Bioactive clerodane diterpenes of giant goldenrod ( Solidago gigantea Ait.) root extract

Giant goldenrod ( Solidago gigantea Ait.) root extract was screened for bioactive compounds by high- performance thin-layer chromatography (HPTLC), coupled with effect-directed analysis including antibacterial ( Bacillus subtilis F1276, B. subtilis subsp. spizizenii, Aliivibrio ﬁscheri and Xanthomonas euvesicatoria ), antifungal ( Fusarium avenaceum ) and enzyme inhibition (acetyl- and butyrylcholinesterases, α - and β glucosidases and α -amylase) assays. Compounds of six multipotent zones (Sg1-Sg6) were characterized by HPTLC-heated electrospray ionization-high-resolution mass spectrometry (HRMS) and HPTLC-Direct Analysis in Real Time-HRMS. Apart from zone Sg3, containing three compounds, a single characteristic compound was detectable in each bioactive zone. The bioassay-guided isolation using preparative-scale ﬂash chromatography and high-performance liquid chromatography provided eight compounds that were identiﬁed by NMR spectroscopy as clerodane diterpenes. All isolates possessed inhibiting activity against at least one of the tested microorganisms. http://creativecommons.org/licenses/by/4.0/


Introduction
A continuously higher qualitative and quantitative supply of agricultural raw materials can meet the increasing demand of the food and feed industry. To improve plant production, appropriate pest management is needed, using effective agrochemicals that are more and more difficult to find due to the emerging (multi)resistance in pathogens against the generally used agents [ 1 , 2 ]. Therefore, many research projects are aimed at discovering effective agrochemicals with new chemical base structure and possibly with low toxicity and fast biodegradability. In the last decade synthetic approaches have not resulted in new antibacterial agents, and the target-based drug discovery also brought disappointment [3] . Purposeful tracking, characterization and isolation of bioactive compounds from natural sources can be achieved by bioassay-guided processes, comprising extraction, fractionation and purification steps, all associated with biomonitoring [ 4 , 5 ]. The high-throughput, relatively cheap effect-directed analysis (EDA) is mination and/or mycelial growth inhibition by separated bioactive compounds. The inhibition zone was indicated by the lack of visible fungal hyphae, however, vital dyes [15,16] or iodine vapor [18] significantly improved the detectability. Conidial suspension of F. avenaceum has been used for dot blot test [18] , which is similar to direct bioautography, but the TLC adsorbent is only used to hold the sample, not to separate its components. Spore germination and hyphal growth are distinct biological processes, thus, to assess inhibition of the fungal growth independently of germination, a hyphal segments (practically, their suspension) are required. The use of mycelial growth inhibitors are useful in plant protection as they prevent local spread of the fungal pathogen. So far, neither an HPTLC adsorbent nor a mycelial suspension have been applied for direct bioautography of Fusarium species.
Solidago gigantea (giant goldenrod) is native to North America. About 250 years ago, it was introduced to Europe as an ornamental and has become an exceptionally successful invasive and competitive species in most European countries with an abundant biomass [19] . It is widespread in whole Europe and a serious invader of abandoned fields, forest edges and river banks [20] . Goldenrod is also a medicinal plant and listed in the European Pharmacopoeia as Solidaginis herba (the whole or cut dried flowering aerial part of either S. gigantea Ait. and/or S. canadensis L.) used to treat disorders of the urinary tract, prostate and kidney. The goldenrod extract was shown to display favourable antibacterial [21] , antifungal [22] , insecticidal [23] and anti-obesity [24] activities that can be attributed to its essential oil [25] , phenolics [26] , saponins [27] and diterpenes [28] .
Recently, S. gigantea root extract was reported to have antihyperglycaemic ( αand β-glucosidase and α-amylase inhibitory) and cholinesterase inhibitory effects in a screening of five goldenrod species [29] . The present study targeted the detailed characterization and bioprofiling of the giant goldenrod root extract using HPTLC-EDA. The discovered active compounds against enzymes, bacteria ( Bacillus subtilis, Aliivibrio fischeri and Xanthomonas euvesicatoria ) and fungus ( F. avenaceum ) were characterized by HPTLCheated electrospray ionization (HESI)-high resolution (HR)MS and HPTLC-direct analysis in real time (DART)-HRMS. The bioassayguided isolated compounds were identified by NMR and their antimicrobial activity was confirmed by HPTLC-antimicrobial tests.

Sample preparation
Roots of Solidago gigantea Ait. were collected in February 2017 (young shoots), August 2017 (full flowering) and July 2019 (full flowering) in the Great Plain, Hungary (N 46 °41 52.1" E 19 °03 3.6" Alt. 90 m). The fresh root was gently washed with water, chopped, dried at room temperature and ground (Bosch MKM60 0 0, Stuttgart, Germany). Powdered samples were macerated in ethanol (150 mg/mL) for 24 h. The filtered crude extract (17 mg dry weight/mL) was used for HPTLC and isolation. Isolated compounds were dissolved in ethanol (2.5 mg/mL).

HPTLC method
Root extracts (1-2 μL/band for antimicrobial tests and 5 μL/band for enzyme tests) and isolated compounds (0.2-0.5 μL/band for antimicrobial tests and 2 μL/band for reagent) were applied as 6-mm bands with 8-10 mm track distance onto the HPTLC plate (Automated TLC Sampler ATS4 or ATS3, CAMAG, Muttenz, Switzerland) at 8 mm distance from the bottom. HPTLC separation was carried out with n -hexane -isopropyl acetate -acetone 16:3:1, V/V/V (MP1) [29] or n -hexane -isopropyl acetateacetic acid 40:9:1, V/V/V (MP2) up to a migration distance of 70 mm (Twin Trough Chamber TTC, CAMAG). Plates were dried in a cold stream of air (5 min). Residues of acetic acid were eliminated by a 20-min drying (Automatic Developing Chamber ADC2, CAMAG) or by potassium hydroxide in the opposite TTC trough for 2 h [30] . The excess of potassium hydroxide was evaporated by a stream of cold air for 15 min. The plate was cut (blade or smartCUT Plate Cutter, CAMAG) into segments for various bioactivity assays or derivatization with vanillin sulphuric acid reagent (40 mg vanillin, 10 mL ethanol and 200 μL concentrated sulphuric acid, heated at 110 °C for 5 min and documented at UV 365 nm and white light illumination in transmittance mode). The chromatograms were detected by a UV lamp and digital camera (Cybershot DSC-HX60, Sony, Neu-Isenburg, Germany) or TLC Visualizer Documentation System or TLC Scanner 4 (both CAMAG).

HPTLC-EDA
Bacterial cell suspensions were prepared and the antibacterial effect was detected, as described in previous methods using B. subtilis F1276 [31] , B. subtilis subsp. spizizenii [32] , A. fischeri [ 33 , 34 ] and X. euvesicatoria [31] . Briefly, the developed, neutralized plates were immersed into the cell suspensions for 6 s. The dark antibacterial zones in the bioautograms of luminescent A. fischeri were instantly documented (BioLuminizer, CAMAG or iBright TM FL10 0 0 Imaging System, Thermo Fisher Scientific, Budapest, Hungary). In the cases of non-luminescent bacteria, bioautograms were visualized after a 2-h incubation (100% humidity at appropriate temperature) by staining with an aqueous MTT solution (1 mg/mL) fol-lowed by a 0.5-h incubation. Colorless (bright) antibacterial zones were obtained against a bluish background.
The reported HPTLC-AChE/BChE [35] , HPTLC-α/ β-glucosidase and HPTLC-α-amylase [29] inhibition assays were applied. For the α-amylase assay, the plate was immersed into the substrate solution (1.4 mg/mL CNP-G3 in ethanol), dried for 2 min in a stream of cold air, immersed in the buffered α-amylase enzyme solution, incubated at 37 °C for 15 min and dried. For the other enzyme assays, each plate was dipped into the respective buffered enzyme solution and incubated for 15-20 min at 37 °C. The AChE/BChE and α/ β-glucosidase autograms were immersed in a solution of the substrate ( α-naphthyl acetate and 2-naphthyl-α/ β-D-glucopyranoside, respectively, followed by a 10-min incubation) and the chromogenic reagent (Fast Blue Salt B) and dried. Documentation was performed at white light illumination in the reflectance mode. Colorless (bright) zones indicated the active compounds against a yellow ( α-amylase) or violet (AChE/BChE and α/ β-glucosidase) background. The detailed enzyme inhibition assay methods are presented in the supplementary data.
The F. avenaceum strain was grown on V8 agar medium (80 mL water, 20 mL Campbell's V8 vegetable juice, 100 μL 1 M potassium hydroxide to adjust to pH 6.5, 0.4 m/m% calcium carbonate, 1.8 g agar) at 20 °C in the dark. Lysogenic broth (LB, 20 mL; 10 g/L tryptone, 5 g/L yeast extract and 10 g/L sodium chloride) was inoculated in a 100 mL Erlenmeyer flask with the fungal culture grown on the V8 agar plate and shaken at 120 rpm at 21 °C for 3 days in the dark. The mycelium was washed with LB to eliminate the conidia and cut to small pieces with a sterile rotor-stator type homogenizer (Model TR-10, Tekmar, Cincinnati, OH) for 2 min two times. The mycelium suspension was diluted to OD 600 0.4 -0.8. Developed HPTLC plates were dipped into the mycelium suspension for 6 s and incubated in a vapor chamber at 21 °C for 48 -72 h. The lack of visible white fungal hyphae indicated the inhibition zones. By spraying aqueous INT solution (1 mg/mL; cca 2 mL/plate) on the bioautograms followed by a 1-h incubation, the contrast was enhanced by revealing bright inhibition zones against a violet background. An aqueous solution of benomyl (1 μL/band; 25 mg/mL, active ingredient of Fundazol 50WP) was applied as positive control (at the edge of the developed plate before immersion in the mycelium suspension).
To evaluate the effects on B. subtilis subsp. spizizenii and AChE, the (bio)autograms were scanned in the fluorescence mode at 546 and 533 nm using the mercury and wolfram lamp (TLC Scanner 4, CAMAG), respectively. Images were processed with ImageJ software (NIH, Bethesda, MA, USA).

HPTLC-HRMS and HRMS-MS
Prewashed plates (with methanol −water 4:1, V / V dried at 100 °C for 20 min) were used and separated zones of interest were marked. For HPTLC-HESI-HRMS, the quaternary pump (Ultimate LPG-3400 XRS, Dionex Softron, Germering, Germany) guided the methanol at a flow rate of 0.1 mL/min through the oval elution head (4 mm x 2 mm) of the PlateExpress interface (Advion, Ithaca, NY, USA) to the HESI-II installed at the hybrid quadrupole-orbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific, Bremen, Germany). Spray voltage was 3.5 kV, capillary temperature was 270 °C, and nitrogen as sheath and auxiliary gas (20 and 10 arbitrary units, respectively) was produced by an SF2 compressor (Atlas Copco Kompressoren und Drucklufttechnik, Essen, Germany). Full scan HRMS spectra were recorded in the negative and positive ionization mode in the range of m / z 50 -750 with a resolution of 280,0 0 0, automatic gain control target of 3 × 10 6 and maximum injection time of 100 ms. Isolated compounds and flash chromatographic fractions were directly injected by flow injection analysis (FIA) in the mentioned HRMS system (spray voltage 3.5 kV, capil-lary temperature 270 °C, sheath and auxiliary gases at 20 and 10 arbitrary units, respectively). Tandem mass spectra were acquired as parallel reaction monitoring with mass isolation of the target molecule (fragmentation/collision energy of 40 -60 eV, resolution of 17,500, automatic gain control target of 2 × 10 5 , maximum injection time of 100 ms and isolation window of m / z 2 for m / z 50). Operation and data processing were performed with Xcalibur 3.0.63 software (Thermo).
A DART interface (IonSense, Saugus, MA, USA) modified for scanning of HPTLC plates [36] was coupled to the mentioned HRMS system. The ion source was operated with an initial needle voltage of 4 kV and grid voltage of 50 V in the positive ionization mode using helium (99.999%) at a flow rate of 3.0 L min −1 and temperature of 500 °C. Spectra were recorded in the full scan ( m / z 100 − 750) at a resolution of 280,0 0 0, automatic gain control target of 5 × 10 4 and maximum injection time of 50 ms. The DART scanning speed was 0.2 mm s −1 . The extracted ion current (EIC) chromatograms were processed with a Gauss smoothing function width of 11 points.

Preparative column chromatography
The extract (10 mL, dried and re-suspended in n -hexane) was fractionated with Combiflash NextGen 300 (Teledyne Isco, Lincoln, NE, USA) flash chromatography system. Separation was performed on a RediSep Rf Gold silica gel column (20-40 μm, 12 g; Teledyne Isco) at a flow rate of 30 mL/min with a gradient of nhexane (A) and acetone (B): 0-0.5 min, 0% B; 0.5-8.5 min 0-30% B. The chromatogram was monitored by absorbance measurement at 215 nm. Compounds in the collected active fractions were further fractionated and purified. HPLC separations (conditions are in Table 1 ) were carried out using an LC-MS-2020 system (Shimadzu, Kyoto, Japan), including binary gradient solvent pump, vacuum degasser, thermostated autosampler, column oven, photodiode detector and electrospray ionization (ESI)-MS system. Instrument control and data acquisition were performed with the LabSolutions 5.42v program (Shimadzu). First, analytical methods were developed that were scaled up using a semi-preparative column. The analytical separation of the fractions (5 μL) on a Gemini C18 column (250 mm x 4.6 mm ID, 5 μm particle size, Phenomenex, Torrance, CA, USA) at 35 °C with a step-wise gradient was detected by MS (nitrogen as nebulizer gas, flow rate 1.5 L/min, drying gas (nitrogen) flow rate 15 L/min, interface temperature 350 °C, heatblock temperature 400 °C, desolvation line temperature 250 °C and detector voltage 4.5 kV). Full scan mass spectra were recorded in the positive and negative ionization mode in the range of m / z 150 -900 with a scan speed of 5000 amu/s. The semi-preparative separation of the fractions (100 μL) on a Gemini C18 column (250 mm x 10 mm ID, 10 μm particle size, Phenomenex) was detected in the UV. Compound Sg6 (90 μL) was purified on a Kinetex pentafluorophenyl (PFP) column (100 mm x 4.6 mm ID, 2.6 μm particle size, Phenomenex) at 35 °C. The fractionation/purification was repeated up to 10-times. The combined fractions were investigated by HPTLC-assays, dried with a rotary evaporator (Rotavapor R-134, Büchi, Flawil, Switzerland) at 40 °C and transferred to NMR spectroscopy.

HPTLC-EDA
Two methods were investigated for the separation of the giant goldenrod root compounds ( Figs. 1 and 2 ) on HPTLC plates silica gel 60 with either n -hexane -isopropyl acetate -acetone 16:3:1 V/V/V (MP1, [29] ) or n -hexane -isopropyl acetate -acetic acid 40:9:1 V/V/V (MP2). Both separations detected at white light illumination after derivatization with the universal vanillin sulphuric acid reagent, provided the six distinguishable colored zones Sg1-Sg6 that were hardly detectable without the derivatization ( Figs. 1 a, 1 b, 2 a and 2 b). MP2 resulted in higher hR F values, which was obvious for Sg5 and Sg6, indicating a potential acidic character. MP2 chromatograms required a neutralization step before EDA. Compounds with antidiabetic (anti-hyperglycaemic) effect could be used for the treatment of type 2 diabetes and obesity that are the most common and increasing chronic diseases in the world. In the HPTLC-EDA screening with MP1 ( Fig. 1 e-k), the zones Sg5 (at hR F 58) and Sg6 (at hR F 37) showed α-glucosidase, β-glucosidase and α-amylase inhibition. The application zone up to hR F 9 was also effective in α-glucosidase and α-amylase assays. Cholinesterase inhibitors are of therapeutic interest in the Alzheimer disease, the most common cause of dementia and one of the major public health issues. Compounds in the zones Sg1 ( hR F 9), Sg4 ( hR F 51), Sg5 and Sg6 inhibited AChE and BChE ( Fig. 1 e and f). However, with MP2 and neutralization, BChE inhibitory zones of Sg1 ( hR F 17) and Sg4 ( hR F 55) were lacking ( Fig. 2 f). Multipotent compounds are generally not favorable for pharmaceutical use, however, these compounds can become lead compounds toward the development of more specific, thus more useful derivatives.
Antibacterial agents are widely required for the fight against infectious plant, animal and human diseases caused by pathogenic bacterial and fungal strains. In the antibacterial assays, the Grampositive B. subtilis subsp. spizizenii soil bacterium, the Gramnegative, naturally luminescent marine A. fischeri and the Gramnegative paprika pathogen X. euvesicatoria were employed. In the B. subtilis assay, Sg1 and compounds at an even lower hR F were only active in the MP2 separation ( Figs. 1 g and 2 g). All marked zones were active against A. fischeri ( Figs. 1 h and 2 h) and X. euvesicatoria ( Fig. 2 i), except for Sg3 at the given amount applied. Additionally, Sg6 showed activity against A. fischeri only on acidfree bioautograms. The positions of the bioactive zones were con-  firmed by densitometry or videodensitometry of the MP2 bioautograms (Fig. S1). The results of the enzyme assays are in agreement with our previous observations [29] , however, the influence of an acidic development and its neutralization on the outcome needs further study.
An HPTLC-antifungal assay using plant pathogenic fungus F. avenaceum was newly developed. F. avenaceum grew well on V8 solid agar medium and the surface of a 5 cm petri dish was totally occupied by its white hyphae within 2-3 days after inoculation by an agar block (5 mm x 5 mm) in the center. Similarly, a dense mycelium suspension was obtained within 3 days by shaking the liquid inoculum (LB medium) at 21 °C. After cutting the mycelium to small fragments, the suspension was initially diluted to reach an OD 600 of 0.4. Dipping a plate into this suspension, an appropriate mycelium growth was optically reached after a 3-days incubation in a vapor chamber at 21 °C ( Fig. 3 ), indicating as mycelium growth inhibition the antifungal activity of zones Sg1, Sg2, Sg3, Sg4 and Sg5. For detection, the three different tetrazolium dyes INT, MTT and TTC were investigated, whereby the fastest coloration was achieved with INT, which colored the hyphae violet ( Figs. 3 and  S2). Compared to the 3-days-old mycelium suspension, the 1-dayold inoculum did not grow as fast on the HPTLC plate, and the 5-days-old was found to be less sensitive (Fig. S3). In agreement to our previous study, no characteristic inhibition was observed using older microbial (bacterial) cells in the stationary and death phases [37] . The staining procedure enabled the reduction of the incubation time to 2 days. The incubation time could not be shortenedby increasing the OD 600 of the mycelial inoculum from 0.2 to 0.8 -whithout a loss in discernability of the inhibition zones. In the final protocol, the mycelium suspension at an OD 600 of 0.4 was incubated for 2 days on the HPTLC plate and then detected by INT staining, followed by a 1-h incubation and documentation under white light illumination in the reflectance mode (Fig. S4).

HPTLC-MS
The bioactive compound zones Sg1-Sg6 were separated with MP1 and characterized by HPTLC-HESI-HRMS and HPTLC-DART-HRMS ( Table 2 (Fig. S6). In the positive and negative ionization mode, also the sodium-methanol-adducts [M + CH 3 OH + Na] + and sodium-acetate-adducts of the respective compounds were observed, which was caused by a too low drying gas flow.
With exception of Sg1, all other compound zones gave mass signals by HPTLC-DART-HRMS (

Isolation of the active compounds
The root extract (10 mL) was fractionated by flash chromatography on a silica gel column, resulting in nineteen fractions (fr1-fr19), which were studied by HPTLC-Vis after derivatization with the vanillin sulphuric acid reagent ( Fig. S8a and b). The six fractions containing the previously characterized bioactive compounds (fr10-fr14 and fr17) were subjected to RP-HPLC-DAD-ESI-MS anal-ysis (Fig. S8c, Table 2 ). The HPLC method 1 ( Table 1 ) was found to be suitable for compound isolation in the Sg1, Sg2 and Sg5 zones, whereby the latter zone was tailing. Acid-free mobile phases were selected to avoid acid-analyte reactions, which might impair the bioactivity result. However, in the case of Sg5, the collected peak was further purified with an acidic conditioning in the HPLC method 4. The Sg6 compound zone was eluted from the C18 column during the washing step. Thus, its retention was achieved on a PFP column (HPLC method 5). The HPTLC separation revealed two co-eluting compounds in each of the HPLC peaks at 10-min (Sg3a and Sg3b) and 12.5-min retention times (Sg3c and Sg4) (Fig. Table 2 Chemical structures, molecular formulae and assignments (using orthogonal techniques) of the eight discovered bioactive compounds in S. gigantea root extract.

Identification and characterization of the isolated compounds
The isolates were analyzed by FIA-HESI-HRMS/MS to successfully confirm that their identity was not changed during the isolation procedure, including fractionation and purification. Fragmentation of the respective sodium adduct of the compounds was not observed except for Sg6 ( Table 2 ), and thus, also the respectively less intense protonated or deprotonated molecules were selected. The intense deprotonated molecules of Sg5 and Sg6 were easily fragmented.
The HPTLC-Vis analysis of the isolates (separated with MP1, Fig. 5 a, and MP2, Fig. S11 and detected after derivatization with the vanillin sulphuric acid reagent) showed that only a single compound was present in all previously assigned zones, except for three compounds (Sg3a-c) detected in Sg3. All isolates were active against B. subtilis , whereby Sg3b had only a weak activity ( Fig. 5 b). Most isolated compounds strongly affected A. fischeri , whereby Sg3b showed a mild effect, and Sg3c and Sg6 no response at all ( Fig. 5 c). All compounds except Sg3b suppressed the growth of F. avenaceum mycelium with varying degrees of effectiveness ( Fig. 5 d). Although for the Sg3 and Sg6 zones of the root extract, no activity against B. subtilis and F. avenaceum , respectively, was evident, their respective isolates did have an antimicrobial effect. This was explained by the fact that their concentrations in the root extract were too low to reach the minimum inhibitory concentration. The eight isolated compounds were most probably responsible for the enzyme inhibiting activity, but this was not confirmed. The purity of the eight isolates was adequate, as confirmed by NMR analysis (Table S1, Figs. S12-S59). The eight compounds were identified as the furan-containing clerodane diterpenoids: a glycol (Sg1, known also as kingidiol, white solid), an epoxy-hemiacetal (Sg2, oil), a dialdehyde (Sg3a, white solid), the clerodane lactone (Sg3b, oil, also known as hautriwaic lactone), an alcohol (Sg3c, oil), a hemiacetal (Sg4, oil), the solidagoic acid A (Sg5, white solid) and the solidagoic acid B (Sg6, white solid), which have been described as components of S. gigantea roots [38][39][40] ( Table 2 ). NMR data in literature supported the identification of the isolated compounds [ 28 , 38 , 39 , 41 , 42 ]. Antimicrobial [43][44][45][46] , cytotoxic [45][46][47] and antiinflammatory effect [48] of several clerodane diterpenes isolated from plants have been described. Among Solidago species, clerodane diterpenes of European goldenrod ( S. virgaurea ) were pub-lished to inhibit Staphylococcus aureus [42] . The activity of three of our isolated compounds has already been studied mainly against insects. Sg5 was inactive, Sg1 caused the retardation of follicle development in mosquito ovaries [28] and displayed moderate toxicity against brine shrimp ( Artemia salina ) [49] . In contrast to Sg1, Sg3b was found as anti-feedant and repellent agent in a study of mealworms ( Tenebrio molitor ) [50] . Antiplasmodial (antimalarial) and antileishmanial (inhibition of the intracellular parasite Leishmania donovani ) effects of Sg3b were also observed, whereby it did not show cytotoxicity against Vero cells (African green monkey kidney fibroblast) [51] . Except for the new antifungal profiling, the HPTLC-bioprofiling of the root extract has been previously reported by us, however, it lacked in identification of the active compounds by HRMS and NMR. To the best of our knowledge, this is the first report about the antimicrobial profile of the eight isolated S. gigantea root diterpenoids. Further investigations are intended to evaluate their possible use as safe and effective pesticides, especially fungicides.

Conclusions
The newly developed HPTLC-F. avenaceum assay was demonstrated for the first time using mycelium suspension. It allowed a cost-effective screening of complex plant extracts for antifungal (mycelium growth inhibiting) compounds in S. gigantea root extract. Further bioprofiling of the extract against various bacterial strains and enzymes by HPTLC-EDA pointed to multi-potent compounds. The comparison of the use of a neutral versus acidic mobile phase demonstrated that the latter can influence the bioassay result. The state of the art combination of HPTLC-bioactivity assays, HPTLC-HESI-HRMS, HPTLC-DART-HRMS, preparative-scale column chromatography (flash chromatography and HPLC) and NMR provided eight multi-potent clerodane diterpenoids in goldenrod. It is the first report on the antifungal, antibacterial and enzyme inhibiting activity of the multipotent isolates, which showed potential as lead compounds especially for various infectious plant diseases.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.