Bio-Guided Isolation of New Compounds from Baccharis spp. as Antifungal against Botrytis cinerea

Baccharis genus Asteraceae is widely used in traditional treatment against fever, headache, hepatobiliary disorders, skin ulcers, diabetes, and rheumatism, as well as an antispasmodic and diuretic. Its phytochemistry mainly shows the presence of flavonoids and terpenoids such as monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Some of them have been evaluated for biological activities presenting allelopathic, antimicrobial, cytotoxic, and anti-inflammatory properties. In this paper, our research group reported the isolation, characterization, and antifungal evaluation of several molecules isolated from the dichloromethane extract from Baccharis prunifolia, Baccharis trinervis, and Baccharis zumbadorensis against the phytopathogen fungus Botrytis cinerea. The isolated compounds have not previously been tested against Botrytis, revealing an important source of antifungals in the genus Baccharis. Six known flavones were isolated from B. prunifolia. The dichloromethane extracts of B. trinervis and B. zumbadorensis were subjected to a bio-guided isolation, obtaining three known flavones, an α-hydroxidihydrochalcone mixture, one labdane, one triterpene, and two norbisabolenes from the most active fractions. The compounds 4′-methoxy-α-hydroxydihydrochalcone (7A), 3β,15-dihydroxylabdan-7-en-17-al (8), and 13-nor-11,12-dihydroxybisabol-2-enone (11) are novel. The most active compounds were the Salvigenin (5) and 1,2-dihydrosenedigital-2-one (10) with an IC50 of 13.5 and 3.1 μg/mL, respectively.


Introduction
The Asteraceae constitutes a defined family among flowering plants distributed primarily in the tropical areas of South America [1]. It has approximately 1500 genera and 25,000 species [2]. The genus Baccharis is represented by more than 500 species which are distributed mainly in Brazil, Argentina, Colombia, Chile, and Mexico [3,4]. Species of the genus Baccharis have been widely investigated for their pharmacological properties, well known by Indigenous populations. Medicinal properties include antidiabetic and antiinflammatory attributes. They are also used to treat liver disease, rheumatism, digestive, hepatic, and renal disorders [3]. Phytochemically, the Baccharis genus produces compounds that have been identified and their biological activities studied [5]. The compounds included in Baccharis are mainly flavonoids [6,7] and terpenoids [8], such as diterpenes [9,10] with clerodane, labdane, and kaurane skeletons. Phenolic compounds and essential oils have been reported in the last years as important sources of natural products with interesting biological activities [11,12].
Baccharis trinervis, B. prunifolia, and B. zumbadorensis are widely distributed from Mexico to Argentina [13,14]. These species are used in the treatment of high fevers, edema, ulcers, and vascular cramps [5]. They are also applied in cases of dizziness, gastrointestinal disorders, and against snake venom [15]. Their different parts have been studied [16][17][18], identifying flavonoids [19] and terpenes [16,20]. Plants used in this work had been collected in Venezuela [21], and it is the first study of the antifungal activity of this species of Baccharis against phytopathogenic fungi. The antifungal activity of this genus has been reported mainly against human fungal infections as a part of its medicinal properties [5,13,[22][23][24]. Nevertheless, the activity against phytopathogen fungi of the genus Baccharis has been poorly documented [25]. The traditional treatment against phytopathogen fungi is the use of chemicals that have some serious restrictions due to the effects on the natural environment. Therefore, the rational control of the diseases that they produce, is one of the biggest challenges facing the agricultural-food industry, which raises the need to discover new antifungal products of plant origin that are friendly to the environment. In this context, Baccharis trimera and Baccharis ochracea essential oils inhibited 100% of the growth of Alternaria alternata [26]; essential oil from Baccharis dracunculifolia was tested against Fusarium graminearum [27]; essential oils of plants, including some species of Baccharis, were tested with good results against Monilinia fructicola [28] and Stemphylium solani [29]; and root extracts from Baccharis salicina decreased the percentage of germination of uredospores of Hemileia vastatrix [30].
Botrytis cinerea is one of the most invasive and most important phytopathogen fungi in terms of economic losses [31][32][33]. It is the causal agent of the grey mold in grapes, one of the most important fruit crops worldwide. Among the possibilities of alternative control is the use of essential oils of Baccharis trimera and Baccharis dracunculifolia tested against B. cinerea and Colletotrichum acutatum that showed effectiveness as preventive and curative treatment [25].

General Procedure
Solvents and reagents were purchased from Sigma-Aldrich, Merck, and EurisoTop ® brands with an analytical grade. Silica gel 60 (63-200 µm; 70-230 mesh) from Merck and columns Sephadex LH-20 from Sigma were used for column chromatography.
HPLC purification was performed with an Elite LaChrom-Hitachi HPLC system equipped with L-2400 UV-Vis detector and L-2490 differential refractor detector.
High-resolution mass spectra (HRESIMS) data were recorded in positive and negative modes using Waters SYNAPT equipment and MassLynx 4.1 software.
The Optical activities were measured with a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (λ = 589 nm) with chloroform as solvent at 25 • C. ECD spectrum was measured on a JASCO J-810 spectropolarimeter at ambient temperature. The ECD curves were simulated using SpecDis 1.51 software [34].

Isolates and Cultures
Baccharis trinervis Pers was collected in July 2012 by the Arenal near Tabay road edges at an altitude of approximately 1500 m above sea level, Libertador Municipality. Baccharis prunifolia Steyerm was collected in July 2008 in Gavidea, located on the outskirts of Rangel Municipality, at an altitude of 2950 m above sea level. Baccharis zumbadorensis Badillo was collected in December 2011 in the Paramo de San José de Acequias, located on the outskirts of Campo Elias Municipality, at an altitude of 3300 m above sea level. A Voucher Specimen of each species (J.M. Amaro, No. 2366, No. 2357, and No. 2349, respectively) was deposited in the Herbarium MERF of the Faculty of Pharmacy-ULA. All the municipalities are in Merida State, Venezuela.
The culture of B. cinerea employed in this work, B. cinerea UCA 992, was obtained from grapes from the Domecq vineyard, Jerez de la Frontera, Cádiz, Spain. This culture of B. cinerea is deposited in the Universidad de Cadiz, Facultad de Ciencias, Mycological Herbarium Collection (UCA). The fungus was grown in an agar-tomato plate to increase the sporulation process and incubated at 25 • C the time needed for the fungus to grow and to produce spores (15 to 20 days).

Extraction and Bio-Guided Isolation
The leaves of the plants were recollected and dried at room temperature. The whole leaves (780 g, 2980 g, and 1750 g for B. trinervis, B. prunifolia, and B. zumbadorensis, respectively) were extracted with dichloromethane at room temperature for 24 h. Next, they were dried again under a hood and then ground. The ground material was extracted in a Soxhlet extractor with methanol as a solvent. The solutions from both extractions were filtered and then concentrated on a rotary evaporator at a temperature not exceeding 40 • C. The extracts obtained were kept at −4 • C.
The total fractions obtained from the CC of the crude extract from Baccharis species were tested against B. cinerea UCA992 by Microplate resazurin assay ( Figure 1, Figure 2, Figure 3 and Figures S1-S3 [35].       The most active fractions were submitted to a deeper analysis as follows: The pooled fraction F (15 g) from B. prunifolia was chromatographed on Sephadex LH-20 with hexane-dichloromethane-methanol mixtures 2:1:1 obtaining 15 fractions. A pure, yellow solid compound 1 was obtained in fraction F6 (0.050 g), and a pure compound 2 was obtained in fraction F9 (0.038 g). In addition, fraction H was chromatographed on preparative TLC with hexane-ethyl acetate mixtures 3:2 to obtain a pure compound characterized as Genkwanin (3) (0.019 g) and Galangustin (6) (0.010 g), both as solids. Compound 4 (0.015 g) was obtained as a solid in fraction G (2.37 g). Finally, the fraction I (2.37 g) was chromatographed on preparative TLC with hexane-ethyl acetate mixtures 3:2 to obtain a solid characterized as 5 (0.052 g).

In Vitro Antifungal Assay
The fungicidal activity of the extracts and pure compounds was tested in vitro against the plant pathogenic fungus Botrytis cinerea UCA992 using different methodologies according to the characteristics of the samples [36]. The estimates of the IC50 values and confidence ranges (95%, 0.0 < 0.05) of each compound were obtained from logarithmic curves by adjusting to a dose-response type curve, as implemented in the program PRISM © statistical analysis (version 5.01). % inhibition = 100 − (Positive control well absorbance) (Negative control well absorbance) × 100

Preparation of Pure Compounds Stock and Broth Microdilution Method Bioassay
The fungicidal activity of the target compounds was tested in vitro against a plant pathogenic fungi Botrytis cinerea UCA992. According to previous reports [37,38], ELISA equipment was employed to measure inhibition in the microplate. All materials were carefully sterilized. The tested compounds were dissolved in dimethyl sulfoxide (DMSO) at 12.5 mg/mL as stock solution. From this solution, a work-solution was prepared, dissolving 13 µL in 1300 µL of Sabourad-glucose broth. Firstly, 100 µL of Sabourad-medium were added to the second and subsequent columns, then 200 µL of work-solution were added to the six first rows of the first column in the plate. Dilutions were prepared taking 100 µL from the first column and mixed to homogeneity with the second one, and this process was repeated for all the columns obtaining a concentration gradient. Next, 100 µL aliquots of a spore solution (5 × 10 4 spores/mL) of the strain B. cinera UCA992 were inoculated in the first three rows of microplates with Sabourad-glucose liquid medium and the corresponding compound in a typical concentration range from 62.50 ppm to 0.061 ppm (concentration range from the first column to the last one), therefore reaching each microplate a total volume of 200 µL. Then, the plate was incubated for 72 h at 28 • C with a fungal control plate (all the microplates with 100 µL of a spore solution (5 × 10 4 ) and 100 µL of Sabouradmedium), to compare with the normal fungal growth and a medium control plate (all the microplates with 100 µL of sterilized water and 100 µL of Sabourad-medium) to eliminate the absorbance relative to the medium. Once the incubation time was completed, the absorbance of the three kinds of plates was measured, and 10 µL of a 0.027 M resazurin solution was added to all the microplates to detect contamination [39,40]. This process was performed at least three times for each compound to gather a statistical data and analyzed by Prism ® to determinate the IC50 value.
For data treatment, we perform a minimum of 9 experiments. It usually takes us three days to complete a study, performing three repetitions per day. Each experiment is submitted to the Grubbs test and then is represented graphically, and an equation associated with the data used to obtain a first approximation of the IC50 value is obtained. Once all the graphs and IC50 values of the "n" experiments are obtained, we discard the extreme values and keep the central values (the volume of "n" data will present a Gaussian curve). The estimates of the IC50 values and confidence ranges (95%, 0.0 < 0.05) of each compound were obtained from logarithmic curves by adjusting to a dose-response type curve, as implemented in the program PRISM © statistical analysis (version 5.01).

Bio-Guided Antifungal Assays: Use of Resazurin as Inhibition Indicator
The minimal inhibitory concentration for microorganism growth (MIC) was determined in triplicate by using the microdilution broth method in 96-well microplates. Samples were dissolved in DMSO at 12.5 mg/mL obtaining a solution of 500 ppm (take 40 µL of stock solution and 960 µL of the medium solution Sabourad-glucose in an Eppendorf). A total of 100 µL of this solution was added into the first well with 100 µL of water or solution of the spores from B. cinerea UCA992 obtaining a 250 ppm concentration. The final DMSO concentration should be less than 2% to not interfere with the assay. Concentrations ranging from 250 to 0.05 ppm were achieved. One inoculated well was included, to allow for control of the adequacy of the broth for organism growth. One non-inoculated well, free of antimicrobial agent, was also employed, to ensure medium sterility. Irgasan was used as positive control. The microplates (96-wells) were incubated at 28 • C for 72 h. After the incubation time, 10 µL of a solution of resazurin (270 mg in 40 mL of distilled and sterilized water) was added to all the microplates to indicate microorganism viability and check non-inoculated well was free of contamination, and then microplates were sealed with a sterile adhesive polyester film (50 µm; VWR ® Microplate Sealing Film) and incubated (28 • C with artificial light) for 24 h more. The MIC values of extract from Baccharis spp. were determined as the lowest concentration in which the resazurin (purple) did not bio-transform to resorufine (red/brown) (see Figure 4) due to the inhibition of B. cinerea growth.

Poisoned Food Medium Assay
The fungicidal properties of most active compounds, 5 and 10, were assessed by the "poisoned food" technique ( Figures S28 and S29) [41]. The bioassay was carried out by measuring radial growth inhibition on an agar medium in a Petri dish in the presence of test compounds at 28 • C. The test compound was dissolved in ethanol, resulting in a final compound concentration of 0.06-30 µg/mL. The final ethanol concentration was identical in the control and treated cultures. The medium was poured into 9 cm diameter sterile Petri dishes, and a 5 mm diameter mycelial disk of B. cinerea cut from an actively growing culture (two days of growth) was placed in the center of the agar plate. Radial growth was measured for three days. Three independent experiments and three replicates per treatment were conducted. The fungicide irgasan was used as a standard for comparison in this test.

Statistical Analysis
The data were analyzed using an ANOVA test with PRISM © statistical analysis software (version 5.01). Dose-response analysis was performed to estimate the IC50 values with 95% confidence ranges.

Statistical Analysis
The data were analyzed using an ANOVA test with PRISM © statistical analysis software (version 5.01). Dose-response analysis was performed to estimate the IC50 values with 95% confidence ranges.
The flavonoids 1, 2, 3, and 4 showed moderate activity. Nevadensin (1) had the lowest activity (IC50 of 57.0 ppm), 4 , 7-dimethoxyapigenin (2); Genkwanin (3) and Cirsimaritin (4) had a similar activity (IC50 of 31.3, 35.9, and 38.9 ppm); all of them have a hydroxyl group in C-5. On the other hand, Genkwanin (3) has a similar structure to 2 with the main difference being the free hydroxyl group presented in C-4 ( Figure 5), so this free hydroxyl group could be related to the activity increase in compound 3 (IC50 of 35.9 ppm) (see Supporting Material).
Salvigenin (5) and Galangustin (6) showed similar activities. In both cases, there is a hydroxyl group in C-5 and a methoxyl group in C-4 (compound 5) and C-8 (compound 6). Salvigenin (5) showed the highest activity of all flavonoid compounds tested. A structural comparison with compounds 3 and 6 shows the importance of the free hydroxyl group in C-4 as seen in the previous case (compounds 2 and 3) and the presence of a methoxyl group in C-7. In conclusion, for this family of compounds, the IC data confirms the importance of presenting at least a free hydroxyl group to increase the polarity, which increases the solubility in polar media (a hydroxyl group in C-5). A hydroxyl group in C-5 and a methoxyl group in C-8 (compound 6) or C-4 (compound 5) could be related to the more efficient structural requirements for a high activity against B. cinerea. Compound 8, a novel compound identified from B. trinervis, showed a lower activity with an IC50 of 70.04 ppm.
Compounds 10 and 11 showed completely different activities (IC50 of 3.1 ppm and 59.1 ppm, respectively); this data is the first report on the fungicide activity of these structures. Compound 10, with an alkene group in C-11-C-12 showed higher activity than the oxidized analog compound 11 (see Figure 8). This fact manifests the importance of the oxidation reaction in B. cinerea as a part of a detoxification pathway [68][69][70].  Figures S23 and S26). The flavonoids 1, 2, 3, and 4 showed moderate activity. Nevadensin (1) had the lowest activity (IC50 of 57.0 ppm), 4′, 7-dimethoxyapigenin (2); Genkwanin (3) and Cirsimaritin (4) had a similar activity (IC50 of 31.3, 35.9, and 38.9 ppm); all of them have a hydroxyl group in C-5. On the other hand, Genkwanin (3) has a similar structure to 2 with the main difference being the free hydroxyl group presented in C-4′ ( Figure 5), so this free hydroxyl group could be related to the activity increase in compound 3 (IC50 of 35.9 ppm) (see Supporting Material).
Salvigenin (5) and Galangustin (6) showed similar activities. In both cases, there is a hydroxyl group in C-5 and a methoxyl group in C-4′ (compound 5) and C-8 (compound 6). Salvigenin (5) showed the highest activity of all flavonoid compounds tested. A structural comparison with compounds 3 and 6 shows the importance of the free hydroxyl group in C-4′ as seen in the previous case (compounds 2 and 3) and the presence of a methoxyl group in C-7. In conclusion, for this family of compounds, the IC data confirms the importance of presenting at least a free hydroxyl group to increase the polarity, which increases the solubility in polar media (a hydroxyl group in C-5). A hydroxyl group in C-5 and a methoxyl group in C-8 (compound 6) or C-4′ (compound 5) could be related to the more efficient structural requirements for a high activity against B. cinerea. Compound 8, a novel compound identified from B. trinervis, showed a lower activity with an IC50 of 70.04 ppm.
Compounds 10 and 11 showed completely different activities (IC50 of 3.1 ppm and 59.1 ppm, respectively); this data is the first report on the fungicide activity of these structures. Compound 10, with an alkene group in C-11-C-12 showed higher activity than the oxidized analog compound 11 (see Figure 8). This fact manifests the importance of the oxidation reaction in B. cinerea as a part of a detoxification pathway [68][69][70].
The most active compounds (5 and 10) were also tested by the poisoned food technique [71,36], obtaining similar results to the previous micro-dilution data ( Figures S28  and S29).  The most active compounds (5 and 10) were also tested by the poisoned food technique [36,71], obtaining similar results to the previous micro-dilution data ( Figures S28 and S29).

Conclusions
Three species from the Baccharis genus (Baccharis prunifolia, Baccharis trenervis, and Baccharis zumbadorensis) were studied in order to isolate new compounds with antifungal activity against the phytopathogen fungus B. cinerea UCA992. This is the first report of biological assays against the phytopathogen Botrytis cinerea which tests these isolated compounds.
For this purpose, the extracts and the fractions from an initial chromatographic analysis were submitted to bio-guided isolation. Phytochemical investigation of the most active fractions of DCM extracts allowed for the identification of twelve compounds. Three of them reported here for the first time: (7A), (8), and (11). All compounds were tested against Botrytis cinerea UCA 992. The most active compounds were Salvigenin (5) with an IC50 of 13.5 ppm and 1,2-dihydrosenedigital-2-one (10) with an IC50 of 3.1 ppm.