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Open Access | Published: 2022 - Issue 5

NEUROPROTECTIVE ROLE OF MICROBIAL BIOTRANSFORMED METABOLITES OF SINAPIC ACID ON REPETITIVE TRAUMATIC BRAIN INJURY IN RATS

Samir M. Osman1, Hesham S. M. Soliman2,3, Fadila M. Hamed1, Diaa A. Marrez4, Amira A. El-Gazar5, Ahmed S. Alazzouni6*, Tamer Nasr7,8, Haitham A. Ibrahim2

 

  1. Department of Pharmacognosy, Faculty of Pharmacy, October 6th University.6th of October, 12566. Cairo, Egypt.
  2. Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Ein Helwan, 11795, Cairo, Egypt.
  3. Pharm D program, Egypt-Japan University of science and technology, New Borg El-Arab City, 21934, Egypt.
  4. Department of Food Toxicology and Contaminants, National Research Centre, Cairo, Egypt.
  5. Department of Pharmacology & Toxicology, Faculty of Pharmacy, October 6th University.6th of October, 12566. Cairo, Egypt.
  6. Department of Zoology, Faculty of Science, Helwan University, Ein Helwan, 11795, Cairo, Egypt.
  7. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, 11795 Helwan, Cairo, Egypt.
  8. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Modern University for Technology and Information, Egypt.

ABSTRACT

Sinapic acid biotransformation was studied and screened using eleven fungal cultures. Penicillium chermesinum AUMC 275, Aspergillus ochraceus AUMC 11328, and Paecilomyces variotii AUMC 5618 showed positive results in the production of metabolites. Sinapic acid biotransformation resulted in the production of gallic acid after 10 days incubation with P. chermesinum, protocatechuic acid after 7 days with A. ochraceus, and ferulic acid after 5 days with P. variotii. The investigated compounds' anti-inflammatory efficacy results, as well as the suggested binding mechanism, affinity, preferred orientation of each docking pose, and binding free energy with the GSK-3β enzyme, were predicted using molecular modeling. The predicted interaction energies for the examined chemicals agreed with the experimental findings. Moreover, we evaluated the neuroprotective potential of the obtained metabolites using the mild repetitive traumatic brain injury (mRTBI) model. Animals were exposed to 5 repetitive hits once per day by weight drop device and were divided into 6 groups (control, mTBI, mRTBI-10, mRTBI-10+Fa, mRTBI-10+PCA, mRTBI-10+GA). All the treatments decreased cortical contents of Tau protein (dementia marker), inflammatory markers (TNF-a and il-6), and signaling molecules such as GSK3B and DKK-1. Furthermore, the histological findings added another neuroprotective potential of obtained metabolites where marked improvements and ameliorative effect on the histoarchtexture of the hippocampus and mild changes in the hippocampus layers by GA, PCA, and FA, respectively. All these effects were mirrored in behavior outcomes, and a significant enhancement in animal behavior was observed. We concluded that GA, PCA, and FA have therapeutic potential for preventing TBI-induced brain injury.

Keywords: Gallic, Protocatechuic, Ferulic acids, Sinapic acid, Neuroprotective, Repetitive traumatic


Introduction

The global health issue of traumatic brain injury (TBI) affects millions of people each year [1, 2]. Chronic brain conditions such as chronic traumatic encephalopathy (CTE) are more likely to develop in later life due to repeated TBI exposure [3]. CTE can arise from contact sports and military contribution [4]. Hence, growing awareness has focused on the neurological sequelae of sports-related head trauma, particularly concussions. Most of them are associated with behavioral deficits such as mood, cognition, and gait disturbances, which are recently categorized as neurodegenerative disorders [5, 6]. However, the whole picture that environs this neurodegenerative disease/syndrome is still unknown. Although some steps/phases have unveiled part of the formidable pathological process after RTBI/CTE, the presence of an effective pharmacological therapy remains challenging. Therefore, one of the current study's aims is to evaluate the potential neuroprotective effect of the natural biotransformed metabolites on the behavioral and biochemical changes developed in the repetitive traumatic brain injury (RTBI) model.

Biotransformation is the process by which organisms or enzyme systems change the structural properties of a chemical substance, resulting in the conversion of one component into another. This method, which bacteria have developed to adapt to environmental changes, is helpful in various biotechnological operations. These reactions are crucial for putting chemical functions into molecules' inaccessible locations and creating unusual structures. Because of its straightforward, affordable, and safe approaches that combine green chemistry with high efficiency, biotransformation has attracted a lot of attention [7].

A minor naturally occurring hydroxycinnamic acid derivative is called sinapic acid. It belongs to the phenylpropanoid family of phenolic compounds, which is thought to have medicinal benefits and is generally harmless. Sinapic acid is a common component of the human diet. It is found in various plants, including fruits, vegetables, cereal grains, oilseed crops, spices, and medicinal herbs. Sinapic acid exhibits a broad range of biological actions, including hepatoprotective, anti-cancer, antioxidant, anti-inflammatory, antibacterial, and anti-inflammatory effects [8].

In this work, we explored the capability of 11 fungal cultures to biotransform sinapic acid into new products. Also, we evaluated the neuroprotective/anti-inflammatory activity of the obtained metabolites, namely gallic acid, protocatechuic acid, and ferulic acid, using the repetitive traumatic brain injury (RTBI) model. This was done by applying 5 repetitive blows on the right anterior frontal area of the right cortex and then left for 10 days to compare the effect of treatments that lasted for 10 days.

Materials and Methods

Instruments and Materials for Microbial Biotransformation

Sabouraud-dextrose agar (SDA) Becton Dickinson and Company, Cockeysville, Maryland 21030 Yeast extract of a high microbial standard (Oxoid LTD, England); microbiological grade peptone (Sigma Chemical Co., USA); agar with potato dextrose (DIFCO, USA); Dextrose, grade AR (Sigma Chemical Co., USA); glycerol, dipotassium hydrogen phosphate, and analytical-grade sodium chloride (ADWIC, Egypt); Mineral agar (Oxoid LTD, England); Sinapic acid originated in St. Louis, USA (Sigma Aldrich); (ScoutTM Pro (OHAUS) model, USA); Digital balance AutoclavePb1 (Germany); bath of sonicated water (Branson 3510E-MTH, Mexico); Incubator with gyratory motion (Lab Line Instrument, USA); Oven with a thermostat, WT-binder 7200 (Germany). Aspergillus alliaceous NRRL315, Aspergillus niger NRRl3, Penicillium chrysogenum ATCC 948, Aspergillus flavus AUMC 4787, Aspergillus awamori AUMC58, Aspergillus ochraceous AUMC11328, Penicillium chermesinumAUMC275, Cunningamella blackeseleeana AUMC5618, Paecilomyces variotii AUMC4807 and Aspergillus versicolor NRRL 1306 were obtained from American Type culture collection (ATCC), Northern Regional Research Laboratories (NRRL) and Assiut University Mycological center (AUMC).

Small Scale Biotransformation of Sinapic Acid

The small-scale screening studies were conducted following a two-stage fermentation technique [9]. The metabolites were found using TLC with the solvent system methylene chloride, methanol (9:1 V/V), and by spraying anisaldehyde/sulfuric acid reagent and vanillin/sulfuric acid.

Large-Scale Biotransformation of Sinapic Acid

After conducting initial exploratory screening tests employing Penicillium chermesinm AUMC275, Aspergillus ochraceus AUMC11328, and Paecilomyces variotii AUMC4807 cultures, which produced positive screening results, large-scale transformation of sinapic acid was carried out. For each experiment, sinapic acid (1 g) was dissolved in 10 ml of DMSO and distributed evenly among 10 flasks (each holding 1000 ml) containing 200 ml of stage II culture. The cultures were then allowed to develop at 25 ± 2 °C on a gyratory shaker at 100 rpm after the appropriate time for each experiment (5, 7, and 10 days). The recovered, filtered liquid media underwent thorough ethyl acetate extraction. The extracts were combined and evaporated to dryness to give 3.4 g (FI) residue forpenicilliumchermesinmAUMC275 experiment, 3 g (FII) for Aspergillus ochraceous AUMC11328 experiment, and 3.6g for (FIII).

The NMR spectra were captured with a Bruker 400 MHz for 1H NMR and a 100.40 MHz for 13C NMR. As an internal reference, chemical shifts were reported as δ ppm relative to tetramethylsilane (TMS). The spectra were conducted in DMSO. Rotational evaporator (BUECHI, Germany). Laminar-flow canopy (EACT 8613, USA). Fluorescence analysis cabinet with a UV lamp and spectroline (R) Model M-10 (USA). Column chromatography was used with silica gel G 60, 70, or 230 mesh (Merck, Germany). Aluminum sheets with silica gel G F254 precoat underwent TLC (Merck, KGaA, Darmstadt, Germany). Anisaldehyde/sulfuric acid reagent was sprayed on developed chromatograms to make them visible. Solvent solutions were used in the development of TLC plates. Methanol and methylene chloride (DCM) are present in the ratios of (S1) (9:1 V/V), (S2) (9.5:0.5 V/V), and (S3) (8:2 V/V).

Isolation and Purification of the Metabolites

Each 1g of substrate for every experiment gives FI (3.4 g), FII (3g), and FIII (3.6 g) was chromatographed separately on a silica gel column (100g 3cm×60cm) using the dry method. Starting with hexane and moving through hexane/DCM mixes with increasing polarity up to 100% DCM and then DCM/MeOH mixtures with increasing polarity up to 50% MeOH, the columns were eluted in a gradient fashion. After purification on a Sephadex column, FI gave a 21% yield, FII offered a 19.5% yield, and FIII gave a 20% yield.

In Vivo Anti-Inflammatory Study

Materials for the Anti-Inflammatory Activity

4% Isoflurane, We obtained adult male Sprague-Dawley rats from the National Research Center (NRC, Giza, Egypt) and DMSO (FINE-CHEM Limited, India).

Tissue Preparation and ELISA Assessment

Animals in the first set (n=6/group) were sacrificed, and the right cerebral cortex was collected and homogenized in phosphate buffer for ELISA measurement. ELISA kits were used for the measurement of the homogenate contents of the dementia marker [phosphorylated tau (p-Tau; MyBioSource, CA, USA)], signaling molecules [glycogen synthase-3beta (GSK-3β; Invitrogen Corporation: Camarillo, CA), Dickkopf -1(DKK-1; MyBioSource, CA, USA), and inflammatory biomarkers [tumor necrosis-alpha factor (TNF-α; Ray Biotech, GA, USA), interleukin-6 (IL-6; R&D Systems, CH)] according to the manufacturer's instructions provided.

Animal Care and Maintenance

Throughout the study, 114 mature male Sprague-Dawley rats (n = 114), weighing 250–300 g, were housed in cages with 5 rats each under standard laboratory conditions (controlled temperature, humidity, ventilation, and a 12-hour light/dark cycle) with unrestricted food and water. Rats were given at least a week to acclimate to the vivarium before any experimental operations. When handling the animals, the Guide for the Care and Use of Laboratory Animals was rigorously followed (NIH publication, 1996).

Induction of Mild Single and Repetitive Trauma

The weight drop device was used to induce the trauma [10], but with a few modifications learned from our earlier research [11]. All of the animals in the six groups had 4% isoflurane anesthesia before being put on the platform directly beneath the weight drop apparatus. Throughout the experiment, isoflurane vaporized at a concentration of 1.5% and was inhaled through a mask to maintain anesthesia. Except for the normal control group, the impact region was chosen to be the right anterior frontal area, 1.5 mm lateral to the midline in the mid-coronal plane. A weight of 75 g was released and dropped with a final impact of 0.5 J onto the skull.

Experimental Design

The animals were separated into six groups (n = 19), with six rats used for biochemical assays, ten rats used for behavioral experiments, and the remaining three rats maintained for histopathological analysis. These sets were chosen based on findings from our earlier study11, in which the brains of animals subjected to behavioral tests lost consistency compared to their mates who were not subjected to these tests. The rats in this group were subjected to 5 days of isofluorane exposure as a negative control. Animals in group 2 were given one blow and slaughtered after 24 hours to act as the mTBI control group. Animals in group 3 were exposed to 1 blow for 5 days and then left for 10 days to compare the effect of treatments that lasted for 10 days after the last fifth blow, and they were signified as mRTBI-10. In group 4, animals in this group were subjected to the 5 hits as those in group 3 and then received 100 mg/kg ferulic acid immediately after the last hit [12], and then left for ten days without further administration of the drug. Group 5, immediately after the 5th hit, protocatechuic acid (30 mg/kg) was given i.p and then continued once per day to reach one week of treatment [13]. In Group 6, gallic acid was injected (10 mg/kg; i.p) 1 h after the 5th the last hit, which continued for 10 days [14].

Open Field Test

At the end of the experiment, animals in the second set were subjected to the open field test, which depends on measuring locomotor activity, emotionality, and exploratory behavior utilizing latency time, ambulation frequency, rearing frequency, and grooming frequency for 3min. Animals were put independently in the central point of the open field mechanical device and permitted to move unreservedly around the open field to investigate nature for three minutes for examination purposes. A camcorder precisely recorded all The actions during these 3 mins, and the accompanying previously mentioned parameters were watched [15].

Molecular Modeling

The crystal structure of Glycogen Synthase Kinase-3 in combination with AR-A014418 as an inhibitor (PDB ID: 1Q5K) was chosen as the template for molecular modeling investigation by Molecular Operating Environment (MOE®). 2014.09 version (Chemical Computing Group Inc., Montreal, Canada). The enzyme was prepared for docking studies by removing chain B of its dimer, water molecules, and ligands that are not involved in binding and adding missing hydrogen atoms. The active docking site was defined using the co-crystallized ligand. The docking methodology employed the Triangle Matcher Placement method and the London dG score function. The protein was minimized with MOE until an RMSD gradient of 0.05 kcal∙mol−1Å−1 was achieved using the MMFF94x force field, and the partial charges were determined automatically.

Statistical Analysis

All data are presented as mean ± S.D. The means of the (n = 6 or 10) were statistically compared using one-way ANOVA, followed by Tukey's post hoc test. GraphPad Prism software (version 5.0 d; GraphPad Software, Inc., San Diego, CA, USA) was used for all statistical tests, and P <0.05 was used as the significance level.

Results and Discussion

Structure Elucidation of Isolated Metabolites

Metabolite 1 was obtained as an off-white amorphous powder (170 mg). The Rf - value was 0.52 (S3), and it appeared as a dark purple spot under short UV light and showed violet fluorescence under long UV light. It gave blue color with FeCl3 Spray reagent.1H NMR (400 MHz, DMSO-d6), δ ppm 6.88 (2 H, s, H-2/6). 13C NMR (100 MHz, DMSO-d6), δ ppm 168.59 (C=O), 146.09 (C-3/5), 138.48 (C-4), 121.80 (C-1), 121.80 (C-2/6). Negative ESI-MS showed a molecular ion peak at 169.11 [M-H], which corresponded to a Mwt of 170. 1H NMR and 13C NMR, and ESI-MS data agreed with the reported data [16]. Accordingly, the compound was confirmed to be gallic acid, see Figure 1a.

Metabolite 2 was obtained as an off-white amorphous powder (167mg). Rf- value 0.66 (S1); it gave dark spots under short UV-light and shiny violet fluorescent spots under long UV light. 1H NMR (400 MHz, CD3OD), δ ppm 7.45 (1 H, d, J= 1.69, H-2), 7.43 (1 H, dd, J= 1.69, 8.34, H-6), 6.79 (1 H, d, J= 8.23, H-5). 13C NMR (100 MHz, DMSO-d6), δ ppm 167.61 (C-7), 149.85 (C-4), 144.79 (C-3), 122.05 (C-6), 121.85 (C-1), 116.58 (C-2), 115.12 (C-5). Negative ESI-MS showed a molecular ion peak at 153.14[M-H]-. All proton and carbon recorded resonances are in good agreement with that of protocatechuic acid (Figure 1a) by comparing data with that isolated before [17].

Metabolite 3 was isolated as an off-white amorphous powder (175 mg). Rf- value 0.64 (S2); it showed a dark purple spot under short UV-light and shiny violet fluorescence under long UV light. 1H NMR (400 MHz, DMSO-d6), δ ppm 7.08 (H, dd, J= 8.19, 1.93, H-6), 7.28 (1 H, d, J= 1.90, H-2), 6.79 (1 H, d, J= 8.06, H-5), 7.49 (1 H, d, J= 15.89, H-7), 6.36 (1 H, d, J= 15.88, H-8), 3.81 (3 H, s, O-CH3). 13C NMR (100 MHz, DMSO+-d6), of δ ppm 168.55 (C-9), 149.64 (C-4), 148.47 (C-3), 145.07 (C-7), 126.33 (C-1), 123.40 (C-6), 116.18 (C-2), 116.06 (C-8), 111.69 (C-5), 56.24 (OCH3). All proton and carbon resonances are in agreement with that of ferulic acid that was isolated and identified before (Rho and Yoon 2017). Negative ESI-MS spectrum confirmed the structure as it showed a molecular ion peak at m/z 193.38 [M-H]- which is equivalent to a molecular formula (C10H10O4) and a molecular mass 194.06, which was consistent with the M.wt of ferulic acid. Accordingly, the compound was identified as ferulic acid (Figure 1a).

Molecular Docking Study

A molecular docking simulation study of gallic, protocatechuic, and ferulic acids, obtained from microbial biotransformation of sinapic acid, was performed to predict the anti-inflammatory activity results. Furthermore, it will aid in understanding the binding mechanisms and diverse interactions between the ligands and the active site of GSK-3β. The most promising compounds are chosen based on the right binding mode and the binding free energy (∆G). Table 1 show the outcomes of the molecular docking, (Figures 1b-1e). Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that controls various signaling pathways. Among the many tasks that GSK-3β controls, inflammation has recently become one of the most intriguing. TNFα, IL-6, and MCP-1 are examples of pro-inflammatory cytokines and chemokines that are expressed due to an increase in NF-κB activity that GSK-3β mediates. The simultaneous reduction in IL-10 expression makes GSK-3β inhibition protective against inflammatory conditions [18, 19]. Gallic acid inhibits the GSK-3β enzyme and Wnt/β-catenin signaling pathway [20]. In focal cerebral ischemia injury, ferulic acid controls the Akt/GSK-3β/CRMP-2 signaling pathway, preventing brain damage, whereas protocatechuic acids cause GSK-3β inhibition [21].

 

 

Metabolite 1 R= OH    Metabolite 2 R= H

Metabolite 3

a)

b)

c)

d)

e)

Figure 1. a) Chemical structure of the isolated metabolites; b) Surface map of AR-A014418 (black) co-crystallized with GSK-3β (1Q5K) (left); and the corresponding interaction diagram (right); c) Predicted binding mode of gallic acid with the active site of GSK-3β; d) Predicted binding mode of protocatechuic acid with the active site of GSK-3β; e) Predicted binding mode of ferulic acid with the active site of GSK-3β.

 

Table 1. The affinity scores and interactions of the tested compounds and reference drug against GSK-3β (in Kcal/mole)

Compound

Score

Interaction

Gallic acid

-5.02

VAL135: Hydrogen –donor

VAL135: Hydrogen –acceptor

Ile62: Arene-H interaction

Protocatechuic acid

-4.55

VAL135: Hydrogen –donor

Ile62: Arene-H interaction

Ferulic acid

-4.31

VAL135: Hydrogen –donor

AR-A014418

-5.21

VAL135: Hydrogen –donor

VAL135: Hydrogen –acceptor

Ile62: Arene-H interaction

 

Anti-Inflammatory Activity

Changes in Cortical Phosphorylated TAU (PTAU)

 

The cortical content of pTau was markedly increased in all the animals exposed to 5 hits and left for ten days compared to single traumatized and healthy animals. Compared to the mRTBI-10 insult, all our drug regimens showed anti-dementia properties by decreasing pTau (Figure 2).

Panel I