ST:LAST_NAME Hamany Djande ST:FIRST_NAME Claude Yasmine ST:ADDRESS 81A Fourth Avenue Westdene ST:EMAIL claudehamany@gmail.com ST:PHONE 0814415123 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Centella asiatica SU:TAXONOMY_ID 48106 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - SKR Continuum #1a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #1a.mzML SUBJECT_SAMPLE_FACTORS - SKR Continuum #2a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #2a.mzML SUBJECT_SAMPLE_FACTORS - SKR Continuum #3a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #3a.mzML SUBJECT_SAMPLE_FACTORS - SKR Continuum #4a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #4a.mzML SUBJECT_SAMPLE_FACTORS - SKR Continuum #5a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #5a.mzML SUBJECT_SAMPLE_FACTORS - SKR Continuum #6a Sample source:Centella callus | Callus type:Centella white callus RAW_FILE_NAME(Raw file name)=SKR Continuum #6a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #1a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #1a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #2a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #2a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #3a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #3a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #4a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #4a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #5a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #5a.mzML SUBJECT_SAMPLE_FACTORS - SKW Continuum #6a Sample source:Centella callus | Callus type:Centella purple callus RAW_FILE_NAME(Raw file name)=SKW Continuum #6a.mzML #COLLECTION CO:COLLECTION_SUMMARY Commercially cultivated C. asiatica was obtained from a local nursery (Gauteng CO:COLLECTION_SUMMARY province, South Africa). A voucher specimen (J. James 1-JRAU) was deposited in CO:COLLECTION_SUMMARY the herbarium of the Botany Department, University of Johannesburg, South CO:COLLECTION_SUMMARY Africa. Callus from stem segments of C. asiatica plants was initiated on CO:COLLECTION_SUMMARY Murashige and Skoog (MS) medium solidified with phytoagar with Murashige and CO:COLLECTION_SUMMARY Skoog (MS) vitamins and hormones (Ducheva, Haarlem, Netherlands). The vitamins CO:COLLECTION_SUMMARY and organics were: 50 mg nicotinic acid, 50 mg thiamine HCl, 10 mg pyridoxine CO:COLLECTION_SUMMARY HCl, 10 mg myo-inositol, 0.1 g casein hydrolysate and 3 g sucrose per 100 mL). CO:COLLECTION_SUMMARY The phytohormones were 2 µM dichloro-phenoxyacetic acid (2,4-D) and 0.5 µM CO:COLLECTION_SUMMARY 6-benylaminopurine (BAP). To obtain callus proliferation, 1 g of callus was CO:COLLECTION_SUMMARY dissected from the ends of the stem segments and aseptically transferred to CO:COLLECTION_SUMMARY Petri-dishes with the medium as described above for further cultivation. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY The cultures were kept in an incubator cabinet with white fluorescent lights (~ TR:TREATMENT_SUMMARY 20 μmol/m2/s) with a 18/6 h light/dark cycle and regulated temperature at TR:TREATMENT_SUMMARY 23°C. Callus was sub-cultured every two weeks and eventually achieved a pure TR:TREATMENT_SUMMARY white appearance. When transferred to a plant growth room with cool white LED TR:TREATMENT_SUMMARY illumination above cultivation shelves (~60 μmol/ m2/s), the edges of the calli TR:TREATMENT_SUMMARY turned a light red colour. Further selection under the LED lights of calli TR:TREATMENT_SUMMARY displaying the red colour eventually resulted in calli with a uniform, purple TR:TREATMENT_SUMMARY appearance (Fig. 1). All calli were sub-cultured on fresh media at two-week TR:TREATMENT_SUMMARY intervals. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Unless otherwise specified, all chemicals used in the research were obtained SP:SAMPLEPREP_SUMMARY from Merck-Sigma-Aldrich, (Modderfontein, South Africa) and all solvents were SP:SAMPLEPREP_SUMMARY analytical grade and obtained from SpS, Romil (Cambridge, UK). For phytochemical SP:SAMPLEPREP_SUMMARY screening, metabolites were extracted based on the metabolites of interest and SP:SAMPLEPREP_SUMMARY as described in each section. Irrespective of the purpose of the extraction, a SP:SAMPLEPREP_SUMMARY 1:2 (w/v) ratio was used, either with analytical grade methanol or ethanol as SP:SAMPLEPREP_SUMMARY stipulated. White and purple callus were used, two weeks following sub-culture. SP:SAMPLEPREP_SUMMARY 1.5 g of the calli were taken and extracted into 3.0 mL (1:2 m/v) analytical SP:SAMPLEPREP_SUMMARY grade solvent. The tissue was homogenised using an Ultra-turrax type shaft SP:SAMPLEPREP_SUMMARY homogeniser (CAT, Berlin, Germany) for 2 min, followed by sonication in an SP:SAMPLEPREP_SUMMARY ultrasonic bath for 15 min. The homogenates were centrifuged at 13,000 g and the SP:SAMPLEPREP_SUMMARY supernatants transferred to 2 mL microcentrifuge tubes. Extractions were SP:SAMPLEPREP_SUMMARY repeatedly performed as described above over a period of one year with SP:SAMPLEPREP_SUMMARY consistent results. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For UHPLC-MS analysis, extracted samples were concentrated to half of the CH:CHROMATOGRAPHY_SUMMARY original volumes at 50 °C in a dry bath in a fume hood and centrifuged again at CH:CHROMATOGRAPHY_SUMMARY 13, 000 g for 10 min before transfer to chromatography vials, capped and stored CH:CHROMATOGRAPHY_SUMMARY for analysis. Samples were analysed using a Waters Acquity Premier UPLC system CH:CHROMATOGRAPHY_SUMMARY (Waters Corporation, Milford, MA, USA) and separation was obtained with a Waters CH:CHROMATOGRAPHY_SUMMARY HSS T3 C18 UPLC column (150 mm x 2.1 mm, 1.8 µm). A solvent mixture was used CH:CHROMATOGRAPHY_SUMMARY consisting of ultra-pure water (solvent A) and UPLC-grade acetonitrile (solvent CH:CHROMATOGRAPHY_SUMMARY B). Both solvents contained 10 mM formic acid. The initial conditions were 95% A CH:CHROMATOGRAPHY_SUMMARY for 1 min, followed by a gradient to 1% A at 16 min. These chromatographic CH:CHROMATOGRAPHY_SUMMARY conditions were maintained for 1 min whereafter the initial conditions were CH:CHROMATOGRAPHY_SUMMARY re-instated. The flow rate was kept constant at 0.4 mL/min and the column CH:CHROMATOGRAPHY_SUMMARY temperature maintained at 60 °C. The runtime was 20 min with injection volumes CH:CHROMATOGRAPHY_SUMMARY varying between 1 and 5 μL. The Waters Sample Manager temperature was CH:CHROMATOGRAPHY_SUMMARY maintained at 6 °C. The Flow rate was 0.4 mL/min. Gradient information: Initial CH:CHROMATOGRAPHY_SUMMARY %A95 %B5 1min %A95 %B5 16min %A10 %B90 16.10min %A1 %B99 17min %A1 %B99 18min CH:CHROMATOGRAPHY_SUMMARY %A1 %B99 18.30min %A95 %B5 20min %A95 %B5 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT Initial %A95 %B5; 1min %A95 %B5; 16min %A10 %B90; 16.10min %A1 %B99; 17min %A1 CH:FLOW_GRADIENT %B99; 18min %A1 %B99; 18.30min %A95 %B5; 20min %A95 %B5 CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Prof Dubery's Lab #MS MS:INSTRUMENT_NAME Waters Synapt-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS For high-definition quadrupole time-of-flight mass spectrometry (HD-qTOF) MS MS:MS_COMMENTS analysis, a Waters Synapt XS system, equipped with a 4 kDalton (Da) quadrupole, MS:MS_COMMENTS was used in sensitivity mode. Accurate mass measurements were obtained using MS:MS_COMMENTS leucine encephalin (554.2615 Da) as the ‘lockmass’ calibrant at a fixed MS:MS_COMMENTS concentration of 200 pg/mL and a constant flow rate of 5 μL/min. The instrument MS:MS_COMMENTS was used in sensitivity mode with an electrospray ionisation (ESI) interface. MS:MS_COMMENTS Analysis was done in positive ionisation mode using a capillary voltage of 0.6 MS:MS_COMMENTS kV, a sample cone voltage of 30 V and source offset of 4 V. Data was collected MS:MS_COMMENTS from 100 to 1500 Da at a scan speed of 0.1 sec. The source temperature was set MS:MS_COMMENTS at 120 °C and a desolvation temperature of 450 °C was used for all the MS:MS_COMMENTS analysis. High purity nitrogen gas was used as the nebulisation gas at a flow MS:MS_COMMENTS rate of 700 L/h and the cone gas flow rate was 50 L/h. MassLynxTM software, V4.2 MS:MS_COMMENTS SCN1028 (Waters corporation, Milford, MA, USA) was used to control the MS:MS_COMMENTS instrumentation and to collect and process all the data. MS:MS_RESULTS_FILE ST003707_AN006082_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END