Comparative study on the chemical composition and biological activity of polyphenolic extracts obtained from Maclura pomifera (Raf.) C.K. Schneid bark and periderm

Abstract

Literature is limited regarding the potential use of the bark of Maclura pomifera as a source of bioactive compounds. The influence of different extraction methods was assessed for the chemical and biological properties of extracts obtained from the bark and periderm of M. pomifera. The extraction process was completed using three methods: microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and sonicator-assisted extraction (SAE). The extracts were characterized by total polyphenol content. The antioxidant capacity was evaluated using DPPH and ABTS methods, the antimicrobial effect was tested against Gram-positive and Gram-negative bacterial strains, and the inhibition of enzyme activity was conducted using α-glucosidase, lipase, and α-amylase assays. The extracts obtained from the periderm exhibited a lower total polyphenolic content compared to those obtained from the bark. The highest antioxidant activity using the DPPH method was observed in the MAE bark extracts and when using the ABTS method, and the highest activity was observed in the UAE bark extracts. Enzymatic activity was most strongly inhibited by the SAE bark extract. The most pronounced antibacterial potential was observed in the MAE and UAE bark extracts, with minimum inhibitory concentration values obtained below 1 mg/mL.

Full Article

Comparative Study on the Chemical Composition and Biological Activity of Polyphenolic Extracts Obtained from Maclura pomifera (Raf.) C.K. Schneid Bark and Periderm

Năstaca-Alina Coman,^a,b Mihai Babotă,^b,e Ingrid Nădășan,^d,* Ștefania Dinică,^e Ruxandra Ștefănescu,^f Oleg Frumuzachi,^c Adrian Man,^g Andrei Mocan,^c,h and Corneliu Tanase ^b,e

Literature is limited regarding the potential use of the bark of Maclura pomifera as a source of bioactive compounds. The influence of different extraction methods was assessed for the chemical and biological properties of extracts obtained from the bark and periderm of M. pomifera. The extraction process was completed using three methods: microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and sonicator-assisted extraction (SAE). The extracts were characterized by total polyphenol content. The antioxidant capacity was evaluated using DPPH and ABTS methods, the antimicrobial effect was tested against Gram-positive and Gram-negative bacterial strains, and the inhibition of enzyme activity was conducted using α-glucosidase, lipase, and α-amylase assays. The extracts obtained from the periderm exhibited a lower total polyphenolic content compared to those obtained from the bark. The highest antioxidant activity using the DPPH method was observed in the MAE bark extracts and when using the ABTS method, and the highest activity was observed in the UAE bark extracts. Enzymatic activity was most strongly inhibited by the SAE bark extract. The most pronounced antibacterial potential was observed in the MAE and UAE bark extracts, with minimum inhibitory concentration values obtained below 1 mg/mL.

DOI: 10.15376/biores.18.4.8104-8119

Keywords: Microwave extraction; Ultrasound extraction; Polyphenols; Antioxidant activity; Antibacterial activity; Enzyme activity; Rhytidome

Contact information: a: Doctoral School of Medicine and Pharmacy, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, Târgu Mures, Romania; b: Research Center of Medicinal and Aromatic Plants, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, Târgu Mures, Romania; c: Department of Pharmaceutical Botany, “Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; d: Residency Department, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mureș, Târgu Mureș, Romania; e: Department of Pharmaceutical Botany, Faculty of Pharmacy, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, Târgu Mures, Romania; f: Department of Pharmacognosy and Phytotherapy, Faculty of Pharmacy, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, Târgu Mures, Romania; g: Department of Microbiology, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mureș, Târgu Mureș, Romania; h: Laboratory of Chromatography, Institute of Advanced Horticulture Research of Transylvania, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania; *Corresponding author: ingrid.nadasan@gmail.com

INTRODUCTION

Lately, interest in researching plant species as sources of natural compounds with therapeutic potential has significantly increased. The number of studies focusing on the assessment of chemical composition and the bioactive potential of secondary products obtained from plant biomass has recorded impressive growth in recent years (Jadoun et al. 2021; Salem and Fouda 2021). Maclura pomifera (Raf.) C.K. Schneid. (Osage orange) is a tree species belonging to the Moraceae family. It is a deciduous species native to the southwestern United States (Texas and Arkansas); however, it is currently widely spread in Europe as well. Compounds isolated from various parts of the Osage orange belong to different classes, such as triterpenes, xanthones, flavonoids, and stilbenes (Cicco et al. 2009; Laczkó-Zöld et al. 2018; Ștefănescu et al. 2022). These secondary metabolites have been associated with numerous pharmacological actions: antibacterial, anti-inflammatory, antitumor, cardioprotective, and anticholinesterase activities (Polbuppha et al. 2017; Khaleghi et al. 2019; Saeed et al. 2023).

Osajin and pomiferin exhibit antibacterial properties, although their antioxidant properties are more significant (Filip et al. 2015). A study examined the anti-aging potential of the methanolic extract from M. pomifera (MP) fruits by measuring antioxidant activities and the inhibition of extracellular matrix-degrading enzymes. The analyzed extracts were promising sources for obtaining anti-aging cosmetic products (Barak et al. 2022). Scientific evidence has demonstrated that MP fruits can be toxic to certain herbivorous mammals. Macluroxanthone and alvaxanthone, extracted from the root bark, have proven highly detrimental to mosquito larvae and, respectively, to goldfish. Like the antifungal substance in the wood, these pigments likely protect the roots against decay and insects (Smith and Perino 1981).

Various species of M. pomifera are used in traditional medicine worldwide, with the fruits and leaves being primarily utilized in this sense. Existing literature to date also describes the potential use of the tree’s bark as a source of bioactive chemical compounds, albeit with limited data to verify this aspect (Filip et al. 2021). In this context, the aim of this study was to assess the influence of different extraction methods on the chemical and biological properties of extracts obtained from the bark and periderm of M. pomifera. As such, this study focused on obtaining extracts through different extraction techniques and evaluating the total polyphenolic content, as well as assessing the antibacterial, antioxidant, and antienzymatic potential of M. pomifera bark and periderm extracts.

MATERIALS AND METHODS

Collection and Processing of Plant Samples

The bark and periderm of Maclura pomifera (Raf.) C.K. Schneid was collected in 2022 from the Medicinal Plants Garden of “George Emil Palade” University of Medicine, Pharmacy, Science, and Technology in Târgu Mureș, Romania. The sampled tree was aged between 20 and 30 years old. For the drying process of the bark and periderm, a Nahita 631 Plus drying oven (Auxilab S.L., Beriáin, Spain) was used, being set at a temperature of 50 °C for 24 h. Afterward, the dried material was ground using a Pulverisette 15 cutting mill (Fritsch GmbH, Idar-Oberstein, Germany). The biomass was used in its raw form without undergoing any other treatments or further processing.

Histological Analysis

The plant material was initially subjected to fixation and preservation by immersion in 70% v/v ethyl alcohol. Subsequently, the samples were processed through sectioning using a microtome blade and a manual microtome, with the sections being retrieved into a container of water. For the clear visualization of the histo-anatomical details, the sections were stained by immersion in an iodine green solution for 10 min, followed by the removal of the excess dye through immersion in water (Tanase et al. 2022). The stained sections were mounted on slides using glycerinated albumin as an embedding medium and analyzed using a Motic B Series microscope equipped with a digital camera (Nikon, Tokyo, Japan).

Extraction Procedure

The extraction process was completed using microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and sonicator-assisted extraction (SAE). The extraction parameters, for the MAE, UAE, and SAE extraction methods, were taken from previously optimized methods on other woody plant matrices. Two solvents with different polarities were used: ethanol-70% which has optimal extraction conditions and water 100% to ensure an economic, sustainable, and ecological extraction with a positive impact on climate change by reducing the use of toxic solvents. A microwave extractor (Ethos X, Milestone, Sorisole, Italy) was used for the MAE. Following that, 10 g of bark/periderm and 200 mL of solvent (water/70% ethanol) were mixed. The aqueous variants were extracted for 29 min and 21 s at a microwave power of 864 W, while ethanol variants were extracted for 17 min and 40 s at a microwave power of 655 W (Nisca et al. 2022). For UAE, 2.5 g of plant material was mixed with 100 mL of solvent (water/70% ethanol). The extracts were then placed in an Elma Transsonics ultrasound bath (Elma Schmidbauer GmbH, Singen, Germany) for 15 min at 70 °C with an ultrasound frequency of 40 kHz (Tanase et al. 2018). For SAE, 5 g of plant material were weighed in a round-bottomed flask and mixed with 100 mL of freshly prepared 40% ethyl alcohol by dilution. The mixture was homogenized and subjected to sonication for 15 min at an amplitude of 40% (Hielscher Ultrasonics, UP 200St, 200 W, 26 kHz, Teltow, Germany). The ultrasonic probe was inserted vertically in the middle of the mixture. To counteract the thermal effect induced by ultrasound exposure and possible interference caused by it (e.g., heating of the extraction mixture, solvent evaporation), the extraction was performed on ice and with constant maintenance of the immersion depth of the extractor probe (Coman et al. 2023).

All extracts were subjected to the lyophilization process using a lyophilizer model BK-FD12S freeze dryer (Biobase Biodustry Co., Ltd., Shandong, China).

The 10 dried extracts of M. pomifera were as follows (Table 1).

Table 1. Characteristics of Solutions Obtained by Various Extraction Methods

Total Polyphenolic Content

To assess the total polyphenolic content (TPC), the Folin-Ciocâlteu method (Cicco et al. 2009) was employed. Gallic acid was used as a reference standard to calculate the polyphenolic content in the samples. Initially, the samples were dissolved in 70% ethanol to obtain working solutions with a concentration of 2 mg/mL. From these working solutions, 400 µL were taken and diluted with 1600 µL of distilled water, resulting in a 1:5 dilution. From the obtained diluted solution, 400 µL were taken and mixed with 400 µL of Folin-Ciocâlteu reagent and 3200 µL of Na₂CO₃ solution (5%). The reaction mixture was stirred and incubated at room temperature in the dark for one hour, and absorbance of the samples was spectroscopically measured at a wavelength of 750 nm using a Specord 200Plus UV-Vis spectrophotometer (Analytik Jena AG, Jena, Germany). To quantify the phenolic compound content, a standard curve was used composed of nine different solutions of gallic acid with concentrations ranging from 0.05 to 0.45 mg/mL. The linear equation for this curve is y = 11.767× x + 0.2737. For each extract, three replicates were performed, and the absorbance of each individual sample was recorded twice, resulting in six absorbance values for each experimental variant. The absorbance of the samples was compared to the absorbance of gallic acid standards, and the total polyphenolic content was expressed in gallic acid equivalents per gram of extract (mg GAE/g) using the calibration curve.

Antioxidant Activity

To evaluate the free radical scavenging activity of the extracts, a microplate reader (Epoch, BioTek, Winooski, VT, USA) was employed. Two in vitro assays were conducted for this purpose: the DPPH test and the ABTS test. Inhibition capacity was calculated using the following Eq. 1,

(1)

where A₀ is the absorbance of the DPPH solution and A₁ is the absorbance of the mixture of the sample and the DPPH solution read at 517 nm after an incubation period of 30 min.

Half-maximal inhibitory concentration (IC₅₀) was calculated using a dose–response curve and was expressed as μg/mL.

Similarly, the assessment of the ABTS free radical neutralization capacity (Ștefănescu et al. 2022) was conducted. Ninety-six-well microplates were used, with each well containing 50 µL of extract at various concentrations along with 200 µL of ABTS methanolic solution. The reaction mixture was then incubated at room temperature for 30 min. Following incubation, the absorbance of the samples was measured at 734 nm. To evaluate the inhibition capacity of the extracts and to calculate the IC₅₀ (the concentration at which 50% of the free radical activity is inhibited), the formula (Eq. 1) described above was utilized.

Enzyme Inhibitory Activity

The potential enzymatic inhibition was tested on three enzymes: α-glucosidase, α-amylase, and lipase.

To determine the α-glucosidase inhibitory capacity (Tanase et al. 2022), the dried extract was first dissolved in phosphate buffer (100 mM, pH = 6.8) supplemented with 5% DMSO (concentrations ranging from 0.002 to 4 mg/mL). Subsequently, 50 µL of the extract was pipetted along with 50 µL of enzyme solution (0.75 U/mL in potassium phosphate buffer, pH = 6.8) and 50 µL of substrate solution (pNPG). After an incubation period of 15 min at 37 °C, the absorbance of the sample was measured at a wavelength of 405 nm. The positive control used in the α-glucosidase inhibition test was acarbose (3.33 to 333.33 µg/mL). The inhibition percentage (%I) of each extract was calculated according to Eq. 2, with the final results being expressed as IC₅₀ values (µg/mL),

(2)

where is the absorbance of the control blank, is the absorbance of the control, is the absorbance of the sample blank, and is the absorbance of the sample.

To assess the inhibitory capacity of the extract’s α-amylase inhibition, the Caraway-Somogyi method utilizing iodine/potassium iodide (IKI) reagent was employed. In a 96-well plate, 25 µL of the extract (previously dissolved in 20 mM phosphate buffer, pH = 6.9, supplemented with 5% DMSO, concentrations ranging from 0.002 to 4 mg/mL) were homogenized with 50 µL of enzyme solution (0.05 mg/mL in phosphate buffer), and the mixtures were pre-incubated for 10 min at 37 °C in darkness. Subsequently, 50 µL of 0.05% starch solution was pipetted into the reaction mixture, and the plate was further incubated for 10 min at 37 °C in darkness. Finally, the catalytic reaction was halted by adding 25 µL of 100 mM hydrochloric acid, followed immediately by the addition of 100 µL of 5 mM IKI solution. The absorbance of the samples was determined at a wavelength of 615 nm. The positive control used in the α-amylase inhibition test was acarbose (3.12 to 200 µg/mL). The inhibition percentage of the samples was calculated according to Eq. 2.

The lipase inhibition assay was based on a previously described protocol using a 96-microplate reader (Moldovan et al. 2023). Each well contained 40 µL of tested sample and 40 µL of type II lipase (L3126, Sigma-Aldrich, Saint Louis, MO, USA) from porcine pancreas (2.5 mg/mL prepared in Tris-Buffer (100 mM Tris–HCl and 5 mM CaCl₂, pH 7.0)). After preincubation of 15 min at 37 °C, 20 µL of 10 mM pNPB solution was added to each well and samples were incubated for another 10 min, at 37 °C. Finally, the absorbance of the samples was measured at 405 nm. Orlistat was used as positive inhibitor, while the results were calculated using Eq. 2.

Antimicrobial Activity

Tests were conducted to evaluate the antibacterial activity of extracts derived from M. pomifera against a broad spectrum of bacteria, including Gram-positive bacteria, such as Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus ATCC 43300, and Enterococcus faecalis ATCC 29212, as well as Gram-negative bacteria, such as Escherichia coli ATCC 25925, Klebsiella pneumoniae ATCC 3883, and Pseudomonas aeruginosa ATCC 27853.

To evaluate the antibacterial activity of MP extracts, the microdilution method in sterile 96-well plates was used. After dissolving the extracts, the authors prepared a series of binary dilutions of the MP extracts in sterile water by sequential transferring of 100 μL of the MP extracts solution from the first column to the subsequent columns of the plate. Simultaneously, a 0.5 McFarland bacterial inoculum using sterile saline solution and freshly obtained cultures of human pathogenic bacteria was prepared. The microorganisms used for antimicrobial evaluations were supplied through the Microbiology Department of the George Emil Palade University of Medicine, Pharmacy, Sciences, and Technology from Târgu-Mureș. From this, 10 μL were mixed with 9990 μL of 2X Mueller Hinton Broth (MHB) and 100 μL of this mixture were transferred into the MP extract dilutions contained in each column of the plate. To ensure proper control, a positive control without the addition of the TS was prepared. Additionally, a negative control consisting of water and 2X MHB to verify sterility was used.

To facilitate result interpretation, 3 μL of a 0.015% resazurin solution was added to each well (Tanase et al. 2019). The plate was then incubated at 37 °C for 2 to 4 h to allow bacterial growth. Colors of resazurin in each well were observed. The last well where the resazurin did not change its color was recorded as the Minimum Inhibitory Concentration (MIC). Before adding the 0.015% resazurin, the wells without visible bacterial growth were identified and 3 μL from these wells were inoculated onto a Sheep Blood Agar culture medium (Oxoid Ltd., Hampshire, United Kingdom). The plates were then incubated at 35 °C for 18 h. The position where no bacterial growth was observed on the Sheep Blood Agar plate was considered the Minimum Bactericidal Concentration (MBC).

Statistical Analysis

The data were presented as mean ± standard deviation. The statistical analysis was performed using the GraphPad Prism 9.4.1 software (GraphPad Software, San Diego, CA, USA). The normality of the sample data was assessed by conducting the Shapiro-Wilk test and visually examining quantile-quantile (Q-Q) plots. Within the analysis, the data underwent a one-way multivariate analysis of variance (ANOVA), followed by the examination of mean group differences using the Tukey post hoc test. Spearman’s correlation coefficients were employed to assess the relationships between total phenolic content (TPC), antioxidant capacity, and enzymatic inhibition. For the chord diagram, the ORIGIN 9 software (OriginLab Corporation, Northampton, MA, USA) was used. Statistical significance was considered at p < 0.05.

RESULTS

Histo-anatomical Analysis of Maclura pomifera Bark

The obtained cross-sectional sections allowed the visualization of the histo-anatomical details of two distinct components of the MP bark (Fig. 1).

Fig. 1. The rhytidome of Maclura pomifera: 1-periderm; 2-sclerenchyma fibers; 3-primary bark; 4-secondary bark; 5-calcium oxalate druses

The well-represented rhytidome can be observed, which includes the outer periderm that has been noticed to be thin and serves to protect the stem externally, and it is composed of 3 to 6 layers of cork (tabular, flattened cells containing tannin), phellogen, and collenchymatous phelloderm. Immediately below the periderm, the primary cortex becomes apparent. It is characterized by multiple layers, rich in strongly lignified sclerenchymatic fibers. The secondary cortex appears as a region radially marked by the medullary rays (lightly stained in green due to its low lignin content); it is also rich in groups of sclerenchymatic fibers, whose cells are highlighted by the darker coloring (most likely due to the contained tannins), and presents numerous large, irregularly shaped druse-type oxalate crystals.

Total Phenolic Content (TPC) and Antioxidant Activity

The total phenolic content (Table 2) varied based on the type of matrix used for the extract preparation, the extraction method employed, and the type of solvent used (p < 0.0001). Regarding the matrix that was used, the extracts obtained from the periderm had a lower total phenolic content compared to those obtained from the rhytidome. It is worth mentioning that the ethanolic extract of periderm obtained through microwave-assisted extraction exhibited a higher phenolic content compared to the aqueous extract obtained from rhytidome using ultrasound. This highlights the enhanced efficiency of microwave-assisted alcoholic extraction compared to aqueous ultrasound extraction, a trend that was also observed for the rhytidome extracts.

Furthermore, the solvents used were also determinants in obtaining polyphenol-rich extracts, although this has not been shown to be statistically significant. Regardless of the applied extraction method, the total amount of polyphenols in aqueous extracts was lower than that of the ethanolic extracts. The use of ethanol also increased the efficiency of the extraction methods used, which was noticeable in both periderm and rhytidome extracts. This effect is due to the intermediate polarity of the ethanol-water mixture, which usually allows the extraction of a wider range of polyphenolic compounds compared to water-only extract.

Table 2. TPC and Antioxidant Activity of Maclura pomifera

Note: dw = dry weight. Different superscript letters (a through f) in the same column mean statistically significant differences at p < 0.05 and the same alphabetical superscript in a column indicates no statistically significant difference; ± standard deviation

Regarding the action of MP rhytidome and periderm extracts on the DPPH radical (Table 2), it was observed that the lowest IC₅₀ values were obtained in ethanolic rhytidome extracts. It is worth mentioning that ethanolic periderm extracts exhibited IC₅₀ values close to those of rhytidome extracts, indicating that polyphenols in these extracts are not the sole responsible metabolites for their antioxidant activity. Therefore, ethanolic extracts obtained through ultrasound-assisted and microwave-assisted extraction proved the most active among the rhytidome extracts. Similar results were obtained for the samples extracted with a sonicator, where the highest IC₅₀ (the inhibition concentration needed to reduce the DPPH absorbance by half) was achieved among rhytidome extracts.

Furthermore, the antioxidant potential of MP rhytidome and periderm extracts was also evaluated using the ABTS method (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). The results obtained indicate a much better ABTS radical neutralization capability for all extracts compared to their activity against the DPPH radical. The variation trend of the IC₅₀ values was similar to the one observed in the complementary test employed; the lowest values were obtained for rhytidome extracts, particularly the ethanolic ones, whereas the inhibitory activity significantly decreased in the series of periderm extracts.

Additionally, as can be observed in Table 2, there were differences of approximately an order of magnitude in antioxidant activity determined against two different radicals (DPPH and ABTS). This is an interesting finding and may indicate various aspects related to the antioxidant behavior of the tested compounds. One of these differences could be antioxidant selectivity. This significant difference in antioxidant activity may suggest that the tested compounds have a strong selectivity for a particular type of free radical. This could be useful in developing specific antioxidants to combat certain types of oxidative stress. Another difference in antioxidant activity may be related to the chemical structure of the tested compounds. Certain functional groups or molecular configurations may be more efficient in neutralizing certain types of free radicals. Additionally, the free radicals used in experiments can have different characteristics and may interact differently with the tested compounds. This can lead to significant variations in antioxidant activity. If the compounds exhibit significantly higher antioxidant activity against a specific type of free radical, this could have implications for the development of treatments or dietary supplements for specific conditions or diseases related to oxidative stress.

Inhibitory Potential Against α-Glucosidase, α-Amylase, and Lipase

The assessment of enzymatic inhibition capacity was tested using an in vitro model of α-glucosidase inhibition as a primary study model. The obtained values highlighted a significantly higher inhibition capacity for rhytidome extracts compared to that of periderm extracts. The effects of the extraction method and solvent were also reflected in relation to the α-glucosidase inhibition, with the most active extracts obtained from rhytidome using MAE and sonicator extraction with ethanol as the extraction solvent. The enzymatic activity was most strongly inhibited by the ethanolic extract obtained from rhytidome through sonicator extraction (IC₅₀ = 2.47 μg/mL), which also had the highest determined TPC. It is worth mentioning that at the opposite end, the aqueous extracts of periderm obtained through ultrasonic and sonicator extraction exhibited the least inhibition of α-glucosidase, which is most likely due to their low polyphenol content (Table 3).

The inhibition of α-amylase was significantly reduced compared to α-glucosidase, with IC₅₀ values for the tested extracts being up to 100 times higher. Furthermore, three of the samples under investigation did not exhibit any inhibition of the enzyme. While the results suggest a lower anti-amylase capacity of the extracts obtained from both MP rhytidome and periderm, it is important to note that the trend of variation described for this activity is similar to that observed in the α-glucosidase inhibition test. Thus, it can be observed that overall, the lowest IC₅₀ values were obtained for the ethanolic extracts of rhytidome processed through MAE and sonication.

Table 3. Enzyme-inhibitory Potential of Maclura pomifera Extracts

Note: dw = dry weight. Different superscript letters (a through e) in the same column mean statistically significant differences at p < 0.05 and the same alphabetical superscript in a column indicates no statistically significant difference; ± standard deviation

Likewise, with respect to the inhibition of α-glucosidase and α-amylase, the effects of the extraction method and used solvent influenced the lipase inhibition potential of tested extracts. Aqueous extract of rhytidome obtained by MAE had the highest potential to inhibit porcine pancreatic lipase, with an IC₅₀ value of 842 µg/mL, while the lowest noticed potential was exhibited by aqueous extract of rhytidome obtained by UAE, with an IC₅₀ value of 2140 µg/mL. While some of the tested extracts exhibited a low-to-moderate lipase inhibition potential, the lipase-inhibition capacity of five extracts could not be established.

From Table 4, a strong and statistically significant correlation (p < 0.05) was apparent between TPC-DPPH, TPC-ABTS, and DPPH-ABTS for all three extraction methods. Between ABTS and α-amylase, as well as between DPPH and α-amylase, a negative correlation was found for extracts obtained through UAE and SAE. Conversely, for extracts obtained through MAE, a strong positive correlation between DPPH and α-amylase was observed, but it did not reach statistical significance. Regarding the relationship between α-amylase and α-glucosidase, as well as between lipase and α-glucosidase, or between lipase and ABTS, a significant correlation was noted, but without reaching statistical significance for extracts obtained through UAE and SAE. Conversely, for extracts obtained through MAE, a negative correlation between these elements was identified.

Evidence for all of these explanations provided in Table 4 can also be observed in Fig. 2. In this figure, the concept of chord diagrams refers to a graphical method used to visualize and illustrate correlations or relationships between different categories. The analyzed categories include TPC, DPPH, ABTS, α-Glucosidases, α-Amylase, and lipase. Additionally, in the figure, one can observe the lines connecting these categories. These lines are drawn between two points to indicate a link or correlation between them. The thickness or intensity of these chord lines may vary to reflect the degree of correlation or connection between categories. Thicker or more intense lines may indicate a stronger correlation.

Therefore, for extracts obtained through UAE and MAE, the thickest line (indicating the strongest correlation) is between DPPH and lipase, whereas for extracts obtained through SAE, the strongest correlation is between ABTS and lipase. However, it is important to mention that the absence of a line indicates that there is no correlation between the respective categories. These diagrams provide a visual and intuitive way to interpret and communicate complex chemistry-related data, thus facilitating the understanding and analysis of these relationships.

Table 4. Spearman’s Rank Correlation Coefficient ρ (p-value) Between TPC, DPPH, ABTS, α-Glucosidases, α-Amylase, Lipase Among Tested Extracts

*Significant result: p < 0.05; ρ = Spearman’s rank correlation coefficient

Fig. 2. Chord diagram presenting the correlation between TPC, DPPH, ABTS, α-glucosidase, α-amylase, and lipase for extracts obtained by: ultrasound-assisted extraction (a), microwave-assisted extraction (b), and sonication-assisted extraction (c)

Antibacterial Activity

The authors tested the antibacterial activity of M. pomifera extracts against Gram-positive bacteria and Gram-negative bacteria. All the results obtained are represented in Table 5. The findings showed that periderm extracts were generally inactive against all tested strains, with only a very weak inhibitory activity against bacterial growth observed for ethanol extract of periderm obtained via UAE. In contrast, rhytidome extracts exhibited slightly increased activity, with the lowest MIC and MBC values observed among ethanol extracts. The most pronounced antibacterial potential was observed for ethanol extracts of rhytidome obtained through MAE and UAE, with MIC values below 1 mg/mL for these extracts.

Table 5. Antibacterial Potential of the Tested Extracts Against Bacterial Strains Evaluated Through MIC and MBC Values

Expressed in mg of lyophilized extract per mL of solution

Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) were the most sensitive to the tested extracts, as most of the extracts inhibited the growth of these strains, except for the MPPA UAE, MPPA MAE, MPPE MAE, and MPP SAE extracts. The MPRE MAE extract was the most effective, inhibiting bacterial growth at a concentration of 0.23 mg/mL (MIC) for Staphylococcus aureus and 0.62 mg/mL for MRSA. For Enterococcus faecalis, the extracts MPRE UAE, MPRE MAE, and MPR SAE only had an inhibitory effect without being bactericidal, as the minimum bactericidal concentration (MBC) was not reached for these extracts.

Regarding the Gram-negative bacterial strains, the authors observed that the antibacterial activity was significantly lower compared to the antibacterial activity against Gram-positive bacteria. For the Escherichia coli ATCC 25922, resistance to almost all tested extracts was observed (MIC and MBC values higher than 5 mg/mL indicate lack of sensitivity), except for the extracts obtained via sonication (MPP-SAE and MPR-SAE). A similar situation was observed for the Klebsiella pneumoniae ATCC 13883, which showed resistance to most of the tested extracts, except for the MPRA UAE and MPRA MAE extracts. None of the tested extracts demonstrated efficiency against the Pseudomonas aeruginosa ATCC 27853, either at equal concentrations or at concentrations lower than 5 mg/mL.

DISCUSSION

Histo-anatomical evaluation of the stem structure of M. pomifera details aspects specific to woody plants belonging to the Moraceae family. From the specialized literature searches, no information was found about the description of the histo-anatomical structure of the bark and periderm. M. pomifera has been used in the past for various purposes, including obtaining natural pigments. Regarding the antioxidant activity of M. pomifera, there is some research suggesting that this plant may contain compounds with antioxidant properties. Antioxidants are substances that help protect cells against oxidative stress, which can contribute to aging processes (Barak et al. 2022) and the development of various conditions, such as cardiovascular diseases, cancer, and neurodegenerative disorders (Alhilal et al. 2023).

Although research on M. pomifera is limited, it was found that MP contains certain classes of compounds that may have antioxidant potential. Among these compounds are osajin and pomiferin, which are known for their antioxidant properties. It is also believed that certain chemicals found in the fruits and leaves of the plant could contribute to its antioxidant effects (Saeed et al. 2023). A study in which osajin (42.9%) and pomiferin (30%) extracted from MP fruits were tested on soybean and fish oils, from which antioxidants and polar components were removed, showed higher antioxidant activity in both oils compared to other commercially available natural antioxidants. Therefore, this study suggests that M. pomifera extracts could be used as an antioxidant for edible oils, though additional safety tests are necessary before implementation (Hwang et al. 2021). Antioxidant activity has also been demonstrated by isolating pomiferin from MP fruits, this molecule being often isolated together with a small amount of osajin, which structurally resembles pomiferin but lacks an aromatic hydroxyl group. Experiments ex vivo on hair follicles showed that doses of pomiferin between 0.05 and 5 ppm had equivalent effects on collagen and elastin expression. Pomiferin could be a chemical substance with potential uses in topical therapies for skin and scalp (Gruber et al. 2014).

Regarding the antibacterial activity of M. pomifera extracts, there are several studies suggesting that certain chemical components found in this plant could have antibacterial potential. Mahmoud (1981) examined the antibacterial activity of the alcoholic fraction of M. pomifera fruit extract using a variation of the microdilution method in an agar medium. It was concluded that two previously isolated isoflavones from this plant, osajin and pomiferin, are responsible for the antibacterial activity of the fruit extract (Mahmoud 1981). Filip et al. (2021) demonstrated that Gram-positive bacteria are often more sensitive to extracts obtained from MP fruits compared to Gram-negative strains. The highest antimicrobial activity was highlighted against the species Listeria monocytogenes and Enterococcus faecalis (Filip et al. 2021). Another study showed that the ethanolic extract from M. pomifera fruits exhibits antibacterial activity against all investigated microbial strains (Canli et al. 2017). The intermediate efficiency of microwave-assisted extraction compared to sonicator-based extraction can be explained by the more intense thermal effects induced by microwave exposure. Although temperature elevation is generally linked to increased extraction yields of bioactive compounds from plant matrices, it can also secondarily induce degradation phenomena, particularly for thermolabile compounds. This can potentially impact the quality of the final extracts (Sridhar et al. 2021).

The description of the α-amylase and α-glucosidase inhibitory capacity of the extracts from M. pomifera bark and periderm represents a novel and original aspect of the present study. The current literature provides information on a moderate potential for α-glucosidase inhibition in extracts obtained through maceration in ethyl acetate from M. fruticosa branches, as well as the efficiency of the flavonoid fraction isolated from immature fruits of M. tricuspida in inhibiting the activity of this enzyme (Polbuppha et al. 2017; Jo et al. 2022). Another study demonstrated that pomiferin significantly stimulated blood insulin levels while simultaneously reducing serum glucose and triglyceride levels in normal rats over a 14-day period following administration of doses at 100 and 300 mg/kg. In comparison to osajin, pomiferin exhibited a higher potential for antidiabetic action (Moon 2014).

Research on M. pomifera continues to evolve, and further investigations are needed to definitively confirm the potential and level of antioxidant activity, antimicrobial activity, and antidiabetic activity of this plant, as well as to gain a deeper understanding of the compounds responsible for these effects.

CONCLUSIONS

1. Sonicator-assisted extraction (SAE) and microwave-assisted extraction (MAE) ensured maximum recovery of total polyphenols from both analyzed matrices compared to ultrasound bath extraction. The choice of solvent was also highlighted as a crucial factor in the quality of the extracts, with ethanol proving suitable for the optimal recovery of polyphenolic phytoconstituents from the matrices under investigation. Regarding the matrix used, rhytidome was found to be much richer in polyphenols, which is associated with the intense metabolic activity present in this tissue compared to periderm, which comprises tissues with low or nonexistent metabolic activity.
2. Tests using the 2,2-dephenyl-1-picrylhydrazyl (DPPH) radical revealed that the lowest IC₅₀ values were recorded for ethanol extracts of rhytidome. Ethanolic extracts of periderm showed IC₅₀ values close to those of rhytidome extracts, indicating that polyphenols in these extracts are not the sole compounds responsible for their antioxidant activity. Furthermore, the results have highlighted a significantly higher 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical neutralization capacity for all extracts compared to the DPPH radical.
3. Regarding the three enzymes analyzed: Ethanol extract of rhytidome exhibited the highest inhibition capacity against α-glucosidase, correlated with its polyphenol content. Lipase inhibition was weaker than that of α-amylase. Aqueous periderm extracts showed the lowest inhibition capacity against both enzymes, likely due to their low polyphenol content.
4. The observed antibacterial effects were moderate to low against the tested bacterial strains.
5. The obtained results support the potential use of M. pomifera rhytidome as a source of polyphenols with biological activity and provide a preliminary basis for further in-depth studies needed to elucidate the mechanisms involved in the biological activity of the phytopreparations derived from these matrices.

ACKNOWLEDGMENTS

This work was supported by the University of Medicine, Pharmacy, Sciences and Technology “George Emil Palade” of Târgu Mureș, Research grant number 164/12/10.01.2023.

REFERENCES CITED

Alhilal, M., Erol, H. S., Yildirim, S., Cakir, A., Koc, M., Celebi, D., and Halici, M. B. (2023). “Osajin from Maclura pomifera alleviates sepsis-induced liver injury in rats: Biochemical, histopathological and immunohistochemical estimation,” Journal of Taibah University for Science 17(1), article ID 2201250. DOI: 10.1080/16583655.2023.2201250

Babotă, M., Frumuzachi, O., Gâvan, A., Iacoviță, C., Pinela, J., Barros, L., Ferreira, I. C. F. R., Zhang, L., Lucini, L., Rocchetti, G., et al. (2022). “Optimized ultrasound-assisted extraction of phenolic compounds from Thymus comosus Heuff. ex Griseb. et Schenk (wild thyme) and their bioactive potential,” Ultrasonics Sonochemistry 84, article ID 105954. DOI: 10.1016/j.ultsonch.2022.105954

Barak, T. H., Kurt Celep, İ., Şentürk, T. B., Bardakci, H., and Celep, E. (2022). “In vitro anti-aging potential evaluation of Maclura pomifera (Rafin.) Schneider 80% methanol extract with quantitative HPTLC analysis,” Turkish Journal of Pharmaceutical Sciences 19(4), 400-407. DOI: 10.4274/tjps.galenos.2021.65087

Canli, K., Bozyel, M. E., and Altuner, E. M. (2017). “In vitro antimicrobial activity screening of Maclura pomifera fruits against wide range of microorganisms,” International Journal of Pharmaceutical Science Invention 6(8), 19-22.

Cicco, N., Lanorte, M. T., Paraggio, M., Viggiano, M., and Lattanzio, V. (2009). “A reproducible, rapid and inexpensive Folin–Ciocalteu micro-method in determining phenolics of plant methanol extracts,” Microchemical Journal 91(1), 107-110. DOI: 10.1016/j.microc.2008.08.011

Coman, N.-A., Babotă, M., Nădășan, I., Nicolescu, A., Pitaru, A.R., Ștefănescu, R., Mocan, A., Frumuzachi, O., Tanase, C. (2023). “The influence of ecological factors on the phytochemical characteristics of Pinus cembra L.,” Applied Sciences 13, article 10184. DOI: 10.3390/app131810184

Filip, S., Djarmati, Z., Lisichkov, K., Csanadi, J., and Jankov, R. M. (2015). “Isolation and characterization of Maclura (Maclura pomifera) extracts obtained by supercritical fluid extraction,” Industrial Crops and Products 76, 995-1000. DOI: 10.1016/j.indcrop.2015.07.066

Filip, S., Đurović, S., Blagojević, S., Tomić, A., Ranitović, A., Gašić, U., Tešić, Ž., and Zeković, Z. (2021). “Chemical composition and antimicrobial activity of Osage orange (Maclura pomifera ) leaf extracts,” Archiv der Pharmazie 354(2), article ID 2000195. DOI: 10.1002/ardp.202000195

Gruber, J. V., Holtz, R., Sikkink, S. K., and Tobin, D. J. (2014). “In vitro and ex vivo examination of topical Pomiferin treatments,” Fitoterapia 94, 164-171. DOI: 10.1016/j.fitote.2014.01.023

Hwang, H., Winkler‐Moser, J. K., Tisserat, B., Harry‐O’kuru, R. E., Berhow, M. A., and Liu, S. X. (2021). “Antioxidant activity of osage orange extract in soybean oil and fish oil during storage,” Journal of the American Oil Chemists Society 98(1), 73-87. DOI: 10.1002/aocs.12458

Jadoun, S., Arif, R., Jangid, N. K., and Meena, R. K. (2021). “Green synthesis of nanoparticles using plant extracts: A review,” Environmental Chemistry Letters 19(1), 355-374. DOI: 10.1007/s10311-020-01074-x

Jo, Y. H., Lee, S., Yeon, S. W., Ryu, S. H., Turk, A., Hwang, B. Y., Han, Y. K., Lee, K. Y., and Lee, M. K. (2022). “Anti-α-glucosidase and anti-oxidative isoflavonoids from the immature fruits of Maclura tricuspidata,” Phytochemistry 194, article ID 113016. DOI: 10.1016/j.phytochem.2021.113016

Khaleghi, A., Naderi, R., Brunetti, C., Maserti, B. E., Salami, S. A., and Babalar, M. (2019). “Morphological, physiochemical and antioxidant responses of Maclura pomifera to drought stress,” Scientific Reports 9(1), article ID 19250. DOI: 10.1038/s41598-019-55889-y

Laczkó-Zöld, E., Komlósi, A., Ülkei, T., Fogarasi, E., Croitoru, M., Fülöp, I., Domokos, E., Ştefănescu, R., and Varga, E. (2018). “Extractability of polyphenols from black currant, red currant and gooseberry and their antioxidant activity,” Acta Biologica Hungarica 69(2), 156-169. DOI: 10.1556/018.69.2018.2.5

Mahmoud, Z. (1981). “Antimicrobial components from Maclura pomifera fruit,” Planta Medica 42(07), 299-301. DOI: 10.1055/s-2007-971646

Moon, H.-I. (2014). “Effect of osajin and pomiferin on antidiabetic effects from normal and streptozotocin-induced diabetic rats,” Natural Product Communications 9(12), 1723-1724.

Nisca, A., Ștefănescu, R., Moldovan, C., Mocan, A., Mare, A. D., Ciurea, C. N., Man, A., Muntean, D.-L., and Tanase, C. (2022). “Optimization of microwave assisted extraction conditions to improve phenolic content and in vitro antioxidant and anti-microbial activity in quercus cerris bark extracts,” Plants 11(3), article 240. DOI: 10.3390/plants11030240

Polbuppha, I., Maneerat, W., Sripisut, T., Limtharakul (née Ritthiwigrom), T., Cheenpracha, S., Pyne, S. G., Muanprasat, C., Seemakhan, S., Borwornpinyo, S., and Laphookhieo, S. (2017). “Antioxidant, cytotoxic and α-glucosidase inhibitory activities of compounds isolated from the twig extracts of Maclura fruticosa,” Natural Product Communications 12(7), article ID 1934578X1701200. DOI: 10.1177/1934578X1701200718

Saeed, S., Khan, M. I. A., Ayesha, S., Zahid, Z., Younis, M., Kashan, M. W., Khan, G. Z., Tahir, M., Khan, S. A., Kamal, M., et al. (2023). “Screening of phytochemicals and anticancer potential of Maclura pomifera leaves extract,” Pakistan Journal of Medical and Health Sciences 17(3), 468-471. DOI: 10.53350/pjmhs2023173468

Salem, S. S., and Fouda, A. (2021). “Green synthesis of metallic nanoparticles and their prospective biotechnological applications: An overview,” Biological Trace Element Research 199(1), 344-370. DOI: 10.1007/s12011-020-02138-3

Smith, J. L., and Perino, J. V. (1981). “Osage orange (Maclura pomifera): History and economic uses,” Economic Botany 35(1), 24-41. DOI: 10.1007/BF02859211

Sridhar, A., Ponnuchamy, M., Kumar, P. S., Kapoor, A., Vo, D.-V. N., and Prabhakar, S. (2021). “Techniques and modeling of polyphenol extraction from food: A review,” Environmental Chemistry Letters 19(4), 3409-3443. DOI: 10.1007/s10311-021-01217-8

Ștefănescu, R., Ciurea, C. N., Mare, A. D., Man, A., Nisca, A., Nicolescu, A., Mocan, A., Babotă, M., Coman, N.-A., and Tanase, C. (2022). “Quercus robur Older bark – A source of polyphenolic extracts with biological activities,” Applied Sciences 12(22), article ID 11738. DOI: 10.3390/app122211738

Tanase, C., Berta, L., Coman, N. A., Roșca, I., Man, A., Toma, F., Mocan, A., Nicolescu, A., Jakab-Farkas, L., Biró, D., et al. (2019). “Antibacterial and antioxidant potential of silver nanoparticles biosynthesized using the spruce bark extract,” Nanomaterials 9(11), article 1541. DOI: 10.3390/nano9111541

Tanase, C., Domokos, E., Coșarcă, S., Miklos, A., Imre, S., Domokos, J., and Dehelean, C. A. (2018). “Study of the ultrasound-assisted extraction of polyphenols from beech (Fagus sylvatica L.) bark,” BioResources 13(2), 2247-2267. DOI: 10.15376/biores.13.2.2247-2267

Tanase, C., Nicolescu, A., Nisca, A., Ștefănescu, R., Babotă, M., Mare, A. D., Ciurea, C. N., and Man, A. (2022). “Biological activity of bark extracts from Northern red oak (Quercus rubra L.): An antioxidant, antimicrobial and enzymatic inhibitory evaluation,” Plants 11(18), article 2357. DOI: 10.3390/plants11182357

Article submitted: August 13, 2023; Peer review completed: September 9, 2023; Revised version received: September 13, 2023; Accepted: September 22, 2021; Published: October 12, 2023.

DOI: 10.15376/biores.18.4.8104-8119