Collection of soil samples and isolation of fungal lipase inhibitor producer(s)
Thirty-nine soil samples were collected from various ecosystems in Egypt: fields, gardens, Nile river bank areas from July 2017–January 2018 (Table 1). Lipase inhibitor active producers were isolated according to the method of Naveen and coworkers with some modifications where: one-gram soil sample was mixed with 100 ml sterile distilled water with shaking at 10×g for 1 h at 28 °C. One ml supernatant was inoculated in duplicate in 50 ml starch casein broth and incubated at 28 °C for 7 days with shaking at 10×g. Following incubation, the mycelial mat was collected by centrifugation at 3000×g for 20 min at 4 °C. The mycelium was methanol extracted (1: 4) and assayed for its lipase inhibitory activity. One ml culture, from the duplicate culture, was spread on starch casein agar and incubated at 28 °C for 2 weeks. Colonies, recovered from samples having PL inhibitory activity, were further sub-cultured on starch casein agar followed by Sabouraud dextrose agar (Difco, USA) . Isolated pure colonies were sub-cultured in starch casein broth for further testing their lipase inhibitor activity.
Colorimetric pancreatic lipase (PL) inhibition assay
Pancreatic lipase (PL) inhibitory activity was measured colorimetrically using the substrate p-nitrophenyl palmitate (PNPP) (Sigma-Aldrich, USA), according to the method of Kordel and coworkers with slight modification where: the enzyme solution was prepared, immediately before use, by dissolving crude porcine PL type II (Sigma-Aldrich, EC 188.8.131.52, USA) in 100 mM Tris (pH 8.2) to get a concentration of 2 mg/ml (200 units/ml). Ten μL methanolic extracts of the suspected cultures were pre-incubated with 40 μL PL solution for 30 min at room temperature before the addition of 20 mM PNPP dissolved in isopropanol . The volume was adjusted to 200 μL using 100 mM Tris (pH 8.2), and the absorbance was measured at 410 nm using a spectrophotometer (Agilent Technologies, USA). The assay was performed in triplicates, and the results were expressed as an average mean value. Orlistat (Marcyrl pharmaceutical industries, Egypt), a known PL inhibitor, was used as a positive control; a control without the inhibitor was tested in parallel. The percentage of PL residual activity was determined for each extract by comparing the activity with and without the tested compounds. Percentage inhibition of lipase activity was calculated using the formula:
Lipase inhibition = (A-B/A) × 100, where A is lipase activity in the absence of inhibitor, B is the lipase activity in the presence of inhibitor .
Confirmatory fluorometric assay for lipase inhibition
The pancreatic lipase inhibitory effect was further confirmed using a fluorometric assay where: 6.0 mg 4-methylumbelliferyl butyrate (MUB) (Sigma-Aldrich, USA) was dissolved in 1000 μl DMSO and five-fold freshly diluted before the measurement. Ten μl 100% methanolic extract of the pure isolate, 90 μl PL enzyme (0.2 μg/ml), and 200 μl 50 mM phosphate buffer solution (pH 7.4) were added to a clear bottom black sides microtiter plate (Corning Incorporated, USA); 50 μl MUB solution was added before measurement. Orlistat was used as a positive control. The emitted fluorescence at 445 nm was measured after excitation at 365 nm with a fluorescence spectrophotometer (Agilent Technologies, USA). The speed of fluorescence development is directly proportional to the product formation, and subsequently to PL activity. The assay was repeated three times, and the PL activity was measured with and without the inhibitor .
Macroscopic and microscopic examination, 18S based rDNA sequencing and phylogenetic analysis of the lipase inhibitor fungal producer
Pure colonies, having lipase inhibitor activity, were sub-cultured on three culture media: potato dextrose agar (PDA) (20% potato infusion, 2% dextrose, and 2% agar); malt extract agar (MEA) (2% malt extract, and 1.2% agar) and Czapek yeast extract agar (CYA) (0.3% NaNO3, 3% sucrose, 0.1% K2HPO4, 0.05% KCl, 0.001% FeSO4 hydrated, 0.05% yeast extract, and 1.5% agar), and incubated at 25 °C for 7 days. Macro and micro-morphological characteristics were studied for identification to species level . Colony morphology, color, size, and texture were examined. The microscopical characteristics: hyphae, conidiophores, and conidia were examined by the wet mount technique at 40X magnification by an Olympus microscope (Olympus Corporation, Japan) .
Genomic DNA extraction was performed as follows: 100–200 mg mycelium was placed in a 1.5 ml Eppendorf tube containing 100–150 μl 0.5 M NaOH, quickly macerated with a micro-pestle (no big chunks) and allowed to stand for 6–10 min. 500 μl Tris-HCl (100 mM, pH 8) was added followed by vortexing. After centrifugation for 10 min at 14,000 rpm, the supernatant was transferred to a new Eppendorf tube and stored at − 20 °C. One μl of the prepared DNA was used as a template in PCR reactions . The PCR reaction was performed in a final reaction volume of 50 μl using the primers: 18S F (5`- tgatccttcygcaggttcac- 3`) and 18S R (5`- acctggttgatcctgccag- 3`) (Invitrogen, USA) at a concentration of 0.5 μM each , 0.5 mM dNTPs (Promega, USA), and 2 U Taq DNA polymerase (Promega, USA) in 1 × PCR buffer (Promega, USA) containing 1.5 mM MgCl2 (Promega, USA), using a Techne thermal cycler (Cole-Parmer, USA). The cycling parameters were as follows: denaturation at 95 °C for 5 min; 30 cycles each 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 2 min; and a final extension at 72 °C for 10 min. The amplified product was purified using Wizard SV Gel and PCR clean up system (Promega, USA), and sequenced using ABI3730XL sequencer (Macrogen, Korea). The obtained sequence was blasted against the nucleotide database using blastn tool of the US National Centre for Biotechnology Information (NCBI) .
Phylogenetic analysis was performed using MEGA-X software . The obtained 18S rDNA sequence of AspsarO and the downloaded sequences of its closely related neighbors were aligned using Clustal W. MUSCLE algorithm was used for trimming and verification of the aligned sequences. Maximum Composite Likelihood was used to compute the evolutionary distances .
Optimization of lipase inhibitor production by AspsarO
Incubation temperature and time
To determine the optimum incubation temperature, a 5 mm mycelial plug of a seven-day-old AspsarO culture was inoculated in 250 ml Erlenmeyer flasks containing 50 ml PDB and incubated at 20 °C, 25 °C, 37 °C, and 42 °C for 6 days with shaking at 5×g. On day six, the lipase inhibitory activity was measured . As for the optimum incubation period, similar AspsarO cultures were incubated at 30 °C for 8 days, and the lipase inhibitor activity was daily monitored starting from day four till the end of the experiment .
Use of different carbon and nitrogen sources
The effect of different carbon sources on lipase inhibitor production was studied by replacing dextrose in the culture medium (PDB) with each of the following sugars: 2% sucrose, 2% lactose, or 2% starch. Also, the effect of supplementing AspsarO cultures with different nitrogen sources was studied by adding 1% of each of the following: yeast extract, peptone, tryptone, or tryptic soy broth to the culture medium (PDP) . A 5 mm mycelial plug of a seven-day-old AspsarO culture was inoculated in 250 ml Erlenmeyer flasks containing 50 ml culture medium and incubated at 30 °C for 6 days with shaking at 5×g. The lipase inhibitor activity was measured on day six .
Plackett-Burman design for optimization of PL inhibitor production
A Plackett-Burman design was used to identify the main variables influencing lipase inhibitor production by AspsarO. Four independent variables, with the possible low (−) and high (+) levels, were assessed for their significance on the inhibitor yield. The tested variables included: carbon source (2% sucrose-2% starch), nitrogen source (1% yeast extract-1% tryptic soy broth), production time (5–6 days), and temperature (25–37 °C). Table 2 shows the tested medium ingredients and incubation conditions of the 16 runs of the assessed factors besides the 17th run under control conditions. Minitab 18 software was used to generate the design and analyze the outputs of the experiments. The calculated E-value magnitude of the tested factor shows its effect or its significance in affecting the response. The positive or negative sign of the E-value is indicative of its positive or negative influence on the responses . All runs were performed in triplicates, and the lipase inhibitory activity was determined .
Production, purification and identification of the lipase inhibitor
To achieve maximum PL inhibitor production, AspsarO was inoculated in potato starch broth containing tryptic soy broth at 37 °C for 6 days. At the end of fermentation, the biomass was separated from eight liters cultures. The broth was subjected to fractionation using methylene chloride (3 × 300 ml). Pooled methylene chloride soluble fractions were evaporated under reduced pressure (Büchi R-100 Rotary Evaporator, Germany) to get a brownish residue (60 mg). The remaining aqueous layer was concentrated under vacuum and applied to a DiAION HP-20 column (5 × 100 cm) (Supelco Analytical, Germany) and eluted with water, 50, and 100% methanol. The collected fractions were dried under vacuum to obtain the dried aqueous (260 mg), 50% (800 mg), and 100% (1.02 g) methanolic fractions. Dried fractions were dissolved in DMSO, and the lipase inhibitor activity was determined. The fractions showing a lipase inhibitor activity were further analyzed by TLC using methanol and chloroform (95: 5 v/v) as a mobile phase on Silica gel 60 TLC plates (Merck, Germany) .
Using a bio-guided approach, the 100% methanolic fraction was further purified to isolate the main lipase inhibitor. It was chromatographed using PuriFlash 4100 (Interchim, France) using 25 g flash cartilage (silica gel 60, 30 um), and eluted with CH2CL2: methanol (9.5:0.5 ~ 90:10 v/v). Twenty ml fractions were collected, and the fractions were monitored using TLC and visualized under UV. Fractions with major spots were collected and evaporated under vacuum to obtain semi-pure fractions (300 mg). They were purified on silica gel columns (2 × 20 cm) using CH2Cl2: methanol (9.5:0.5 v/v) as eluent to get a pure compound (10 mg off-white powder). The identity of the compound was assessed using 1H and 13C NMR, and the obtained data was compared to literature .
Molecular docking study
The Molecular Operating Environment (MOE, 2015.10) software was used in all molecular modeling studies. All minimizations were done, using MOE, until an RMSD gradient of 0.05 kcal∙mol− 1 Å− 1 was reached using a MMFF94x force field. Partial charges were calculated automatically. The X-ray crystallographic structure of human pancreatic lipase (PDB ID: 1LPB) was downloaded from the protein data bank . We removed the water molecules and ligands not involved in binding from the co-crystallized enzyme. The enzyme was prepared, for docking, using the Protonate 3D protocol in MOE with default settings. The co-crystallized ligand was used to define the binding site. Triangle Matcher placement method and London dG scoring function were used in docking .
Determination of IC50
Dried 100% methanolic fractions of AspsarO and kojic acid were dissolved in DMSO to get the following concentrations: 1, 5, 10, 25, 50, and 100 μg/ml. Similar concentrations of orlistat (reference standard) were also prepared. The PL inhibitor activity of all the prepared concentrations was measured . IC50 was calculated by plotting log (dose)-response inhibition curve using the equation “log (inhibitor) vs. response” with GraphPad Prism software .
Evaluating the AspsarO lipase inhibitor in a high fat diet (HFD) induced obesity animal model
Six weeks old male Sprague-Dawley rats weighing from 125 g to 165 g were purchased from New veterinary center (Cairo, Egypt). Rats were kept in the laboratory animal housing at the faculty of Pharmacy, Cairo University, following the recommendations of the guide for care and use of laboratory animals. They were randomly assigned to polycarbonate cages, with bedding of husk, and 12-h light/dark cycles; feed and water were given ad libitum. Environmental conditions were maintained at a temperature of 22 °C ± 2 °C and relative humidity of 60% ± 10%. All animal procedures were performed as per the international ethical guidelines and the National Institute of Health guide concerning the care and use of laboratory animals.
Acute toxicity testing
The acute toxicity test of the dried 100% methanolic fraction of AspsarO was performed as per the organization for European economic cooperation (OECD) guidelines No. 420. Increasing doses of 100, 400, 800, and 1000 mg/kg of the tested extract in distilled water, were administered as a single dose (one ml) by oral gavage to four rats; one rat received distilled water and served as a control. Pharmacotoxicity signs like changes in the skin, fur, eyes, respiratory and central nervous systems, and any changes in behavior or physical activities were observed at 10 min, 30 min, 60 min, 120 min, 4 h, and 6 h after treatment. Treated animals were daily monitored during the period of the study for mortality, and any pharmacotoxicity signs .
Following 1 week of acclimatization with pelletized commercial diet, we randomly divided the rats into three groups of five rats each. The groups were as follows: group on a normal diet, a HFD-fed group, and a HFD-fed group receiving 100 mg/kg/day of the dried 100% methanolic fraction of AspsarO dissolved in water. The extract was administered as a single daily oral dose for 28 days. HFD was prepared by mixing 35% ghee with the ground standard diet. The food intake and body weight were monitored every 48 h .
Following 4 weeks of treatment, blood samples were collected from 12 h fasted rats by retro-orbital puncture, and serum was separated by centrifugation at 2000×g for 10 min. Serum total cholesterol (TC) and triglycerides (TGs) were determined using commercial kits (Roche, Germany) and Cobas 8000 automated analyzer (Roche, Germany). Rats were sacrificed by cervical dislocation under anesthesia, and the livers were dissected and weighed [38, 59].
We used the GraphPad Prism 7 software for statistical analysis, including t-test, One-way ANOVA, and Dunnett’s multiple comparisons post-test at a p-value < 0.05. Results were expressed as mean ± standard deviation (SD) (n = 3). Also, unpaired Student’s t-test and One-way ANOVA followed by Sidak’s or Holm-Sidak’s multiple comparisons tests were used for analyzing the animal model experiments. The results were expressed as mean ± standard error (SEM) (n = 5) and were considered significantly different at a p-value < 0.05 (GraphPad Prism, version 7, GraphPad, La Jolla, CA).