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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                                                         Research Article 

Sterculia setigera hydroethanolic extract protects brain tissues ex vivo against lipid peroxidation and possesses in vitro antioxidant and anti-inflammatory properties

Yendubé T. Kantati* , Magloire K. Kodjo, Bignoate Kombate, Lucie A. Togbossi, Kwashie Eklu-Gadegbeku, Messanvi Gbeassor

Physiopathology Bioactive Substances and Innocuity Research Unit (PBSI). Laboratory of Physiology / Pharmacology. Faculty of Sciences, University of Lomé – TOGO. 01BP 1515

INTRODUCTION

Despite decades of active research, the identification of neuroprotective molecules/treatment remains a priority objective to provide therapeutic solutions to the millions of people worldwide suffering from neurodegenerative diseases. Considering that oxidative stress and inflammation remains major deleterious process in brain degeneration^1,2, plants which often contain antioxidants and anti-inflammatory compounds may represent good candidates for neuroprotection studies. In a previous ethnopharmacological survey, we found that in Togo, local populations used Sterculia setigera Del. (Sterculiaceae) to manage central nervous system (CNS) disorders in general and neurodegenerative diseases in particular³. This plant is a wild tree commonly found in the Sudano-Sahelian zone of Africa. In Togo, S. setigera is more present in the savannas of the north where its name in Moba is “Natoufégligue”. It is called “ponsemporgo” in Mooré (Burkina-Faso), “kukkuki” in Hausa (Niger, Nigeria), “kôgurani” in Bambara (Mali) and “mbep” in Wolof (Senegal). Ethnobotanical studies report the use of this gum for the treatment of snake bites, jaundice and bronchitis, when several studies have detected the presence of active metabolites in stem barks of S. setigera such as tannins, saponins, glycosides and anthraquinone⁴. Traditional healers have mentioned the use of S. setigera leaves extracts for the treatment of epileptic conditions⁵ and for the treatment of memory loss and dementia³. However, the potential neuroprotective properties of S. setigera have not been tested. The aim of this study was to investigate the neuroprotective potential of S. setigera leaves hydroethanolic extract in ex vivo and in vitro conditions.

MATERIALS AND METHODS

Plant material and extraction

Plant material 

Sterculia setigera’s leaves were harvested in Dapaong (TOGO) in the month of October 2021. A voucher specimen was deposited, after identification, in the herbarium of the Laboratory of Botany and Plant Ecology (University of Lomé-TOGO) under the number 08655TG.

Extraction  

400 g of the powder were macerated in a volume of 4 L of an ethanol-distilled water mixture (80:20, v/v) for 72 hours with an intermittent manual stirring. The macerate obtained was filtered and evaporated under vacuum at 45°C using a rotary evaporator. The evaporated extract was lyophilized to obtain a dry extract stored in the refrigerator at 4°C until use.

Ex vivo neuroprotection by inhibition of lipid peroxidation

Brain homogenates

Animals (wistar rats) were anesthetized with ether before being decapitated. Brains were quickly removed, rinsed in an ice-cold normal saline solution (NaCl 9g/L). 4 mg of brain tissues were then ground in 1 mL of 150 mM Tris-HCl solution pH=7.4 for lipid peroxidation assay, and in 0.2 M Tris-HCl solution pH = 8.2 for Glutathione assay. The homogenates were stored at -20°C until use.

Ascorbic acid/Fe ²⁺ (AA-FeCl₂) induced lipid peroxidation in rat brain homogenate 

The anti-lipid peroxidation effect of S. setigera extract was studied using colorimetric method⁶ of Kpemissi et al. (2019). The investigational tubes contained 500 µL of brain homogenate, 200 µL of Tris-HCl buffer (pH 7.4), 100 µL of 0.1 mM ascorbic acid, 100 µL of 4 mM FeCl₂ and 100 µL of different amount of S. setigera extract or quercetin. The samples were kept at 37 °C for one hour. After the incubation time, the malondialdehyde (MDA) concentration in the samples was estimated as previously described using 1,1,3,3-tetra-methoxypropane to make a standard curve (Kpemissi et al., 2019) at concentration points: 0.625; 1.25; 2.5; 5 and 10 nM. Absorbances were read at 586 nm using a UV-VIS spectrophotometer (Genesys 20, Thermo Scientific).

In vitro anti-inflammatory tests

Inhibition of egg albumin denaturation

This assay was realized using egg albumin denaturation method⁷. The reaction mixture was composed of 0.2 mL of fresh chicken’s egg albumin, 2.8 mL phosphate buffer saline (pH 6.4) and 2 mL of various concentrations (25 - 400 µg/mL) of CM extract or standard drugs. All the samples were set aside at 37 °C for 25 minutes followed by heating for 5 minutes at 70 °C. The cooled solutions were centrifuged at 3,000 rpm for 10 minutes. Then the absorbance of supernatant solutions was measured at 660 nm. The % of inhibition and IC50 values were calculated using the equation:

% inhibition = ((A0 - Ae) *100) / A0.

Where A0 represents the absorbance of control tube without extract or Diclofenac, and Ae represents the absorbance in the presence of the extract or Diclofenac. The tests were repeated three times.

Inhibition of red blood cells hemolysis 

The anti-inflammatory activity was assessed using red blood cells (RBCs) membrane stability method⁸ according to the protocol of Shinde et al. (1999). The equal volume of rat blood and 0.9% w/v normal saline was taken in heparinized tubes. The blood was centrifugated for 10 min at 3,000 rpm. Then the suspended cells were washed carefully with saline three times. Finally, 10% v/v cell suspension was prepared using saline and used in heat-induced hemolysis assay. Briefly, the assay mixture contained 1mL of RBCs suspension and 1mL S. setigera extract or standard drug solutions (50- 1,000 µg/mL). The tube samples were kept at 70 ˚C in the bath for 30 minutes. After cooling and centrifuging for 5 minutes at 3,000 rpm, the absorbance was measured at 560 nm using UV spectrophotometer (Dadoriya et al., 2020). In addition, the % inhibition of hemolysis and IC50 values were calculated using the same equation described in the assay of egg albumin denaturation. 

In vitro antioxidant tests

DPPH radical scavenging activity

2,2-diphenyl-1-picryl-hydrazyl (DPPH) test was realized⁹ according to the method of Sen M and Dastida (2010). To 1.5 mL of the DPPH solution (100 µmol/l), 0.25 mL of methanol (white) or 0.25 mL of methanolic solution of S. setigera (25; 50; 100 and 200 µg/mL) were added into tubes. Ten minutes (10 min) after incubation at room temperature, absorbances were red at 517 nm. Ascorbic acid (200 µg/mL) served as reference antioxidant. The percentage inhibition of the DPPH radical was calculated using the equation:

% inhibition = ((A0 - Ae) *100) / A0.

Where A0 represents the absorbance of DPPH alone, and Ae represents the absorbance in the presence of the extract or ascorbic acid. The tests were repeated three times.

Ferric ions reducing power assay

The ability to reduce ferric ions was measured using the method described by Nisha et al. (2012) and slightly modified¹⁰. The FRAP reagent was generated by mixing 300 mM sodium acetate buffer (pH 3.6), 10.0 mM TPTZ (tripyridyl triazine) solution, and 20.0 mM FeCl₃.6H₂O solution in a 10:1:1 volume ratio. S. setigera at different concentrations (25; 50; 100; 200; 500 μg/mL) were then added to 3 ml of FRAP reagent and the reaction mixture was incubated at 37 °C for 30 min. The increase in absorbance at 593 nm was measured. The higher the absorbance of the reaction mixture, the greater the reducing power. Ascorbic acid was used as a positive control. The tests were repeated three times.

Determination of total antioxidant capacity (TAC)

The phosphomolybdenum method was used to access the total antioxidant capacity of the extracts¹¹. A 0.3 mL of extract was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. Then, the absorbance of the solution was measured at 695 nm using a UV-VIS spectrophotometer (Genesys 20, Thermo Scientific) against blank after cooling to room temperature. Methanol (0.3 mL) in the place of extract was used as the blank. The total antioxidant activity is expressed as the number of microgram equivalents of ascorbic acid or vitamin E used as reference antioxidants. The calibration curve was prepared by mixing ascorbic acid or vitamin E (0; 25; 50; 100; 200, 500 and 1000 µg/mL) with methanol.

Quantitative dosage of phytochemicals

Quantitative determination of total flavonoids

The determination of total flavonoids was carried out according to the colorimetric method, basing on their ability to chelate metals including aluminum¹². To 2 mL of the ethanolic solution of S. setigera (1 mg/mL) or of rutin (1 mg/mL), were added 2 mL of aluminum chloride (20 mg/mL) and 6 mL of sodium acetate (50 mg/mL). The blank was made in the same way without S. setigera, replaced by 2 mL of ethanol. After an incubation time of 150 minutes, absorbances were read at 440 nm. The total flavonoid content of S. setigera was determined from the linear regression equation of the calibration curve established with rutin (0-100 µg/mL) and expressed in µg of rutin equivalent per milligram of dry extract (µg Eq/mg of extract). Rutin served as a reference compound. The tests were repeated three times.

Quantitative determination of total phenols and tannins

The determination of total phenols was based on the Folin-Ciocalteu method¹³. Total phenols were assayed after fixing the tannins with PVPP (polyvinylpolypyrrolidone) following two steps. Firstly, to tubes containing 10 mg of PVPP diluted in 500 µL of methanol, 500 µL of S. setigera 1 mg/mL were added. The mixture thus obtained was homogenized and incubated on ice for 30 minutes. After centrifugation, 200 μL of the supernatant were transferred into dry tubes for the assay with the Folin-Ciocalteu reagent. The blank didn’t contain S. setigera, replaced by 500 µL of methanol. Secondly, Folin-Ciocalteu (200 µL) was added to 200 µL of S. setigera (1mg/mL) or to 200 µL of the Gallic Acid solutions (200, 150, 100, 50, 25 and 0 µg/mL) or 200 µL of the solution obtained during the previous step (extract + PVPP). The mixtures were incubated at room temperature for 15 minutes. After incubation, 800 μL of sodium carbonate solution (700 mM) were added to the mixtures. The blank was prepared with 200 µL of Folin-Ciocalteu, 200 µL of methanol and 800 µL of the sodium carbonate solution (700 mM). Absorbances were read at 735 nm. The quantity of total phenol is expressed in mg equivalent of Gallic Acid/g of extract. The total amount of tannin was calculated using the formula:

DOT = DOExt – (DOExt + PVP)

Where DOT = DO tannins; DOExt = DO extracted; DOExt + PVP = DO extracted + PVP

RESULTS

Lipid peroxidation inhibition 

Peroxidation of lipids contained in brain homogenates have significantly raised in the concomitant presence of Ascorbic acid and FeCl₂ (AA-Fecl₂) as shown by Figure 1. Incubation with total hydroethanolic extract of S. setigera or standard antioxidant Quercetin have reversed the lipoperoxidation (-78.28 % and -86.23 % at 200 µg/mL, P<0.001 respectively).

Figure 1: Inhibition of lipid peroxidation in brain homogenates by S. setigera

Values are expressed as Means ± SEM, n = 3. Units: nM/mg brain samples. ***P<0.001: vs Control; # P<0.05 and ###P<0.001: vs AA-FeCl₂. 

Anti-inflammatory activities

Table 1 shows that the extract inhibited the denaturation of chicken egg albumin and stabilized the red blood cell membrane against hemolysis induced by heat. The IC50 found for S. setigera (139.68 ± 0.36 µg/mL) when inhibiting RBCs hemolysis is very close to that of Diclofenac (128.41 ± 0.21 µg/mL).

Table 1: Inhibition of albumin denaturation and RBCs hemolysis by S. setigera

Values are expressed as Means ± SEM, n = 3. Units: IC50 are expressed as µg/mL of S. setigera (µg/mL).

DPPH radical scavenging activity

Figure 2 shows the free radical scavenging activity of the hydroethanolic extract of S. setigera and standard Ascorbic acid (AA). Among these two compounds, Ascorbic acid activity (95.48 ± 0.08 %) was higher than that of the extract (92.67 ± 0.17%) at the same concentration of 200 μg/mL. The IC50 of S. setigera extracts (21.25 ± 0.41 µg/mL) closely resembled that of the standard (20.60 ± 0.26 µg/mL).

Figure 2: Determination of DPPH radical scavenging activity of S. setigera

Values are expressed as Means ± SEM, n = 3. Units: µg/mL

Ferrous reducing antioxidant capacity

A higher absorbance indicates a higher Ferrous reducing power (FRAP). Figure 3 indicates moderate to high FRAP with increased concentration of the extracts. At 500 µg/mL, the absorbance of S. setigera hydroethanolic extract was 4.77 ± 0.40.

Figure 3: Absorbance of FRAP of S. setigera extract at different concentrations

Values are expressed as Means ± SEM, n = 3. Units: µg/mL.

Total antioxidant capacity 

The TAC of S. setigera hydroethanolic extract expressed as equivalent of standards used (Vitamin E and Ascorbic acid) are presented in Table 2.

 Table 2: TAC of S. setigera extract

Values are expressed as Means ± SEM, n = 3. Units: TAC are expressed as Vitamin E Equivalent (µg Vit.EE/mg) and Ascorbic acid Equivalent (µg AAE/mg) (µg/mL).

Total phenolic, flavonoid and tannins contents

Table 3 shows the total polyphenols, flavonoid and tannins contents in the hydroethanolic extracts of S. setigera expressed as Gallic acid Equivalent (GAE) and Rutin Equivalent (RE). Flavonoids (172.38 ± 8.36 µgRE/mg) are well represented. 

Table 3: Polyphenol contents of S. setigera extracts 

Values are expressed as Means ± SEM, n = 3. Units: total phenols and tannins are expressed as µg Gallic acid equivalent/mg of S. setigera (µgGAE/mg), while flavonoids are expressed as µg Rutin equivalent/mg of S. setigera.

DISCUSSION

Free radicals’ production is one of the well-known outcomes of brain’s high consumption of oxygen and glucose^14,15. This important demand of oxygen in particular for ATP production, as neuronal activity fails quickly when privates in transient ischemia, leads unfortunately to the rise of free radical and non-radicals, notably superoxide anion (O₂^.-), hydrogen peroxide (H₂O₂) and hydroxyl (.OH)¹⁶. Such species, usually considered to constitute the “dark side” of O₂ biochemistry, can initiate disruptive peroxidation reactions with various substrates important to the survival of cells such as proteins, lipids and nucleic acids. Lipids peroxidation especially may then be used as a good biomarker of brain oxidation status. In our ex vivo studies, ascorbic acid/Fe²⁺-induced lipid peroxidation in wistar rat’s brain homogenates was significantly reduced by S. setigera hydroethanolic extract. Malondialdehyde (MDA), the end-product of lipid peroxidation, was significantly lowered by concomitant incubation of brain homogenates with extract at doses ranging from 50 to 200 µg/mL. This significant antioxidant neuroprotection afforded by S. setigera hydroethanolic extract could be linked to its antioxidant capacities, as confirmed by in vitro antioxidant tests. In fact, the extract showed a strong scavenging activity, with an inhibitory concentration (IC₅₀) of DPPH free radicals very close to that of the standard antioxidant used, ascorbic acid. S. setigera also demonstrated its capability to reduce ferric ions, well known as prooxidant. Through Fenton-type reactions, the conversion of Fe²⁺ into Fe³⁺ can generate free radicals, leading to increased peroxidation and lipids damages¹⁷. Then, by reducing ferric ions in FRAP test, S. setigera confirms its good antioxidant capacities and gives credence to its folkloric usage. 

Moreover, the S. setigera hydroethanolic extract has proved in this study it’s in vitro anti-inflammatory capacities. The capability of this extract to counteract RBCs hemolysis in particular was remarkable, with an IC₅₀ close to that of standard anti-inflammatory drug Diclofenac. Elsewhere, Henneh et al. (2018) also showed that the stem bark extract of Sterculia setigera (30, 100 and 300 mg/kg, p.o.) exhibits in vivo anti-inflammatory activity by reducing rats paw oedema in the carrageenan and prostaglandin E2-induced inflammation tests¹⁸. Theses antioxidants and anti-inflammatory activities should be linked to its phytochemical composition, namely the presence of flavonoids in high concentration in our study. Flavonoids are natural components that received great attention in the last decades. They are well known to prevent oxidative stress and cell death by scavenging ROS, chelating metal ions and quenching singlet oxygen¹⁹. Catechin for example, an abundant flavonoid, has been shown to have an important antioxidant and anti-inflammatory²⁰. As oxidative stress and inflammatory processes are among the most deleterious in neurodegeneration, our results showing antioxidant, anti-inflammatory and antilipoperoxidative effects, make S. setigera a good candidate for neuroprotective studies and neuroactive drug discovery. 

CONCLUSION

S. setigera hydroethanolic extract protected the brain homogenate against lipid peroxidation induced by ascorbic acid/Fe²⁺ mixture ex vivo. The plant extract also exhibited in vitro antioxidant and anti-inflammatory properties. These preliminary observations confirm the use of this plant in traditional medicine strategies against central nervous system disorders in general and neurodegenerative diseases in particular. Further in vivo investigations are needed to reveal its neuroprotective effects in models of animal’s neurodegeneration and its chemical composition for potential drug discovery.

ACKNOWLEDGMENT

This study was not funded.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

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