Asian Pacific Journal of Tropical Biomedicine

: 2023  |  Volume : 13  |  Issue : 2  |  Page : 60--69

Antioxidant, antimicrobial, and α-glucosidase inhibitory activities of saponin extracts from walnut (Juglans regia L.) leaves

Youssef Elouafy1, Adil El Yadini1, Salma Mortada2, Mohamed Hnini3, Hicham Harhar1, Asaad Khalid4, Ashraf N Abdalla5, Abdelhakim Bouyahya6, Khang Wen Goh7, Long Chiau Ming8, My El Abbes Faouzi2, Mohamed Tabyaoui1,  
1 Laboratory of Materials, Nanotechnology and Environment, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Morocco
2 Laboratories of Pharmacology and Toxicology, Pharmaceutical and Toxicological Analysis Research Team, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Rabat, Morocco
3 Center of Plant and Microbial Biotechnology, Biodiversity and Environment, Microbiology and Molecular Biology Team, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Morocco
4 Substance Abuse and Toxicology Research Center, Jazan University, Jazan 45142, Saudi Arabia; Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center for Research, Khartoum, Sudan
5 Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia
6 Laboratory of Human Pathologies Biology, Faculty of Sciences, Mohammed V University in Rabat, Morocco
7 Faculty of Data Science and Information Technology, INTI International University, Nilai, Malaysia
8 School of Medical and Life Sciences, Sunway University, Sunway City, Malaysia

Correspondence Address:
Abdelhakim Bouyahya
Laboratory of Human Pathologies Biology, Faculty of Sciences, Mohammed V University in Rabat
Long Chiau Ming
School of Medical and Life Sciences, Sunway University, Sunway City


Objective: To investigate the relationship between triterpenoid saponin content and antioxidant, antimicrobial, and α-glucosidase inhibitory activities of 70% ethanolic, butanolic, aqueous, supernate and precipitate extracts of Juglans regia leaves. Methods: Triterpenoid saponins of different Juglans regia leaf extracts were measured by the vanillin method. Antioxidant activity was evaluated against DPPH and ABTS free radicals. We also assessed α-glucosidase inhibitory and antimicrobial activities of the leaf extracts. Pearson«SQ»s correlation coefficient was evaluated to determine the correlation between the saponin content and biological activities. Results: The butanolic extract was most effective against DPPH with an IC50 of 6.63 μg/mL, while the aqueous extract showed the highest scavenging activity against ABTS free radical with an IC50 of 42.27 μg/mL. Pearson«SQ»s correlation analysis indicated a strong negative correlation (r = -0.956) between DPPH radical scavenging activity (IC50) and the saponin content in the samples examined. In addition, the aqueous extract showed the best α-glucosidase inhibitory activity compared with other extracts. All the extracts had fair antibacterial activity against Bacillus subtilis, Escherichia coli, and Klebsiella pneumoniae except for the aqueous extract. Conclusions: Juglans regia extracts show potent antioxidant, antimicrobial, and α-glucosidase inhibitory activities. There is a correlation between saponin levels in Juglans regia leaf extracts and the studied activities. However, additional research is required to establish these relationships by identifying the specific saponin molecules responsible for these activities and elucidating their mechanisms of action.

How to cite this article:
Elouafy Y, El Yadini A, Mortada S, Hnini M, Harhar H, Khalid A, Abdalla AN, Bouyahya A, Goh KW, Ming LC, Faouzi ME, Tabyaoui M. Antioxidant, antimicrobial, and α-glucosidase inhibitory activities of saponin extracts from walnut (Juglans regia L.) leaves.Asian Pac J Trop Biomed 2023;13:60-69

How to cite this URL:
Elouafy Y, El Yadini A, Mortada S, Hnini M, Harhar H, Khalid A, Abdalla AN, Bouyahya A, Goh KW, Ming LC, Faouzi ME, Tabyaoui M. Antioxidant, antimicrobial, and α-glucosidase inhibitory activities of saponin extracts from walnut (Juglans regia L.) leaves. Asian Pac J Trop Biomed [serial online] 2023 [cited 2023 Mar 29 ];13:60-69
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Significance: Triterpenoid saponins are glycosides with a wide range of bioactive structures and biological activities, and can be found in many medicinal plants. However, no studies have been done on Juglans regia to identify or even quantify the content of these molecules in this plant. This study shows the relationship between triterpenoid saponin content and various biological activities in different leaf extracts of Juglans regia. It provides evidence for the potential medicinal properties of Juglans regia leaves, including antioxidant, antimicrobial, and α-glucosidase inhibitory properties, and a solid foundation for further research on the potential uses of Juglans regia leaves as a natural source of medicinal compounds.

 1. Introduction

Morocco’s flora is abundant with many medicinal plants which are largely employed as traditional medicines to combat numerous illnesses, including cardiovascular and hypertension problems, diabetes, rheumatism, stomach pain, as well as many other diseases[1],[2].

Juglans regia (J. regia) is among the commonest plants in Morocco, covering more than 7600 hectares[3]. This plant is composed of several families of bioactive molecules, such as polyphenols, flavonoids, saponins, phytosterols, tocopherols, and various other molecules that make J. regia a vast diversity of medicinal properties[4],[5],[6],[7].

On the other hand, saponin compounds have attracted huge interest over the past few decades, as they have proven their ability to treat several health problems[8]. Various studies have been devoted to investigating the anticancer activity of saponin molecules isolated from different species of plants[9],[10],[11]. Other studies have proven the anti-inflammatory activity of glycyrrhizin[12],[13], which is a natural triterpenoid saponin isolated from Glycyrrhiza glabra root[14]. Furthermore, other authors have focused on hepatoprotective[15], and antidiabetic activities of saponins[16].

Given the relationship between J. regia and the pharmacological properties of saponins, as well as the valorization of this plant, this article will quantify triterpenoid saponin contents, polyphenols, flavonoids, condensed tannins, as well as the total sugar contents of different extracts of J. regia leaves. Additionally, we aimed to investigate the antioxidant, α-glucosidase inhibitory, and antimicrobial activity of the plant extracts.

 2. Materials and methods

2.1. Plant materials

J. regia leaves were harvested in July 2022 at Demnate (31° 43' 52" N, 7° 02' 10" W) in Azilal province, Morocco. The leaves were cut into tiny pieces and dried in light-proof conditions at room temperature (22 ± 1) °C for 72-96 h. Afterward, the leaves were ground until obtaining a fine powder.

2.2. Saponins extraction process

[Figure 1] illustrates the extraction protocol of saponins from J. regia leaves which was performed according to Chua’s protocol with some modifications[17].{Figure 1}

The dried leaf powder was firstly delipidated in a Soxhlet cartridge using n-hexane as a delipidating solvent to delimit the apolar part of the plant. After delipidation, the cartridge was dried overnight in an oven at 25 °c to remove traces of hexane. On the following day, the residue was removed from the cartridge and underwent reflux extraction with EtOH/water solution (70:30) for 2 h at a temperature of 80 °C. After that, the solution was filtered and concentrated using a rotary evaporator (Heidolph Hei-VAP Precision motor, Germany) to obtain a crude extract of 70% EtOH, which was used afterward in the fractionation procedure. The crude ethanolic extract was diluted in distilled water which was then fractionated using the liquid-liquid extraction technique with butanol, and this process was repeated three times.

After the liquid-liquid extraction and completion of the separation process, the aqueous phase was lyophilized using a freeze-dryer (VaCo 2, Zirbus technology GmbH, Germany), while the butanol phase was concentrated using a rotary evaporator (Heidolph Hei-VAP Precision motor, Germany). The crude butanol extract was reconstituted in 99.8% methanol, and upon addition of diethyl ether, a precipitate was formed carrying all compounds non-soluble in diethyl ether, this precipitated fraction was filtered using a Fritted glass funnel, and then rotavaporized to remove any trace of solvent.

2.3. Determination of the total polyphenols content

Quantification of the total phenols content of the crude ethanolic 70% extract was performed by the Folin-Ciocalteu method according to the protocol described by Soto-Maldonado et al. with minor modifications[18]. Shortly, 2 500 μΕ of 10% Folin-Ciocalteu in distilled water was added to 2000 μΕ of Na2CO3 (7.5%) and 500 μΕ of the ethanolic extract prepared previously at a concentration of 1000 μg/mΕ. After 15 min of incubation at 45 °C, the absorbance was measured at 756 nm against a blank solution, using a UV-visible spectrophotometer (Model UV-5800PC UV/VIS Spectrophotometer, manufactured by Shanghai Metash Instruments CO., LTD). The blank contained the same volume of Folin-Ciocalteu and Na2CO3 and we replaced the volume of ethanolic extract of J. regia leaves with 500 μΕ of EtOH/ H2O (70:30). The results were expressed as mg equivalent of gallic acid (GAE) per gram of crude extract.

2.4. Determination of the total flavonoids content

Quantification of the total flavonoid content of the crude ethanolic 70% extract was performed by the aluminum trichloride method according to the protocol described by El-Guezzane et al. with minor modifications[19]. Briefly, 1 mL of the ethanolic extract (70%) at a concentration of 1000 μg/mL was diluted with 6.4 mL of distilled water, and then 0.3 mL of NaNO2 solution (5%) was added. Afterward, 0.3 mL of AlCl3 (10%) was added to the mixture after 5 min, then 2 mL of NaOH (1 M) was also added after another 5 min. The absorbance was measured at 510 nm against a blank solution and the results were expressed as mg equivalent of quercetin (QE) per gram of crude extract.

2.5. Determination of the total condensed tannin content

The transformation of condensed tannin into anthocyanidols using hydrochloric acid and vanillin was used to quantify the total condensed tannin concentration according to the protocol described by Cesprini et al[20]. Briefly, 25 μL of the 70% ethanolic extract solution (1000 μg/mL) was added to 1.5 mL of 4% methanol vanillin solution and 750 μL of concentrated hydrochloric acid. Thereafter, the absorbance was measured at 500 nm after 15 min against a blank solution, and the results were expressed as mg equivalent of catechin (CE) per gram of crude extract.

2.6. Determination of the total sugar content

Quantification of the total sugar content of the five extracts was performed by the phenolic sulfuric acid method according to the protocol described by El Moudden et al[21]. Briefly, 1000 μL of each sample (1000 μg/mL) was added to 1000 μL of phenol (5%) and 5000 μL of concentrated sulfuric acid. The mixture was left for 10 min and then incubated for 20 min in a water bath at 30 °C. The results were expressed as mg glucose equivalent (GE) per gram of crude extract.

2.7. Determination of the triterpenoid saponin content

The content of triterpenoid saponins was measured by the vanillin method[17]. Briefly, 250 μL of each sample was added to 250 μL of 8% ethanolic vanillin solution and 2500 μL of concentrated sulfuric acid. The mixture was incubated for 10 min in a water bath at 60 °C and then placed in ice water for 5 min in order to stop the reaction. Thereafter, the mixture absorbances were measured at 544 nm and the results were expressed as mg of oleanolic acid equivalent (OA) per gram of extract.

2.8. 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity

The antioxidant activity was evaluated by DPPH assay according to the protocol described by Nounah et al[22]. Briefly, 500 μL of different concentrations of each extract (5-100 μg/mL) were added to 500 μL of 0.2 mM DPPH ethanolic solution, vortexed, and incubated in the dark at room temperature for 30 min, and then the absorbance values were measured at 517 nm against a blank containing 500 μL of DPPH solution and 2500 μL of pure ethanol. The results were expressed as the amount of concentration required μg/mL) to reduce 50% of the free DPPH radical (IC50).

2.9. 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity

Equal volumes of 7 mM ABTS and 2.4 mM potassium persulfate solutions were vortexed and left in the dark for 16 h at room temperature. The resulting solution was adjusted with ethanol absolute to achieve an optical density of 0.700 ± 0.020 at 734 nm. Afterward, 1 800 μL of the adjusted solution was then added to 200 μL of different concentrations (5-100 μg/mL) of each extract and the absorbances were measured after 30 min of incubation at 734 nm[23]. The results were expressed as the amount of concentration required μg/mL) to reduce 50% of the free ABTS radical (IC50).

2.10. α-Glucosidase inhibitory activity

The glucosidase assay is essentially based on the inhibition of the enzyme glucosidase prepared in a sodium phosphate buffer solution (pH = 6.4), which can hydrolyze 4-nitrophenyl-α-D-glucopyranoside. The formation of a yellow solution is a sign of the hydrolysis reaction. It is inversely proportional to the inhibition capacity. The appearance of a light yellow or transparent mixture indicates a higher inhibition capacity of the extract. The assessment of α-glucosidase inhibitory activity was performed following a protocol previously described[24]. Briefly, 100 μL of 0.1 M sodium phosphate buffer solution (pH = 6.4) that contains the α-glucosidase enzyme solution, was incubated along with 150 μL of each sample at 37 °c for 10 min. Afterward, 200 μL of 1 mM 4-nitrophenyl-α-D-glucopyranoside, prepared in the same buffer solution, was incubated at 37 °C for 30 min. Then 1000 μL of Na2CO3 was added to stop the reaction and the optical densities were measured at 405 nm. Acarbose was used as a positive control.

2.11. Antimicrobial activity evaluation

The antimicrobial activity of J. regia leaf extracts was assessed using the agar well diffusion method[25]. Similar to the procedure used in the disk diffusion method, the Mueller Hinton agar plate surface was inoculated by spreading a volume of the microbial inoculum over the whole agar surface (20-100 μL) depending on the growth of each strain. Then, a well with a diameter of 6 to 8 mm was aseptically drilled with a sterile tip, and a volume (10 μL) of J. regialeaf extract was introduced into the well. The plates were allowed to diffuse and incubated for 24 h at 37 °C. The inhibition zone was measured and the assay was performed in triplicate. Tetracycline (30 μg, Sigma) was used as a reference antibiotic.

Microbial susceptibility tests using agar dilution and minimum inhibitory concentration (MIC) were performed to evaluate the antibacterial activity of the prepared extracts of J. regia leaves against a Gram-positive bacterium Bacillus subtilis (B. subtilis) (MW471619) and two Gram-negative bacteria, Escherichia coli (E. coli) ATCC 11775, and Klebsiella pneumoniae (K. pneumoniae)(MW524112). All materials were steam-sterilized at 120 °C for 20 min. A 20 mL sterile culture medium was inoculated with tested bacteria. These strains in this study have been determined to be the most representative bacteria. Considering their antibiotic resistance, these are the most common bacteria detected in a clinical setting in health care institutes. They are usually used to evaluate antimicrobial capacity and provide an appropriate evaluation grid to determine the efficacy of collected plant extracts.

2.12. Statistical analysis

The statistical significance of the data was verified using a two-way ANOVA test with the aid of GraphPad Prism 9 software. The Bonferroni’s multiple comparisons test was applied at a confidence level of 95.0% to determine the significance of the results. The data were expressed as mean±standard error of the mean of triplicate experiments (n = 3). Statistically significant differences were considered at P-values less than 0.05.

 3. Results

3.1. Total polyphenol, flavonoid, and condensed tannin content

The secondary metabolites were quantified before the fractionation protocol, therefore, the assays were performed for the 70% ethanolic leaf extract. The total polyphenol content was (147.15 ± 0.34) mg GAE/g while the total flavonoid and condensed tannin content were (22.17 ± 0.15) mg QE/g and (201.02 ± 0.26) mg CE/g crude extract, respectively.

3.2. Total sugar content

The total sugar contents of different J. regia leaf extracts are summarized in [Table 1]. The study found that the total sugar content of different J. regia leaf extracts is dependent on the fractionation solvent used. The highest amount of total sugar was recorded in the precipitate extract with (304.47 ± 2.11) mg GE/g, followed by the ethanolic, aqueous and butanolic extracts (P < 0.001). In contrast, the supernate extract had the lowest sugar content of (92.13 ± 0.93) mg GE/g.{Table 1}

3.3. Triterpenoid saponin of J. regia leaf extracts

The total triterpenoid saponin contents of different J. regia leaf extracts are reported in [Table 1]. The butanolic, precipitate, and supernate extract had relatively higher triterpenoid saponin content of (214.49 ± 8.91), (205.46 ± 4.11), and (200.41 ± 7.99) mg OA/g, respectively, with no significant difference (P > 0.05). The aqueous extract showed the lowest triterpenoid saponin content. These results indicate that less polar compounds, including saponins, may disperse in organic phases while the polar compounds remain in the aqueous extract.

3.4. Antioxidant activity of J. regia leaf extracts against DPPH and ABTS

[Table 2] shows the IC50 values of J. regia extracts against DPPH and ABTS free radicals, and it could be seen that all these extracts had excellent antioxidant activity with IC50 values not exceeding 75 μg/mL in both DPPH and ABTS assays.{Table 2}

The butanolic, supernate, and precipitate extracts showed significant antioxidant activity among all extracts while the aqueous extract presented the weakest antioxidant activity. The results of the ABTS test showed that the fractionation process increased the antioxidant capacity of J. regia leaves. All extracts after the fractionation process recorded lower IC50 values than the 70% ethanolic extract with an IC50 of (72.26 ± 1.80) μg/mL. The aqueous extract showed the highest scavenging activity in the ABTS assay with an IC50 of (42.27 ± 0.80) μg/mL. The precipitate, butanolic, and supernate extracts also showed high IC50 values compared to those recorded in the DPPH assay, but still presented considerable antioxidant activity towards the ABTS radical.

3.5. α-Glucosidase inhibitory activity of J. regia leaf extracts

IC50 values of different extracts against α-glucosidase are reported in [Table 2], and the percentages of inhibition of each extract at different concentrations are illustrated in [Figure 2]. J. regia leaf extracts showed excellent antidiabetic activity, even at low concentrations, less than 250 μg/mL, with IC50 values extremely close to the positive control acarbose [IC50= (18.01 ± 2.00) μg/mL], except for the supernate extract which had the highest IC50 value of (81.99 ± 1.12) μg/mL. Both the aqueous and precipitate extracts exhibited strong α-glucosidase inhibitory activity with IC50 values of (11.00 ± 1.03) μg/mL and (13.66 ± 1.18) μg/mL, respectively, with no significant difference (P > 0.05).{Figure 2}

3.6. Antimicrobial activity of J. regia leaf extracts

[Table 3] depicts the zones of inhibition (ZOI) corresponding to the MICs of J. regia leaf extracts. Our findings revealed that the antimicrobial activity of J. regia leaves was relatively high compared to the reference antibiotics (Tetracycline 30 μg). All our extracts showed antimicrobial activity against the investigated strains, except for the aqueous extract which had no antimicrobial activity in the studied concentration range (100-5000 μg/mL).{Table 3}

The 70% ethanolic extract showed the largest ZOI of 5.33 mm against B. subtilis compared with the standard control (4 mm), with an MIC of 1000 gg/mL, followed by the precipitate extract (ZOI = 3.67 mm/MIC = 1000 μg/mL). All the extracts (except for the aqueous extract) inhibited E. coli better than the standard control. The precipitate and the supernate extracts were most effective against E. coli with ZOI of 5.33 and 5.67 mm, respectively, while the butanolic and 70% ethanolic extracts recorded ZOI of 4.00 and 4.33 mm, respectively. In addition, the 70% ethanolic and supernate extracts showed the most significant ZOI of 7.67 and 6.67 mm, respectively against K. pneumoniae [Table 3] and [Figure 3].{Figure 3}

3.7. Pearson ’s correlation coefficient

The Pearson’s correlation coefficient is a metric utilized to quantify the linear association between two variables. As shown in [Figure 4], a heat map displays the correlation coefficients between antioxidant, antidiabetic, and antimicrobial activities, and the contents of saponin and sugar. Of particular significance was the strong negative correlation (r = -0.956) between DPPH IC50 and saponin contents, which demonstrates that an increase in saponin content leads to a decrease in IC50 against DPPH, thereby enhancing antioxidant activity. Another noteworthy correlation was evident between DPPH IC50 and the zone of inhibition of E. coli, with an rvalue of -0.919, signifying a robust relationship between antioxidant and antimicrobial activities. Furthermore, the correlation between saponin content and the zone of inhibition of E. coli, with an r value of 0.863, underscores the impact of saponin content on antimicrobial activity.{Figure 4}

 4. Discussion

The polyphenols, flavonoids, and tannins of J. regia are widely investigated for their medicinal properties[26]. J. regia leaf extracts are rich in secondary metabolites[27],[28], which gives them therapeutic potential against a wide range of diseases, including antihypertensive activity, lipid-lowering effect, protection of the liver and kidneys, and anti-cancer activity[4].

In terms of the total sugar content, the results of the study indicate that J. regia leaves contain a considerable quantity of total sugars in all different extracts, which can be explained by the fact that the sugars (carbohydrates) are formed through photosynthesis of the leaves[29]. The dependence of the total sugar content upon the fractionation solvent can be attributed to the different solubility properties of the saponin molecules in different solvents, leading to varying levels of sugar extraction. The high total sugar content in the precipitate extract can also suggest that this extract may have a higher saponin content than the other extracts, and the lowest sugar content at the supernatant can be explained by the insolubility of the saponin molecules in diethyl ether taken with them a considerable amount of sugar linked by glycosidic bonds. This study also suggested that the total sugar content could be used as an indication of the saponin content, since these sugar molecules are the hydrophilic part of the saponins, and they can be built up from one or more glycosidic bonds at different places of the saponin aglycone[30].

Recently, triterpenoid saponins have attracted remarkable interest due to their bioactivity and structural diversity[8],[31]. They represent a large family of amphiphilic glycosides with lipophilic (triterpenoid-aglycone) and hydrophilic (sugar) parts[8] and offer a diverse range of pharmacological properties including cardiovascular effects[32], anti-inflammatory activity[33] and potential anticancer properties[34],[35]. The result of this study suggests that the butanolic, precipitate, and supernate extracts could be a good source of triterpenoid saponins. Although a few studies have focused on phytochemical screening of J. regia saponin[36],[37], none have been conducted to characterize or identify these molecules. This opens the door for further research to develop new approaches for the purification and characterization of saponins from J. regia leaf extracts.

Concerning the antioxidant activity, the results of this study indicate that the antioxidant activity of J. regia leaf extracts is influenced by the choice of fractionation solvent used. The butanolic, supernate, and precipitate extracts were found to have the highest antioxidant activity against the DPPH radical, which is consistent with the higher content of triterpenoid saponins in these extracts, the same applies to the aqueous extract which has the lowest triterpenoid saponin value, and the weakest antioxidant activity [IC50 = (74.29 ± 0.46) μg/mL]. These results suggest that there is a strong correlation between saponin content and antioxidant activity by DPPH. There is also a strong negative correlation between the IC50 value of the DPPH assay and saponin content (r = –0.956), proving that higher saponin content correlates with the lowest IC50 values, indicating higher antioxidant activity.

The results of the ABTS test showed that the fractionation process increases the antioxidant capacity of J. regia leaves, and all extracts after the fractionation process showed lower IC50 values than the 70% ethanolic extract. However, the aqueous extract, which had the weakest activity against the DPPH radical, showed the highest scavenging activity in the ABTS assay, which confirms that we cannot assert the inactivity of an extract based on a single free radical test.

However, there are no similar studies that applied the same fractionation protocol on J. regia leaves to compare our results with them, but there are several studies that investigate the antioxidant activity of J. regia leaves that our results support[4].

α-Glucosidase is an acidic enzyme responsible for the hydrolysis of oligosaccharides into glucose within the intestine leading to increase blood glucose levels[38]. The results of this investigation indicate that the aqueous extract with the highest antioxidant activity against ABTS radical, also shows the highest α-glucosidase inhibitory activity, meaning that the antioxidant activity may influence the antidiabetic behavior of our extract. Furthermore, both extracts (aqueous and precipitate) exhibited stronger activity than the positive control, acarbose. The same finding has been reported by several researchers in the phytochemical area[39],[40]. Zhang et al. found that the hydroalcoholic extract (EtOH/Water) of propolis shows more potent α-glucosidase inhibitory activity than acarbose[41]. A recent study in 2020 also revealed that walnut septum acetone extract has a stronger α-glucosidase inhibitory activity than acarbose (IC50 = 0.14 mg/mL versus 0.80 mg/mL)[42]. There is a strong negative correlation between IC50 values of α-glucosidase inhibitory activity and sugar content (r = -0.889), suggesting that natural sugars from J. regia may have the capacity to increase α-glucosidase inhibitory activity by interacting with the α-glucosidase enzyme, inhibit them, and consequently decrease blood glucose levels, but further in-depth studies are required to validate this assumption. Overall, J. regia leaves showed outstanding antidiabetic activity as evidenced by excellent α-glucosidase inhibitory activity, making this walnut by-product a promising source of bioactive compounds for pharmacological purposes.

This paper proposes the use of J. regia leaves, a by-product of walnut cultivation, as an antimicrobial material. J. regia extracts showed interesting antimicrobial potency against a wide variety of microbial pathogens[4]. According to the study by Boulfia et al, it can be said that the leaves of J. regia have potent antibacterial activity against B. subtilis, better than that of the bark extracts with MICs of 5000 pg/mL in the ethanolic and diethyl ether extracts, and 2500 μg/mL in the acetone extract[43] and these MICs are significantly higher than those found in J. regia leaf extracts. In the case of Gram-negative bacteria, E. coli and K. pneumoniae, they were found to be more sensitive to J. regia extract than B. subtilis with MICs not exceeding 500 μg/mL. The 70% ethanolic and supernate extracts showed the most significant ZOI of 7.67 and 6.67 mm, respectively against K. pneumoniae. These findings are very important since K. pneumoniae is one of the major contributors to healthcare contamination in humans, including pneumonia, urinary tract infections, sepsis, and meningitis[44]. Our results were better than what Oliveira et al., reported in their study on the green husk of J. regia from different cultivars[45], which were incapable to inhibit the growth of E. coli at the high concentration studied (100 mg/mL). The results of the present study provide evidence for the potential use of J. regia leaves as an antimicrobial agent. Pearson’s correlation coefficient demonstrated the bacterial strains studied are all positively correlated between B. subtilis and E. coli (r = 0.820), between B. subtilis and K. pneumoniae (r = 0.969), and between K. pneumoniae and E. coli (r = 0.894). It also showed a strong positive correlation between triterpenoid saponin content and E. coli ZOI (r= 0.863), as well as a strong negative correlation between DPPH IC50 and E. coli and K. pneumoniae ZOI (r = -0.919 and -0.791 respectively), suggesting that triterpenoid saponin compounds may influence antimicrobial activity as they did with antioxidant activity. However, further studies are needed to identify the molecules present in our extract and test them individually to prove this assumption.

In conclusion, the butanol, supernate, and precipitate extracts showed the highest levels of triterpenoid saponins with the most significant antioxidant activity against the DPPH free radicals. All our extracts exhibited considerable antioxidant activity with IC50 values not exceeding 75 μg/mL in both DPPH and ABTS tests. Furthermore, the aqueous and precipitate extracts had a potent ability to inhibit α-glucosidase enzyme better than acarbose. J. regia leaves also showed a relatively high antibacterial activity compared with a reference antibiotic tetracycline. Overall, the results of this investigation showed that the triterpenoid saponins of J. regia leaves have both biochemical and pharmacologically potent activities.

Conflict of interest statement

The authors declare no conflict of interest.


The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work (Grant Code: 22UQU4331128DSR77).


The study was supported by the Deanship of Scientific Research at Umm Al-Qura University (Grant code: 22UQU4331128DSR77).

Authors’ contributions

MT and LCM were responsible for conceptualization, as well as project administration, and AEY provided supervision. YE, SM, and MH were responsible for data curation and formal analysis, while AEY, HH, and AB conducted investigations. AB and LCM also contributed to the drafting and critical revision of the manuscript. MEAF, AB, KWG, and AK were responsible for validation, and YE, AK, ANA, and KWG contributed to visualization. YE, and MH were responsible for writing the original draft, while AB, HH, AK, ANA, KWG, and MEAF contributed to the review and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.


1Kachmar MR, Naceiri Mrabti H, Bellahmar M, Ouahbi A, Haloui Z, el Badaoui K, et al. Traditional knowledge of medicinal plants used in the northeastern part of Morocco. Evid Based Complement Alternat Med 2021; 2021. doi: 10.1155/2021/6002949.
2Bouyahya A, el Omari N, Elmenyiy N, Guaouguaou FE, Balahbib A, Belmehdi O, et al. Moroccan antidiabetic medicinal plants: Ethnobotanical studies, phytochemical bioactive compounds, preclinical investigations, toxicological validations and clinical evidences; challenges, guidance and perspectives for future management of diabetes worldwide. Trends Food Sci Technol 2021; 115: 147-254.
3Houmanat K, Abdellah K, Hssaini L, Razouk R, Hanine H, Jaafary S, et al. Molecular diversity of walnut (Juglans regia L.) among two major areas in Morocco in contrast with foreign varieties. Int J Fruit Sci 2021; 21(1): 180-192.
4Bourais I, Elmarrkechy S, Taha D, Mourabit Y, Bouyahya A, El Yadini M, et al. A review on medicinal uses, nutritional value, and antimicrobial, antioxidant, anti-inflammatory, antidiabetic, and anticancer potential related to bioactive compounds of J. regia. Food Rev Int 2022. doi: 10.1080/87559129.2022.2094401.
5Elouafy Y, el Yadini A, el Moudden H, Harhar H, Alshahrani MM, Awadh AAA, et al. Influence of the extraction method on the quality and chemical composition of walnut (Juglans regia L.) oil. Molecules 2022; 27(22): 7681.
6Elouafy Y, el Idrissi ZL, el Yadini A, Harhar H, Alshahrani MM, al Awadh AA, et al. Variations in antioxidant capacity, oxidative stability, and physicochemical quality parameters of walnut (Juglans regia) oil with roasting and accelerated storage conditions. Molecules 2022; 27(22): 7693.
7Nasar S, Murtaza G, Mehmood A, Bhatti TM, Raffi M. Environmentally benign and economical phytofabrication of silver nanoparticles using Juglans regia leaf extract for antibacterial study. J Electron Mater 2019; 48(6): 3562-3569.
8Du JR, Long FY, Chen C. Research progress on natural triterpenoid saponins in the chemoprevention and chemotherapy of cancer. Enzymes 2014; 36: 95-130.
9Xu MY, Lee DH, Joo EJ, Son KH, Kim YS. Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells. Food Chem Toxicol 2013; 59: 703-708.
10Mo S, Xiong H, Shu G, Yang X, Wang J, Zheng C, et al. Phaseoloideside E, a novel natural triterpenoid saponin identified from entada phaseoloides, induces apoptosis in Ec-109 esophageal cancer cells through reactive oxygen species generation. J Pharmacol Sci 2013; 122(3): 163-175.
11Qin H, Du X, Zhang Y, Wang R. Platycodin D, a triterpenoid saponin from Platycodon grandiflorum, induces G2/M arrest and apoptosis in human hepatoma HepG2 cells by modulating the PI3K/Akt pathway. Tumor Biol 2014; 35(2): 1267-1274.
12Wang XR, Hao HG, Chu L. Glycyrrhizin inhibits LPS-induced inflammatory mediator production in endometrial epithelial cells. Microb Pathog 2017; 109: 110-113.
13Li Y, Sun F, Jing Z, Wang X, Hua X, Wan L. Glycyrrhizic acid exerts anti-inflammatory effect to improve cerebral vasospasm secondary to subarachnoid hemorrhage in a rat model. Neurol Res 2017; 39(8): 727-732.
14Ayangla NW, Kumar V, Gupta RC, Dey A, Dwivedi P, Pandey DK. Response surface methodology and artificial neural network modelling for optimization of solid-liquid extraction and rapid HPTLC analysis of glycyrrhizin in Glycyrrhiza glabra root. S Afr J Bot 2022; 148: 11-20.
15Zheng YF, Wei JH, Fang SQ, Tang YP, Cheng HB, Wang TL, et al. Hepatoprotective triterpene saponins from the roots of Glycyrrhiza inflata . Molecules 2015; 20(4): 6273-6283.
16Choudhary N, Khatik GL, Suttee A. The possible role of saponin in type-II diabetes-A review. Curr Diabetes Rev 2021; 17(2): 107-121.
17Chua LS, Lau CH, Chew CY, Dawood DAS. Solvent fractionation and acetone precipitation for crude saponins from Eurycoma longifolia extract. Molecules 2019; 24(7): 1416.
18Soto-Maldonado C, Caballero-Valdés E, Santis-Bernal J, Jara-Quezada J, Fuentes-Viveros L, Zúñiga-Hansen ME. Potential of solid wastes from the walnut industry: Extraction conditions to evaluate the antioxidant and bioherbicidal activities. Electron J Biotechnol 2022; 58: 25-36.
19El-Guezzane C, El-Moudden H, Harhar H, Chahboun N, Tabyaoui M, Zarrouk A. A comparative study of the antioxidant activity of two Moroccan prickly pear cultivars collected in different regions. Chem Data Collec 2021; 31: 100637.
20Cesprini E, de Iseppi A, Giovando S, Tarabra E, Zanetti M, Šket P, et al. Chemical characterization of cherry (Prunus avium) extract in comparison with commercial mimosa and chestnut tannins. Wood Sci Technol 2022; 56(5): 1-19.
21el Moudden H, el Idrissi Y, el Yadini A, Harhar H, Tabyaoui B, Tabyaoui M. Effect of filtration of olive mill wastewater on the phenolic composition and its influence on antioxidant activity. Pharmacol Online 2019; 2: 161-176.
22Nounah I, Hajib A, Harhar H, el Madani N, Gharby S, Guillaume D, et al. Chemical composition and antioxidant activity of Lawsonia inermis seed extracts from Morocco. Nat Prod Commun 2017; 12(4): 487-488.
23Elouafy Y, Mortada S, El Yadini A, Hnini M, Aalilou Y, Harhar H, et al. Bioactivity of walnut: Investigating the triterpenoid saponin extracts of Juglans regia kernels for antioxidant, anti-diabetic, and antimicrobial properties. Prog Microbes Mol Biol 2023; 6(1). doi: 10.36877/pmmb. a0000325.
24Mortada S, Brandán SA, Karrouchi K, El-guourrami O, doudach L, El Bacha R, et al. Synthesis, spectroscopic and DFT studies of 5-methyl-1H-pyrazole-3-carbohydrazide N -glycoside as potential anti-diabetic and antioxidant agent. J Mol Struct 2022; 1267: 133652.
25Chavez-Esquivel G, Cervantes-Cuevas H, Ybieta-Olvera LF, Castañeda Briones MT, Acosta D, Cabello J. Antimicrobial activity of graphite oxide doped with silver against Bacillus subtilis, Candida albicans, Escherichia coli, and Staphylococcus aureus by agar well diffusion test: Synthesis and characterization. Mater Sci Eng C 2021; 123: 111934.
26Sharafati-Chaleshtori R, Sharafati-Chaleshtori F, Rafieian M. Biological characterization of Iranian walnut (Juglans regia) leaves. Turk J Biol 2011; 35(5): 635-639.
27Moravej H, Salehi A, Razavi Z, Moein MR, Etemadfard H, Karami F, et al. Chemical composition and the effect of walnut hydrosol on glycemic control of patients with type 1 diabetes. Int J Endocrinol Metab 2016; 14(1): e34726.
28Karakaya S, Koca M, Yeşilyurt F, Hacimüftüoğlu A. Antioxidant and anticholinesterase activities of Juglans regia L. growing in turkey. Ankara Univ Eczacilik Fak Derg 2019; 43(3): 230-238.
29Wang SW, Pan CD, Zhang CF, Chen H. Characteristics of carbohydrate assimilation and distribution in walnut (Juglans regia L.). Hortic Sci Technol 2021; 39(2): 152-166.
30Colson E, Savarino P, Claereboudt EJS, Cabrera-Barjas G, Deleu M, Lins L, et al. Enhancing the membranolytic activity of Chenopodium quinoa saponins by fast microwave hydrolysis. Molecules 2020; 25(7): 1731.
31Mohan VR, Tresina PS, Daffodil ED. Antinutritional factors in legume seeds: Characteristics and determination. Encycl Food Health 2016. doi: 10.1016/B978-0-12-384947-2.00036-2.
32Aravinthan A, Kim JH, Antonisamy P, Kang CW, Choi J, Kim NS, et al. Ginseng total saponin attenuates myocardial injury via anti-oxidative and anti-inflammatory properties. J Ginseng Res 2015; 39(3): 206-212.
33Ji DB, Xu B, Liu JT, Ran FX, Cui JR. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells. Mol Carcinog 2011; 50(12): 945-960.
34Ming ZJ, Hu Y, Qiu YH, Cao L, Zhang XG. Synergistic effects of β-aescin and 5-fluorouracil in human hepatocellular carcinoma SMMC-7721 cells. Phytomedicine 2010; 17(8-9): 575-580.
35Shen DY, Kang JH, Song W, Zhang WQ, Li WG, Zhao Y, et al. Apoptosis of human cholangiocarcinoma cell lines induced by β-escin through mitochondrial caspase-dependent pathway. Phytother Res 2011; 25(10): 1519-1526.
36Mehta J, Shahista S. Studies on the screening of phytochemical, antioxidant and antibacterial activities of certain medicinal plants of Kashmir. Int J Biol Res Dev 2019; 2(2): 1-14.
37Harouak H, Ibijbijen J, Nassiri L. Chemical profile of Tetraclinis articulata (Vahl) Masters, and Juglans regia L. and Olea europaea L. var. Sylvestris used against oral diseases: In vitro analysis between polyphenolic content and aqueous extraction optimization. Heliyon 2021; 7(5): e07118.
38Sayah K, Marmouzi I, Naceiri Mrabti H, Cherrah Y, Faouzi MEA. Antioxidant activity and inhibitory potential of Cistus salviifolius (L.) and Cistus monspeliensis (L.) aerial parts extracts against key enzymes linked to hyperglycemia. Biomed Res Int 2017; 2017: 1-7.
39He H, Lu YH. Comparison of inhibitory activities and mechanisms of five mulberry plant bioactive components against α-glucosidase. J Agric Food Chem 2013; 61(34): 8110-8119.
40Figueiredo-González M, Grosso C, Valentão P, Andrade PB. α-Glucosidase and α-amylase inhibitors from Myrcia spp.: A stronger alternative to acarbose? J Pharm Biomed Anal 2016; 118: 322-327.
41Zhang H, Wang G, Beta T, Dong J. Inhibitory properties of aqueous ethanol extracts of propolis on alpha-glucosidase. Evid Based Complement Alternat Med 2015; 2015. doi: 10.1155/2015/587383.
42Rusu ME, Fizesan I, Pop A, Mocan A, Gheldiu AM, Babota M, et al. Walnut (Juglans regia L.) septum: Assessment of bioactive molecules and in vitro biological effects. Molecules 2020; 25(9): 2187.
43Boulfia M, Lamchouri F, Toufik H. Mineral analysis, in vitro evaluation of alpha-amylase, alpha-glucosidase, and beta-galactosidase inhibition, and antibacterial activities of Juglans regia L. bark extracts. Biomed Res Int 2021; 2021. doi: 10.1155/2021/1585692.
44Petrosillo N, Taglietti F, Granata G. Treatment options for colistin resistant Klebsiella pneumoniae: Present and future. J Clin Med 2019; 8(7): 934.
45Oliveira I, Sousa A, Ferreira ICFR, Bento A, Estevinho L, Pereira JA. Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks. Food Chem Toxicol 2008; 46(7): 2326-2331.