1. Introduction
Ergot alkaloids (EAs) are secondary metabolites produced by a wide range of fungi, predominantly Claviceps spp. (e.g., C. purpurea), a parasite of wheat, rye, and other Gramineae grasses. EAs are synthesized within the sclerotia (ergot) that form in the ears of host rice plants. EAs were discovered as the causative compound of wheatgrass poisoning, the main symptom of which is cramps and miscarriages [1] [2].
EAs and their analogs are classified into three types: Clavine-type, peptide-type, and simple amides of the lysergic acid-type, and more than 70 compounds are currently known [3]. EAs and their analogs are compounds with many pharmacological activities; for example, ergotamine is used to treat migraine, ergometrine is used to prevent postpartum hemorrhage after childbirth, and bromocriptine and pergolide are used to treat Parkinson’s syndrome [4]. In particular, the market size of ergot-derived dopamine agonists in Japan was approximately 1.5 billion yen in FY2017 and a stable supply is required [5]. EAs and their analogs are thus essential compounds for the development of new drugs and clinical applications, including antibiotics, and treating cancer, psychiatric disorders, and coronavirus infection [6] [7] [8] [9].
EAs have a sterically complex chemical structure and industrial chemical synthesis has not been achieved due to economic disadvantages [10]. The industrial method for producing EAs is to extract them from ergot on rye artificially cultivated with C. purpurea in the field, or by the liquid culture of the mycelial phase of Claviceps spp. [11] [12]. The field production is greatly influenced by weather conditions, but liquid culture production covered these shortcomings. In addition, solid media changed the EAs composition ratio and increased the amount of EAs production [13]. The production of EAs by cultivating Claviceps spp. on solid media has been reported using wheat, rye, or sugarcane dregs but few studies on cereal culture have been published and the process is poorly understood [13] [14]. Furthermore, the ability to produce EAs in the mycelial phase varies significantly between species and strains of Claviceps [15].
This study searched for Claviceps strains cultured artificially in the mycelial phase and the produced EAs were identified. We cultured selected strains on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium.
Twenty-two strains of Claviceps spp. isolated in Japan were examined to produce EAs in the mycelial phase of liquid media. The results showed that C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. We report the types and total amounts of EAs produced by the mycelial phase of C. purpurea var. agropyri cultured on cereal media made from white rice, brown rice, or rye.
2. Materials and Methods
2.1. Experimental Instruments
Analytical TLC was performed on silica gel 60 F254 (Merck Ltd., Darmstadt, Germany). HPLC analysis was performed using an 1100 series HPLC system (binary pump: G1312A, autosampler: A1329A, column compartment: G1316A, UV detector: G1314A, Agilent Technologies, Inc., CA, USA) equipped with an Inertsil ODS-3 column (3 μm, 2.1 × 150 mm) (GL Science Inc., Tokyo, Japan). Open column chromatography for compound isolation was performed using 100 g of Sephadex LH-20 (GE Healthcare, IL, USA) in a 3.5 × 80 cm glass column. Preparative HPLC was performed on a Shimadzu LC-20AT and SPD-10AV instrument with a GL Science InertSustain C18 (5 μm, 10 × 250 mm) column. NMR spectra were recorded on an ECA-600II (1H: 600.17 MHz; 13C: 150.91 MHz) (JEOL, Tokyo, Japan). Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative to residual solvent signals ((δH 7.26)/(δC 77.0) for CDCl3, (δH 3.31)/(δC 49.0) for CD3OD as internal standards). Mass spectra were measured on a JMS-T100LP (JEOL, Tokyo, Japan).
2.2. Materials
Twenty-two strains of the genus Claviceps were isolated from parasites of 18 grass hosts from 1990 to 2019 in 12 prefectures in Japan (Table 1). Ergotamine tartrate standard was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
2.3. Fermentation and Extraction
2.3.1. Fermentation and Extraction of Claviceps Spp. in T25 Culture
The culture conditions using a liquid medium were based on the method of Amici et al. [16]. Claviceps species were pre-cultured on T2 agar (sucrose 100 g, l-asparagine 10 g, yeast extract 0.1 g, Ca(NO3)2 1 g, KH2PO4 0.25 g, MgSO4-7H2O 0.25 g, KCl 0.12 g, FeSO4-7H2O 0.02 g, ZnSO4-7H2O 0.015 g, agar 20 g, purified water to 1 L, 25% NH3 aq. pH 5.2). Fermentation was conducted using seed medium (sucrose 100 g, citric acid 10 g, yeast extract 0.1 g, Ca(NO3)2 1 g, KH2PO4 0.5 g, MgSO4-7H2O 0.25 g, KCl 0.12 g, FeSO4-7H2O 0.007 g, ZnSO4-7H2O 0.006 g, purified water to 1 L, 25% NH3 aq. pH 5.2) and fermentation T25 medium (sucrose 300 g, citric acid 15 g, yeast extract 0.1 g, Ca(NO3)2 1 g, KH2PO4 0.5 g, MgSO4-7H2O 0.25 g, KCl 0.12 g, FeSO4-7H2O 0.007 g, ZnSO4-7H2O 0.006 g, purified water to 1 L, 25% NH3 aq. pH 5.2).
First, the mycelia were cultured on T2 agar for 21 days at 25˚C, then two pieces of cultured agar (1 × 2 cm2) were transferred into 500 mL Erlenmeyer flasks containing 100 mL of seed culture medium and grown at 25˚C and 150 rpm for six days. Next, 20 mL of the seed culture medium was transferred into 1000 mL Erlenmeyer flasks containing 170 mL of fermentation medium and grown at 25˚C and 150 rpm for 12 days.
T25 culture filtrate was adjusted to pH 8.5 with saturated aqueous Na2CO3 and ergot alkaloids were extracted twice using liquid/liquid extraction with an equal volume of chloroform. The chloroform extract was evaporated to dryness in a vacuum evaporator.
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Table 1. Sample overview of Claviceps spp. and the yield of extract from T25 culture used in the study.
-: No growth; n.d.: No data.
2.3.2. Fermentation and Extraction of C. purpurea var. agropyri on Cereal Culture
C. purpurea var. agropyri was pre-cultured on potato dextrose agar (PDA, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Fermentation was conducted using M102 medium (sucrose 30 g, malt extract 20 g, peptone 2 g, yeast extract 1 g, MgSO4-7H2O 0.5 g, KCl 0.5 g, KH2PO4 1.0 g, purified water 1 L, NaOH pH 6.0) and cereal medium (dry cereal (rye, brown rice): 150 g, tap water: 90 mL; white rice: 30, 60, 90, 120, 150 mL) [17].
First, C. purpurea var. agropyri was pre-cultured on PDA and grown for 21 days at 25˚C, then two pieces of cultured agar (1 × 2 cm2) were transferred into 500 mL Erlenmeyer flasks containing 200 mL of M102 medium and grown at 25˚C and 150 rpm for seven days. Next, 10 mL of cultured M102 was transferred into 500 mL Roux flasks containing cereal medium and grown at 25˚C with shaking for 28 days.
Ergot alkaloids were extracted from the cereal cultures with 300 mL of chloroform: 25% NH3 aq. (500:1), then the chloroform extract was evaporated to dryness in a vacuum evaporator. The amount of extract obtained from each culture was 547.8 ± 296.5 mg for white rice, 829.6 ± 144.3 mg for brown rice, and 257.6 ± 9.4 mg for rye.
2.4. Isolation of Pyroclavine (1), Festuclavine (2) and Agroclavine (3)
C. purpurea var. agropyri was incubated using 10 Roux flasks containing rice medium for 28 days at 25˚C, then the cultures were extracted with chloroform. The crude chloroform extract (7.6 g) was obtained by evaporation under vacuum, suspended in 100 mL of 4% tartaric acid aqueous solution, and partitioned twice with an equal volume of n-hexane. The pH of the remaining water layer was adjusted to 8.5 with saturated Na2CO3 aqueous solution, and alkaloids were extracted twice with an equal volume of chloroform. The solvent was removed under vacuum, and the chloroform extract (273.3 mg) was separated into ten fractions by open column chromatography using Sephadex LH-20. The mobile phases were n-hexane:chloroform = 1:4, chloroform:acetone = 3:2, chloroform:acetone = 1:4, acetone, and MeOH, in turn. The volume of each solvent used was 200 mL, and 100 mL fractions were collected. Fractions 4 and 5 (total 92.3 mg) were purified by reverse-phase (RP)-HPLC (solvent: 30% acetonitrile containing 0.05% TFA, room temperature, flow rate: 2 mL/min) to obtain pyroclavine (1: 5.4 mg), festuclavine (2: 1.8 mg) and agroclavine (3: 10.1 mg).
2.5. TLC Analysis
Chloroform extracts of T25 culture were dissolved in chloroform at 50 mg/mL and ergotamine tartrate was dissolved at 1 mg/mL, then 2 μL aliquots were spotted on a TLC plate and developed with chloroform/methanol/acetic acid (90/15/0.1). Chromatographed ergot alkaloids were observed by spraying the plates with van Urk’s reagent.
2.6. HPLC Analysis
Compounds 1 - 3 were dissolved in 5% acetonitrile at 100 μg/mL to provide a standard solution, which was diluted stepwise to provide a dilution series of 12.5, 2.5, 0.5, and 0.1 µg/mL. The chloroform extracts were dissolved in 5% acetonitrile to 1 mg/mL, centrifuged at 7200 g for 5 min, and 10 µL of the supernatant was used as the analysis sample. The HPLC mobile phases were 0.1% formic acid solution (A) and acetonitrile containing 0.1% formic acid (B). The gradient was 0 - 1 min 5% B, 1 - 10 min 5% - 20% B, 10 - 28 min 20% - 30% B, 28 min 20% - 30% B, 28 - 34 min 95% B, 34 - 43 min 5% B. An ODS column was used at 40˚C and a flow rate of 0.2 mL/min. A calibration curve was constructed by linear approximation using the obtained area values. The approximations were compound 1:y = 0.000001817x + 0.02460 with an R2 value of 0.9997, compound 2:y = 0.000004338x + 0.002143 with an R2 value of 1.0000, and compound 3:y = 0.000001982x + 0.05990 with an R2 value of 0.9998.
3. Results
3.1. Culture of Claviceps Spp. in T25 Liquid Medium
Twenty-two strains were cultured in T25, which has already been reported to produce EAs during culture. All strains except C. purpurea var. sasae and C. microspora grew in T25 (Table 1).
3.2. Detection of EA-Producing Strains by TLC Analysis and Identification of Compounds
The EAs detected in chloroform extracts of the T25 culture media were confirmed by TLC analysis with van Urk’s color reagent, using ergotamine tartrate as a color control. C. purpurea var. agropyri produced compounds showing blue-violet spots 1 (Rf 0.07), 2 (Rf 0.11), and 3 (Rf 0.24) with the same color as ergotamine tartrate (Rf 0.54) (Figure 1(A)).
The molecular formulae of these alkaloids are C16H20N2 (1), C16H20N2 (2) and C16H18N2 (3) as determined by ESI-MS analysis. Compounds 1 - 3 were identified as pyroclavine (1) [18], festuclavine (2) [19], and agroclavine (3) [20] by 1H-, 13C-NMR spectroscopy after isolation by preparative HPLC (Figure 1(B), Tables 2-4, Figures S1-S6).
3.3. Production of 1 - 3 on Cereal Medium by C. purpurea var. agropyri
C. purpurea var. agropyri produced EAs (1 - 3) in the mycelial phase cultured in a T25 medium. The production of 1 - 3 on cereal medium (white rice, brown rice, rye) was calculated using the absolute calibration curve method (Figure 2(A)). C. purpurea var. agropyri produced 1 - 3 on all cereal media, with agroclavine (3) as the major EA (Figure 2(B) and Figure 2(C)). The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. C. purpurea var. agropyri produced the highest amount of EAs in white rice culture. The total amount of 1 - 3 in white rice medium (150 g of white rice per Roux flask) was about 34 times higher than that in T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).
3.4. Effects of Moisture Content of White Rice Medium on the Production of 1 - 3 by C. purpurea var. agropyri
C. purpurea var. agropyri efficiently produced 1 - 3 in white rice medium culture. Osmotic pressure (water activity) of the liquid medium affects the production of EAs in the mycelial phase of Claviceps spp. [21] [22]. Therefore, we examined the relationship between the water content of white rice medium and the
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Table 2. 1H- and 13C-NMR data of 1 and pyroclavine [18].
*NMR data were measured using CD3OD.
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Table 3. 1H- and 13C-NMR data of 2 and festuclavine [19].
*NMR data were measured using CD3OD.
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Table 4. 1H- and 13C-NMR data of 3 and agroclavine [20].
*NMR data were measured using CDCl3.
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Figure 1. The TLC analysis of chloroform extracts of T25 culture media and the structures of compounds 1 - 3. (A): TLC analysis of chloroform extracts of T25 culture media. Solvent system: CHCl3/MeOH/CH3COOH (90/15/0.1), ergotamine tartrate: 2 μg/spot, chloroform extract: 100 μg/spot, reagent: van Urk’s reagent, a: ergotamine tartrate, b: C. purpurea, c: Claviceps sp1 ex Alopeculus, d: Claviceps sp2 ex Dactylis, e: Claviceps sp3 ex Phalaris, f: Claviceps sp4 ex Phalaris, g: Claviceps sp5 ex Phragmites, h: C. purpurea var. agropyri, i: C. litoralis, j: Claviceps sp6 ex Phragmites, k: C. paspali, l: C. queenslandica MAFF 306124, m: C. queenslandica MAFF 247574, n: C. panicoidearum, o: C. microspora var. kawatanii, p: C. sorghicola MAFF 247566, q: C. sorghicola MAFF 306571, r: Claviceps sp7 ex Miscanthus, s: C. africana, t: C. bothriochloae, u: C. yanagawaensis, (B): The structures of 1 - 3.
production of 1 - 3 by C. purpurea var. agropyri. The production of EAs 1 - 3 was maximal at 90 mL of water per 150 g of white rice (Figure 3).
4. Discussion
The purpose of our study was to identify Claviceps sp. that can be cultured in the mycelial phase, determine their ability to produce EAs, and cultivate the strains on cereal media (white rice, brown rice, and rye), and examine their ability to produce EAs on each medium.
Twenty-two strains of Claviceps spp. isolated in Japan were examined for their production of EAs in the mycelial phase using a liquid medium. The results showed that C. purpurea var. agropyri, isolated from Elymus tsukusiensis var. transiensis as a host plant, produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase.
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(C)
Figure 2. Production of 1 - 3 by C. purpurea var. agropyri fermentation. (A) Culture conditions for C. purpurea var. agropyri, a: T25 culture, b: white rice culture, c: brown rice culture, d: rye culture. [(B), (C)] Amount (μg) of 1 - 3 produced in T25 culture, white rice, brown rice, and rye cultures by C. purpurea var. agropyri. Data are shown as mean ± standard deviation (SD) of the mean (N = 3).
Clavine-type EAs 1 - 3 were first isolated by Abe et al. from Claviceps sp. parasitic on Elymus tsukusiensis var. transiensis [23] [24] [25]. Previous studies reported various physiological activities of agroclavine (3), including partial agonist or antagonist effects on adrenergic receptors, dopamine receptors, and serotonin receptors [26]. Furthermore, the carcinocidal effect of agroclavine (3) was enhanced by derivatization [7].
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Figure 3. The change in the total amount of 1 - 3 in 150 g white rice with 30, 60, 90, 120, 150 mL water. Data are shown as mean ± SD of the mean.
However, 1 - 3 are difficult to obtain and there are no commercially available standards. C. fusiformis, isolated in South Asia and Africa, is a representative Clavine-type EA-producing fungus whose host plant is Pennisetum typhoideum Rich [27] [28] [29]. C. fusiformis belongs to section Pusillae within the genus Claviceps, and C. purpurea belongs to section Purpurea in the taxonomic study by Píchová et al. [30]. In that study, Clavine-type alkaloids were isolated from a phylogenetically distinct species of fungi from which Clavine-type alkaloids had previously been isolated, suggesting that C. purpurea var. agropyri could be a useful new Clavine-type EA-producing fungus distinct from C. fusiformis.
When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The highest production of 1 - 3 was found in white rice culture. This is the first report on the production of EAs in white rice medium. The total amount of 1 - 3 in white rice medium (150 g of white rice per Roux flask) was about 34 times higher than that in T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask), indicating the usefulness of white rice medium.
Osmotic pressure (water activity) of the liquid medium affects the production of EAs in the mycelial phase of Claviceps spp. [21] [22]. The production of EAs by C. purpurea var. agropyri was affected by the moisture content of the white rice medium. C. purpurea var. agropyri produced the highest amount of 1 - 3 when 90 mL of water was added to 150 g of white rice. These data suggest that water content is a major factor affecting the production of EAs in white rice medium. An osmosensor of filamentous fungi is the group III histidine kinase upstream of a mitogen-activated protein kinase (MAPK) cascade [31]. The production of aurofusarin of Fusarium graminearum is regulated by the group III histidine kinase FgOs1 [32]. On the other hand, the type III histidine kinase CpHK1 in Claviceps purpurea does not directly regulate EAs production, and the relationship between osmotic pressure and EAs production remains unclear [22].
5. Conclusion
This study showed that white rice medium is effective for the production of the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) by C. purpurea var. agropyri. Further improvement of cultural conditions, such as water content, may increase the production of EAs in white rice medium.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 16K07238.
Supporting Information
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Figure S1. 1H-NMR spectrum of 1 (600.17 MHz) in CD3OD.
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Figure S2. 13C-NMR spectrum of 1 (150.91 MHz) in CD3OD.
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Figure S3. 1H-NMR spectrum of 2 (600.17 MHz) in CD3OD.
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Figure S4. 13C-NMR spectrum of 2 (150.91 MHz) in CD3OD.
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Figure S5. 1H-NMR spectrum of 3 (600.17 MHz) in CDCl3.
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Figure S6. 13C-NMR spectrum of 3 (150.91 MHz) in CDCl3.