Phenanthrene-enriched extract from Eulophia macrobulbon using subcritical dimethyl ether for phosphodiesterase-5A1 inhibition

General materials

Dimethyl ether or DME (Tamiya 420D, commercial grade) was used for extraction and purchased from Siam Tamiya Co., Ltd., Thailand. The cGMP, crude snake venom from Crotalus atrox, histone from calf thymus, bovine serum albumin (BSA), ethylene glycol tetra-acetic acid (EGTA), imidazole, Tris ((trishydroxymethyl)aminomethane), magnesium chloride (MgCl₂), DEAE-Sephadex, phenylmethylsulfonyl fluoride (PMSF) were bought from Sigma‐Aldrich (St Louis, MO, USA) [³H]cGMP and scintillation cocktail Ultima gold was purchased from Perkin Elmer (Waltham, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (Pen‐Strep), and Geniticin (G418) were purchased from Gibco by Life Technologies (Paisley, Scotland). Lipofectamine 2000 (Invitrogen) was purchased from ThermoFischer Scientific (Waltham, MA, USA). A Hipure plasmid Maxiprep kit was bought from ThermoFischer Scientific. Human embryonic kidney (HEK)293 cell lines were purchased ATCC (Virginia,USA). Genistein (A) (purity >98%) was purchased from Apex Biotechnology (Boston, USA). Sildenafil citrate (purity >98%) was purchased from the European Directorate for Quality of Medicines and Health care (EDQM), Council of Europe (Strasbourg, France). ACN, water and MeOH (LC‐MS grade) were purchased from RCI Labscan, (Bangkok, Thailand). Formic acid (AR grade) was obtained from Merck (Darmstadt, Germany). The organic solvents (analytical grade) were purchased from Burdick & Jackson (B&J) (UK). TLC aluminium sheets and silica gel 60 F254 were purchased from Merck (Darmstadt, Germany).

Plant material

E. macrobulbon was collected in Prachinburi province, Thailand. It was identified by Asst. Prof. Dr. Anupan Kongbangkerd, Faculty of Sciences, Naresuan University. The herbarium specimen (No. 002716) is kept in the Biology Department, Faculty of Sciences, Naresuan University, Thailand, which is in compliance with the Convention on Biological Diversity and the Convention on Trade in Endangered Species of Wild Fauna and Flora. The fresh roots were chopped and air-dried at 55 °C for 3 days. The dried plant was ground into fine powder (4 kg) and sieved (150–170 µm) and stored in a desiccator at room temperature until use.

Isolation of the main bioactive compound from E. macrobulbon

The isolation of HDP followed previous reports with some modifications¹⁴. In brief, dried powders of E. macrobulbon (4 kg) were macerated two times with 95% EtOH (28L), then filtered and the solvent was removed under reduced pressure to provide 450 g of crude extract (11.2% yield). The extract (384.4 g) was dissolved in 100% MeOH and partitioned twice with hexane. The hexane part was discarded and the MeOH part was diluted with DI water to give 20% MeOH and partitioned twice with DCM. The DCM portion was dried under reduced pressure to yield 19.9 g of crude extract. The DCM residue was mixed with silica gel and loaded on to a silica gel chromatography column (i.d. 103 × 40 mm). The mobile phase for gradient elution was 100%DCM to 0.5–4% MeOH in DCM. Eighteen fractions were collected (EMD-1-18). The target compound was monitored to reference standard of HDP by TLC using DCM:MeOH (9.5:0.5 %v/v) as the mobile phase (the Rf value was around 0.3). The fraction of EMD-14 was obtained 0.49 g and chosen for further isolation. EMD-14 (0.24 g) was dissolved in methanol and subjected in a Sephadex LH-20 column (i.d. 1.5 × 200 cm) eluting with 100% MeOH to yield 19 fractions. Three fractions (EMDLH14-10 to EMDLH14-12) were pooled and evaporated and recrystallized with MeOH/DCM to give 0.19 g of crystalline bioactive compound (HDP). The spectroscopic data of ¹H-NMR and MS were in agreement with those reported in the literature¹⁴. The purity and spectroscopic data of HDP are described in supplementary materials, Figs. S2, S3 and S4. The isolated HDP was used as a reference standard to quantitatively control the quality of the extracts using LC-MS.

Methods of classical solvent and ultrasound-assisted extraction

Classical solvent extraction: fine powder of E. macrobulbon root (10 g) was macerated in different solvents, (i) 95% EtOH, (ii) EtOAc, or DCM. The sample-to- solvent ratio (w/w) was varied from low to high (1:6.25, 1:10 and 1:20), each maceration period was either 24, 48 or 72 h.

Ultrasound-assisted extraction; the fine powder (10 g) was macerated with different organic solvents, EtOH, EtOAc and DCM at a fixed sample-to-solvent ratio of 1:10 at 40 °C for 40 min. The ultrasound frequency was set at low to high intensity (100 kHz to 1 MHz) (Transonic, Themo Fisher Scientific, Göteborg—Sweden). Whenever the extraction process reached the time course, the extraction samples were filtered (Whatman paper 2 µm) and then dried under reduced pressure to provide the crude extract. The extract was then dried over a desiccant for 48 h and weighed.

Subcritical fluid dimethyl ether extraction; the dried powder (5 g) was mixed with the required volume of water or co-solvent and the mixture was placed in cellulose thimble (30 × 100 mm) along with a magnetic bar of 15.9 × 8 mm (length × diameter). The DME extractor was applied for this work and the apparatus was schematically presented in reference of³⁸. The thimble was then placed in an extractor (100 ml total volume of stainless-steel with a closed system). Liquefied DME was filled into the extractor at the required solvent to solid weight ratio. The extraction was carried out at a controlled temperature and stirring speed of 500 rpm required time (see below). After extraction, DME and the liquid sample were passed through a stainless steel filter (5 µm pore diameter, Swagelok). The chamber was inverted to a 75 ml Erlenmeyer flask. The remaining liquid sample was then dried over desiccant for 48 h, the amount weighed and the yield determined.

Optimization of dimethyl ether extraction

The extraction conditions were optimized by comparing;

1. (i)

   The amount of solvent as ratio of dried sample to DME solvent (w:w) was varied from 1:2, 1:4, 1:6, 1:8, 1:10, 1:12 and 1:13.5. The optimum ratio was selected for the extraction period.

2. (ii)

   The extraction period was varied (20, 30, 40, 60, 90 and 120 min). The minimum time necessary to achieve asymptotic HDP content was chosen.

3. (iii)

   The extraction temperatures were set at 30 ± 1, 35 ± 1, 40 ± 1, and 50 ± 1 °C.

4. (iv)

   The amount of co-solvents, water of 0%, 10%, 20%, 40%, 60%, 100% and 200%, or EtOAc of 0%, 10%, 40%, 100%, 200% and 500% of the powder weight.

The PDE5A1 inhibitory bioactivity, %HDP content and chemical profile were determined for all extracts. The extraction efficiency was also evaluated using the following parameters;

1. (1)

   Y; Percentage of process yield (%w/w)

2. (2)

   B; Percentage of HDP content in the extract (%w/w)

3. (3)

   t; Extraction period (min)

4. (4)

   v; Solvent amount

5. (5)

   Y/v and Y/t

6. (6)

   B/v and B/t

7. (7)

   Extractable HDP amount to dried plant (mg/kg)

These parameters were determined for all extraction methods, classical solvent extraction, ultrasound-assisted extraction, and subcritical fluid dimethyl ether extraction.

Quantitative determination of HDP content in E. macrobulbon extracts using LC–MS

A method for the determination of HDP in E. macrobulbon samples by LC–MS was developed and validated. An Agilent 1260 infinity Series HPLC coupled to an Agilent-6540 Q-TOF-MS spectrometer was used. The chemical constituents were separated on an EC-C18 (50 × 3 mm, 2.7 cm) column. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B). The following gradient system began from 0 to 5 min, 40% and 5–8 min, 20% B with a post run 2 min. The injection volume was 5 µl, flow rate was 0.3 ml/min and the column was maintained at 35 ºC. The optimized MS conditions were: drying gas flow 10 L/min, drying gas temperature 350 °C, nebulizer 30 psig, capillary voltage 3500V, skimmer 65 V, and octapole RFV 750 V. The ESI negative ionization in the Scan and SIM mode was used. The validation data was analyzed by Agilent MassHunter Quantitative Analysis Software Version B.05.02/Build 5.2.365.0. The analytical method was validated and standard curve HDP was established. The stock solution of HDP standard was freshly prepared by dissolving in 100% MeOH to obtain stock concentration of 100 µg/ml. This solution was further diluted with MeOH to make standard concentrations for the creation of calibration curves (0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 µg/ml). Samples were dissolved in 100% MeOH giving solutions of 5 mg/mL, then diluted to 50 µg/ml. All samples and standards were filtered through nylon syringe filters (0.45 μm pore size) before injection. All analyzes were performed in triplicate.

During the analysis, the stability of the LC–MS system was checked by using QC1 (concentration of 1.5 µg/ml) before starting each experimental batch. In addition, QC1 was added for the injections at the beginning, middle and end of the experiment to evaluate the LC–MS system and the stability of HDP throughout the analysis of the sample batch.

Qualitative analysis of E. macrobulbon extracts by LC-ESI-QTOF-MS

Conditions for LC–MS to measure secondary metabolites in E. macrobulbon samples were determined using a Zorbax Eclipse Plus C18 (4.6 × 100 mm, 3.5µm) column and gradient elution with 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B). The elution program ran for 0 min, 5%B; 0–6 min, 35%B; 6–10 min, 50%B; and 10–18 min, 20% B with a follow-up time of 2 min (post-run). The flow rate was 0.6 ml/min, the injection volume was 10µl, and the column temperature was maintained at 35 °C.

The MS condition was: negative ESI ionization in scan and SIM mode; drying gas flow 10 L/min at 350 °C; nebulizer 30 psig; capillary voltage 3500 V; skimmer 65 V; octapole RFV 750 V; and fragmentor in negative mode used 250 V. The mass range was set at 100–1200 m/z and the collision energy of target MS/MS was operated at 10, 20, and 40V, respectively. Data from LC–MS/MS were acquired using Agilent LC-MS-QTOF MassHunter Data Acquisition Software version B.05.01 and Agilent MassHunter Qualitative Analysis Software B 06.0 for structure elucidation. For the structure elucidation, compounds were compared with previous literature data with ion molecular mass and fragmentation pattern or with MassHunter Metlin Metabolite PCD/PCDL database (Agilent Technologies), from Scifinder (https://scifinder.cas.org), Chemspider (http://www.Chemspider.com) and/or Massbank (http://www.massbank.jp).

Samples from a suitable extraction condition were prepared at 5 mg/ml in 100% MeOH and diluted to 50 µg/ml. They were then filtered through nylon syringe filters with a pore size of 0.45 μm before injection into the LC system.

Preparation of phosphodiesterase-5 (PDE5-A1)

HEK293 cells were grown in DMEM supplemented with 10% FBS, in 75 mm flasks at 37 °C in a humidified 5%CO₂. A human PDE5A1 plasmid, a gift from Professor Dr Joseph A. Beavo, University of Washington, Seattle, WA, USA, were sub‐cloned into a pcDNA3 vector containing an ampicillin resistant gene. The human PDE5-A1 plasmid was scaled up and purified using Hipure plasmid Maxiprep kit (Invitrogen‐PureLink). HEK293 cells were transfected with human PDE5A1 plasmid using Lipofectamine-2000 following the company protocol. After 2 days of transfection, PDE5-A1 expression was induced by a selective antibiotic (Geneticin (G418, Gibco), 1 mg/ml) for 7 days. The surviving cells were sub‐cultured in DMEM, supplemented with 10% FBS in 175 mm flasks at 37 °C in a humidified 5% CO₂ atmosphere, and the cells further cultured until they reached 90–100% confluence. The cells were then harvested using a scraper and lysed by sonication in 1 ml of Tris buffer [50 mM Tris pH 7.5, 2 mM EDTA, 1mM dithiothreitol (DTT) and 1:100 of 100 mM PMSF]. The homogenate was centrifuged at 4 °C for 20 min and the supernatant was used as a source of PDE5A1. A PDE5 inhibitor, sildenafil, was used to confirm the presence of PDE5A1 enzymatic activity.

Measurement of PDE5-A1 enzyme activity

To assess PDE5A1 inhibition, a reaction mixture comprising 20 µl of reagent A (100 mM TrisHCl (pH 7.5), 100 mM imidazole, 15 mM MgCl₂, 1.0 mg/ml BSA and 2.5 mg/ml snake venom), 20 µl of 10 mM EGTA, 20 µl of PDE5A1 solution, and either 20 µl of test sample or solvent (5% DMSO) only as a control. The reaction was started by adding substrate 20 µl of 5 µM [³H]cGMP (~50,000 cpm) and performed at 30 ºC for 40 min. Then, 100 µl of 50% DEAE resin was added to the reaction. After shaking for 10 min, the resin was allowed to settle (20 min), the supernatant was treated with a second cycle of 50% DEAE resin. This supernatant (100 µl) was shaken with 200 µL of MicroScint-20 and tritium counted on a TopCount NXT scintillation counter (PerkinElmer, USA) for 2 h. The PDE5A1-hydrolyzed <25% of the substrate. Each was performed in duplicate in 96-well plates^27,28.

In preliminary screening, samples of plant extract and pure compound were tested at final conc of 50 µg/ml and 10 µM respectively. All samples were dissolved in DMSO and diluted with water. DMSO was limited to 1% in the final assay medium. When PDE5A1 inhibition was >80%, samples were further diluted and re-analyzed. IC₅₀s were calculated using Prism software (Graph Pad Inc., San Diego, CA). Sildenafil was used as the positive control.

Data analysis

The %PDE5A1 inhibition was calculated and plotted against log10[sample] and thereafter, half maximum inhibitory concentrations (IC₅₀) were interpolated by Graph-Pad Prism v. 8 (San Diego, USA). Data were processed by analysis of variance (ANOVA) or Tukey’s multiple comparison tests. Results were considered significant where P<0.05. Means and SDs were all calculated from at least three determinations of each sample.

Ethics statement

The research did not include human subjects or animal experiments.