Abstract
Plants represent a great source of medicines, and for their components to be discovered, extraction processes must be developed, especially methods based on green technology. Supercritical fluid extraction (SFE) was employed as a green method for Moringa oleifera extraction in the present investigation. The maximum yield of extraction was obtained at 25 MPa. Moreover, the extraction at 25 MPa induced the release of various phenols and flavonoids, as analyzed via high-performance liquid chromatography. The investigation revealed the concentrations of chlorogenic, gallic, rosmarinic, and coumaric acids to be 150.59, 89.90, 44.75, and 29.41 µg/mL, respectively at 25 MPa. However, their concentrations were 0.73, 1.53, 0.24, and 0.04 µg/mL, respectively at 15 MPa; vs. 4.73, 2.62, 1.06, and 0.50 at 35 MPa, respectively. Totals of saponin, flavonoid, phenolic, tannins, and alkaloid were recorded in maximum yield at 25 MPa. Moringa oleifera extracted at 35 MPa reflected highest inhibition zones of 27 ± 0.1, 30 ± 0.2, and 30 ± 0.1 mm against C. glabrata, C. tropicalis, and C. albicans, correspondingly. α-Amylase and α-glucosidase activities were greatly suppressed by the M. oleifera extract at 25 MPa with less IC50 (12.97 µg/mL and 6.0 µg/mL), than the IC50 (53.46 and 22.02 µg/mL) at 15 MPa, compared with acarbose IC50 (5.52 and 2.64 µg/mL), correspondingly.
Download PDF
Full Article
Evaluating the Anti-yeast, Anti-diabetic, Wound Healing Activities of Moringa oleifera Extracted at Different Conditions of Pressure via Supercritical Fluid Extraction
Mutasem S. Almehayawi,a Mohammed S. Almuhayawi,b Shams R. Abo El-Fadl,c Mohammed K. Nagshabandi,d Muyassar K. Tarabulsi,d Samy Selim,e,* Yasir S. Alruwaili,e,f Ehab M. Mostafa,g Soad K. Al Jaouni,h,* and Tarek M. Abdelghany i,*
Plants represent a great source of medicines, and for their components to be discovered, extraction processes must be developed, especially methods based on green technology. Supercritical fluid extraction (SFE) was employed as a green method for Moringa oleifera extraction in the present investigation. The maximum yield of extraction was obtained at 25 MPa. Moreover, the extraction at 25 MPa induced the release of various phenols and flavonoids, as analyzed via high-performance liquid chromatography. The investigation revealed the concentrations of chlorogenic, gallic, rosmarinic, and coumaric acids to be 150.59, 89.90, 44.75, and 29.41 µg/mL, respectively at 25 MPa. However, their concentrations were 0.73, 1.53, 0.24, and 0.04 µg/mL, respectively at 15 MPa; vs. 4.73, 2.62, 1.06, and 0.50 at 35 MPa, respectively. Totals of saponin, flavonoid, phenolic, tannins, and alkaloid were recorded in maximum yield at 25 MPa. Moringa oleifera extracted at 35 MPa reflected highest inhibition zones of 27 ± 0.1, 30 ± 0.2, and 30 ± 0.1 mm against C. glabrata, C. tropicalis, and C. albicans, correspondingly. α-Amylase and α-glucosidase activities were greatly suppressed by the M. oleifera extract at 25 MPa with less IC50 (12.97 µg/mL and 6.0 µg/mL), than the IC50 (53.46 and 22.02 µg/mL) at 15 MPa, compared with acarbose IC50 (5.52 and 2.64 µg/mL), correspondingly.
DOI: 10.15376/biores.19.3.5961-5977
Keywords: Moringa oleifera; SFE-CO2; Anti-yeast; α-Amylase; α-Glucosidase; Wound healing
Contact information: a: Department of Emergency Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia; b: Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia; c: Medicinal and Aromatic Plants Department, Horticulture Research Institute, Agriculture Research Center, P.O. Box 12619, Giza, Egypt; d: Department of Medical Microbiology and Parasitology, Faculty of Medicine, University of Jeddah, Jeddah, Saudi Arabia; e: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka 72388, Saudi Arabia; f: Sustainable Development Research and Innovation Center, Deanship of Graduate Studies and Scientific Research, Jouf University, Sakaka, Saudi Arabia; g: Department of Pharmacognosy, College of Pharmacy, Jouf University, Sakaka 72341, Saudi Arabia; h: Department of Hematology/Oncology, Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia; i: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 11725, Egypt; * Corresponding author: sabdulsalam@ju.edu.sa (S.S.), saljaouni@kau.edu.sa (S.K.A.), tabdelghany.201@azhar.edu.eg (T.M.A.)
INTRODUCTION
Natural products of plant origin perform a critical job in the management of several diseases in humans as a harmless substitute to drugs from chemical industries (Al-Rajhi et al. 2022; Qanash et al. 2022; Al-Rajhi and Abdelghany 2023a,b; Qanash et al. 2023). Moringaceae is a family of one genus, Moringa that has 13 species indigenous to numerous countries (Stephenson and Fahey 2004). Among the 13 species, Moringa oleifera is the most studied and utilized species. Moringa oleifera Lam. is a fast-developing tree that is native to India and is well known for its therapeutic and dietary properties for centuries (Rani et al. 2028). Scientists have recently learned that M. oleifera has rich bioactive potential and phytochemical content (Dzuvo et al. 2021; Khalid et al. 2023), leading to increased usage of its extracts and oils in merchantable properties, such as therapeutical, nutraceutical, pharmaceutical, and cosmetic formulations (Muhammad et al. 2013). Preliminary investigations have shown that the various parts of plant; leaves, roots and seeds reveal characteristic compositions and properties (Alawlaqi et al. 2023; Al-Rajhi et al. 2023a,b).
Studies show that several polyphenols, primarily phenolic acids, flavonoids, and tannins were included in M. oleifera; and to detect these compounds, advanced techniques of chromatography comprising gas chromatography-mass spectrometry, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry are applied. For example, leaves of M. oleifera contained significant quantities of myricetin, rutin, kaempferol, lutein, and quercetin constituents, which perform vital functions like antioxidant, inflammatory, and anti-diabetic activities that improve the viability and propagation of the cellular response to injuries of tissue (Baldisserotto et al. 2018). Profiling M. oleifera leaf extract by HPLC showed that it contains p-coumaric, gallic, ferulic, chlorigenic acid, and sinapic acids that offer antioxidant functions to stabilize free radicals (Khalid et al. 2023). Many revisions have reported the hypoglycemic influence of M. oleifera, which might be because of the occurrence of fiber and quercetin-3-glucoside components, possibly by stopping glucose application and reducing the rate of gastric emptying (Ndong et al. 2017); improving secretion of insulin, and enhancing glycogen production, besides glucose uptake in the liver as well in muscles (Olayaki et al. 2015; Muhammad et al. 2016). Moringa oleifera leaves are acknowledged to decline blood glucose concentration, modeled in type 2 diabetics control (Ndong et al. 2017). Another study reported that uptake of M. oleifera leaf extract depresses levels of sugar in blood within 3 h once consumption (Mittal et al. 2007).
Diverse pharmacological utilizations were linked with M. oleifera extracts as mentioned in several scientific reports; for instance, Moringa oleifera methanolic extract has shown wound healing properties in infected wounds via methicillin-resistant bacteria in rats infected with diabetes (Al-Ghanayem et al. 2022). In a wound healing investigation according to Rathi et al. (2006), the healing process of the aqueous extract of M. oleifera leaves was documented and showed a strong improvement in skin and granuloma breaking strength, wound occlusion rate, hydroxylproline content, and decrease in scar zone, besides granuloma dry weight. Different compounds present in the M. oleifera extract were determined and applied locally as an ointment that enhanced the contraction of the wound and reduced the time of epithelization. Its efficacy was attributed to the rise in collagen formation, activities of antioxidant enzyme, and density of capillary (Al-Ghanayem et al. 2022).
Candida species are pathogenic microorganisms of yeasts developing mucocutaneous and systemic illnesses in humans, mostly in diabetic patients, low oral hygiene (immunocompromised patients), xerostomia, transplant recipients, malnutrition, and malignancy (Ndong et al. 2017). According to Rathi et al. (2006) four yeasts namely Cryptococcus neoformans, with three species of Candida including C. tropicalis, C. parapsilosis, and C. glabrata, are considered the greatest principal pathogenic yeasts for humans. Enormous species of Candida show as unaffected by antifungal constituents (Mittal et al. 2007). Thus, there is a demand for new constituents to inhibit pathogens of yeasts plus better effectiveness and less poisonousness. Moringa oleifera extracts showed fungistatic traits that elevate the metabolism change of numerous Candida isolates comprising C. albicans; M. oleifera has improved metabolism alterations (stress response) of cells of C. albicans, which get together with the capability to prevent growth and formation of its biofilm up to 72 h (Gani et al. 2023). Moreover, in vivo employment of M. oleifera extracts showed high fungitoxic impact that completely prevented the growth of G. candidum at 100 mg/mL. The effectiveness of M. oleifera extract was due to the occurrence of saponins, phenols, tannins, flavonoids, and terpenoids, besides glycosides (Chiejina and Onaebi 2016). With regard to Candida tropicalis, flavonoids, steroids, tannins, alkaloids etc., present in the ethanolic and aqueous extracts leaves of M. oleifera exhibited highly antifungal properties (Patel et al. 2014). Al-Ghanayem et al. (2022) documented the application of M. oleifera extract in the healing of diabetic rat wounds diseased with methicillin-resistant bacteria. Merits of M. oleifera can be attained by utilizing the fresh part of plant or via implying extraction methods. Ordinary techniques of extraction, including maceration, Soxhlet, and hydrodistillation entail different drawbacks like long extraction time, extracts containing residual toxic solvents, and thermal disintegration of target constituents (Chemat et al. 2019). New safe and more environmentally friendly alternatives have increasingly been a demand that have made scientists suggest green tools as a solution to dominate the environmental and quality matters. These technologies, such as extraction with pressurized hot-H2O (Matshediso et al. 2015), microwave-assisted (Rodríguez-Pérez et al. 2016a), and ultrasound (Zhong et al. 2018), usually provide high yields of the total extraction.
Supercritical fluid extraction (SFE) with carbon dioxide (SFE-CO2) has been applied on a large-scale as a substitute green technology. It offers extracts with developed compound selectivity that can be improved by checking the temperature and pressure. The SFE-CO2 shows dispersal coefficients alike to gases and slight viscosities alike to solutions, these properties causing a decline in solvent surface tension and rise in the solvent diffusion into the tissues (Zorić et al. 2022). SFE-CO2 has been earlier utilized to get extracts of M. oleifera leaves (Rodríguez-Pérez et al. 2016b; da Silva et al. 2022). The goals of this paper were to estimate SFE-CO2 of the obtained extracts from M. oleifera leaves and profile them utilizing HPLC assay to define the present bioactive ingredients to stand up for their potential use as anti-yeast, anti-diabetic, antimicrobial, and healing efficacy.
EXPERIMENTAL
Plant Samples
Moringa oleifera leaves were brought from the National Research Center, Cairo, Egypt during October 2023. The revised species has been identified via the deceased Prof. Dr. Abo El-Fetoh Mohamed Abd-Allah at National Research Center, Egypt. Plant leaves were air-dried for 48 h, and subsequently ground to powder, sieved, and kept in paper bags at room temperature in dark until further use.
Plant Extract via Supercritical CO2 Extraction (SFE-CO2)
The SFE-CO2 method was used to obtain plant extracts by utilizing the unique properties of supercritical fluids. In this case, carbon dioxide is pressurized and heated to its supercritical state, where it exhibits both gas and liquid-like properties. This allows it to effectively extract desired compounds from plants without leaving behind any residual solvents. The first step is to set up the supercritical CO2 extraction equipment, which typically consists of a high-pressure vessel, a pump, a heat exchanger, and a collection vessel. The equipment must be designed to handle the high pressures and temperatures included in the procedures. The powder of M. oleifera leaves is loaded into the container of extraction. The vessel is then sealed, ensuring that it is airtight to prevent any CO2 leaks during the extraction process. CO2 is introduced into the system through the pump, and pressure is gradually increased to reach the supercritical state. The temperature is also carefully controlled to maintain the desired supercritical conditions. Supercritical CO2 acts as a solvent and penetrates the plant material, dissolving and carrying away the desired compounds so adjustments can be made to optimize the extraction process. After the extraction process, the SFE-CO2 containing the dissolved compounds is depressurized and directed to a collection vessel. As the pressure decreases, the SFE-CO2 reverts to a gaseous state, leaving behind the extracted compounds. The collected extract may undergo further processing steps to remove any residual CO2 or impurities, such as filtration, evaporation, or winterization, depending on the specific requirements of the extract.
Assessment of Total Contents of Flavonoid, Phenolic, Tannin, Saponins, and Alkaloid of Plant Extract
Total phenolic content was estimated via a colorimetric technique using Folin-Ciocalteu reagent (Sembiring et al. 2018). The AlCl3 calorimetric technique was utilized to detect the total content of flavonoid (Sembiring et al. 2018). Total content of alkaloid was assessed according to approach of Shamsa et al. (2008). Total saponins content (TSC) was estimated using Vanillin-Sulphuric acid method (Anh et al. 2018). Tannin content was estimated via acidified vanillin method according to Broadhurst and Jones (Broadhurst and Jones 1978).
Analysis of Plant Extract via HPLC
The HPLC investigation of M. oleifera leaves was performed utilizing an instrument (Agilent 1260 series). Separation was achieved using an Eclipse C18 column (4.6 mm × 250 mm in diameter, 5 μm). The mobile phase consisted of both (A) water and (B) 0.05% trifluoroacetic acid in acetonitrile at constant movement rate of 0.9 mL/min. The mobile phase was sequentially programmed with a linear gradient as follows: 0 min for 82% A; 0 to 5 min for 80% A; 5 to 8 min for 60% A; 8-12 min for 60% A; 12-15 min for 82% A; 15 to 16 minutes for 82% A and 16 to 20 min for 82% A. The multi-wavelength detector was monitored at 280 nm. The injection volume was 5 µL for each solution of tested sample. The column temperature was preserved at 40°C.
Anti-yeast Activity of Plant Extract
Using the cup-plate agar diffusion technique, the anti-yeast potential of the M. oleifera extracts was evaluated against Candida albicans with code number ATCC 10231, C. glabrata with code number RCMB 027016, and C. tropicalis with code number ATCC 10243. After standardising the used yeasts to the 0.5 McFarland scale, they were poured into petri dishes and sown in a molten, sterile Sabouraud dextrose medium. Four cups were cut from agar layer and taken out once the mixture solidified using a sterile cork borer with a 6-mm radius. Each cup was packed with 100 µL of the extract (20 µg/mL) using an automatic microlitre pipette. The cups were then refrigerated at 2 °C for 20 min to permit the tested extract to permeate over the surface of agar and an overnight incubation period at 25 °C. An accurately calibrated millimetre ruler was used to record the inhibition zones that were visible (Abdelghany et al. 2021).
Anti-diabetic Activity via Inhibition of α-Amylase and α-Glucosidase Activities in vitro
Pistia-Brueggeman and Hollingsworth’s procedure (Pistia-Brueggeman and Hollingsworth 2001) was minor change to test the utilized extract for inhibition of α-glucosidase activity. In summary, 0.1 M phosphate buffer (125 μL) adjusted at 6.8 of pH was mixed with 50 μL of the tested M. oleifera extract at different doses (from 1.97 up to 1000 μg/mL) and incubated for 20 min at 37 °C along through 10 μL of the α-glucosidase. After the period of incubation, one M p-nitrophenyl-α-D-glucopyranoside (p-NPG) (20 μL) was added as enzyme substrate to start the enzyme reaction, and afterward the mixture of reaction was left for 30 min. Then, it was fortified with 50 μL of Na2CO3 (0.1 N), the enzyme reaction was blocked, and the wavelength at 405 nm was measured utilizing a spectrophotometer model Biosystem 310 Plus. The inhibition of α-glucosidase activity was estimated via Eq. 1
(1)
where Aun is the untreated α-glucosidase absorbance, while Atr is treated α-glucosidase absorbance by the extract of M. oleifera. One unit of the enzyme (E) was described as the amount of E required for the making of one μmol of p-nitrophenol got from the p-NPG/ minute.
A modified version of Wickramaratne et al. (2016) procedure was employed to test the used extract for α-amylase inhibitory potential employing the 3,5-dinitrosalicylic acid (DNSA) procedure. To dissolve the tested extract, 10% DMSO was used in total. After that, it was further dissolved in a buffer consisting of NaCl (0.006 M) adapted to pH 6.9 and Na2HPO4 (0.02 M)/NaH2PO4 (0.02 M), yielding doses started from 1.9 up to 1000 μg/mL. A total of 200 μL of the tested sample and α-amylase (2 units/mL) were joined to create the reaction mixture, which was subsequently incubated at 30 °C for 10 min. Next, the reaction mixture was added to suspended starch (200 μL) in water (1%), which was left for 3 min. To halt the reaction, 200 μL of DNSA reagent was inserted to the mixture and allowed to boil at 90 °C in a water bath for 10 min. After cooling of the reaction mixture up to 25 °C, it diluted utilizing 5 mL of H2O. At 540 nm, the wavelength was then determined. The tested extract was mixed with 200 μL of buffer to create activity (100%) of α-amylase control, while the tested extract was used to make the blank reaction without the α-amylase solution. Equation 2 was utilized to compute the α-amylase inhibition,
(2)
where Atr is the absorbance of enzyme treated with the M. oleifera extract, and Aun is the absorbance of α-amylase without treatment (control).
Using a regression equation, the extract dose required to inhibit 50% of the activity of enzymes (IC50, μg/mL) was determined by plotting doses (1.95 to 1000 μg/mL) and developing a percentage of inhibition.
Scratch Wound Closure Assay for Measuring the Healing Properties of Plant Extract
Examination of the scratch wound was performed using a multi-well plate. Ten µg/mL of fibronectin extracellular matrix substrate was coated. The plate was then kept at 37°C for a 2-hour incubation period. After release of unbound extracellular matrix, phosphate-buffered saline (PBS) was used as a wash. Trypsin was employed to remove the developed cells from the plate containing the tissue culture. Cells were grown on a scratched research plate and then incubated to allow the cells to proliferate and form a uniform monolayer. A monolayer cell containing a uniform monolayer was rubbed with the pipette tip. Following the scratching, the cell layer was gently rinsed to remove any detached cells. Next, a fresh medium containing verified cells was employed. The plate was placed in a cell culture incubator at 37 °C for 24 up to 48 hours. Post the incubation period, the cell layer was washed with PBS. The cells were then fixed using 3.7% paraformaldehyde for 15 minutes. Subsequently, the cells were stained with 1% crystal violet in ethanol for 10 minutes. A phase-contrast microscope was utilized to examine the cell culture (Martinotti and Ranzato 2020).The subsequent Eqs. 3 to 5 were utilized to calculate the analysis as follows:
(3)
(4)
(5)
Statistical Analysis of Findings
The data were collected three times, and the outcomes are expressed as mean ± SD (standard error). CoStat software (version 14, IBM Corp., Armonk, NY, USA) as One-way investigation of variance (ANOVA) was employed to statistical investigations
RESULTS AND DISCUSSION
Solvent extraction is the foundation of the most popular procedures for the extraction of significant constituents of plants. The main drawback of this extraction, though, is that the extracts contain solvent residue. To recover the extract without solvent residue, SFE-CO2 has recently drawn rising consideration relative to traditional ways such as solvent extraction and hydro-distillation. The SFE-CO2 operates at low temperatures, preserving the thermally labile constituents in the extracts, which is another advantage it has over traditional extraction methods. It can be seen from Table 1, maximum yield (0.850 g) of the M. oleifera extract was obtained at 25 MPa, followed by 35 MPa (0.448 g), and 15 MPa (0.272 g) pressure (Table 1). Through SFE, an earlier study (Zhao and Zhang 2013) reported that extract yield from the oil of M. oleifera leaves increased with pressure ranged from 30 to 50 MPa, where the maximum yield (6.34%) was recorded at 50 MPa at 60 °C. The current results were also in parallel with other findings (Chen et al. 2022) but with extraction of Moringa seed oil.
Table 1. Extraction Yield from 5.0 g Dry Powder of M. oleifera Leaves Exposed to Different Conditions of Pressure Bar
Table 2. Phenols and Flavonoid Compounds of M. oleifera Extract at Different Levels of Pressure Values of Extraction
The HPLC analysis of extract showed that extraction at 25 MPa increased the releasing numbers and concentration of phenolic and flavonoid constituents (Table 2 and Fig. 1). For example, the concentrations of gallic acid, chlorogenic acid, coumaric acid, and rosmarinic acid were 89.90, 150.59, 29.41, and 44.75 µg/mL with increasing percentages of 98.3%, 99.52%, 99.86%, and 99.46%, respectively, when compared with their concentrations at 15 MPa (Table 2 and Fig. 2). Moreover, besides hesperetin, two unknown compounds with retention times (RT) 7.84 and 9.95 appeared only at 25 MPa. Remarkable detection of rutin (44.85 µg/mL) was observed at 35 MPa compared with their concentrations 29.40 and 1.01 µg/mL at 25 and 15 MPa, respectively (Table 2 and Fig. 3). The obtained outcomes showed that extraction at 25 MPa is a promising condition to liberate active compounds. Methyl gallate, caffeic acid, syringic acid, and ferulic acid were not detected at 15 MPa, but they appeared at high pressures of 25 and 35 MPa. These results were consistent with some other reports. Verma et al. (2009) via HPLC showed existence of flavonoids, such as rutin, kaempferol, and quercetin, in addition to phenolic acids such as chlorogenic, gallic, ferulic, and ellagic acids, in M. oleifera extract. According to Braham et al. (2020), chlorogenic acid and quercetin represented the most detected compounds in the hydroacetonic and hydroethanolic extracts of M. oleifera. Moreover, Qadir et al. (2022) reported that the RP-HPLC investigation of M. oleifera leaves extract reflected that the major compounds were quercetin followed by ferulic acid, p-coumaric acid, and gallic acid. The differences in the number and concentrations of detected constituents may be dependent on the cultivation conditions, extraction methods, and used solvents. Moringa oleifera leaves were extracted at varying pressures (15 to 25 MPa) and temperatures (35 to 80 °C) (da Silva et al. 2022), where 20 MPa and 60 ℃ were the best conditions for obtaining the maximum quantity of extract yield, total phenolic compounds, and bioactive constituents. Further, tannins (TanE)/gm, alkaloid (µg/g), saponin (mg AE/g), flavonoid (mg QuE/g), and phenolic (mg Ga)/g) of M. oleifera were detected in highest concentrations at 25 MPa. Moreover, their concentrations at 35 MPa were more than that at 20 MPa (Table 3).
Table 3. Total Phytochemical Content of M. oleifera Extract at Different Pressure Values
Fig. 1. HPLC chromatograms of identified phenols and flavonoids in the extracts at 25 MPa
Fig. 2. HPLC chromatograms of identified phenols and flavonoids in the extract at 15 MPa
Fig. 3. HPLC chromatograms of identified phenols and flavonoids in the extract at 35 MPa
Moringa oleifera extract possesses anti-yeast activity at all extraction conditions; however, extract at 25 MPa exhibited more inhibition zones of 27 ± 0.1, 30 ± 0.2, and 30 ± 0.1 mm against C. glabrata, C. tropicalis, and C. albicans, respectively, than the inhibition zones for extracts obtained at 15 or 35 MPa. Moreover, the effect of positive control was less than the effect of extract at 25 MPa (Table 4 and Fig. 4). According to Aisha et al. (2016) various fungi, primarily C. albicans, Rhizopus stolonifer, and Aspergillus niger, were repressed via ethanolic extracts of M. oleifera, but C. albicans was the most susceptible to the extract. The inhibition of fungi was dependent on the used solvent, for example the extracted M. oleifera by petroleum ether did not reflect prevention of C. albicans and A. niger growth unlike its reaction to a polar solvent (Moyo et al. 2012). Strong antifungal efficacy of M. oleifera extracted by methanol was observed on mycelial development, spore germination, and B. cinerea ultrastructure at MIC concentration (Ahmadu et al. 2020). Chlorogenic acid that was detected with high concentration in the extract of M. oleifera at 25 MPa in the present study played a vital role as an anti-yeast (Geotrichum candidum and C. albicans) agent in a recent investigation (Alsalamah et al. 2023).
Table 4. Activity of M. oleifera Extract Obtained at Different Levels of Pressure
Fig. 4. Impact of various pressures 15 (A), 25 (B), and 35 (C) MPa on the anti-yeast activity of M. oleifera extracted (PC, Nystatin as positive control; NC, DMSO as negative control)
The need to investigate natural and biodegradable agents for wound healing in lieu of traditional therapies is growing. Because of the existence of bioactive components, like flavonoids, phenols, triterpenes, and alkaloids, medicinal plants are used to treat wounds. In a recent study, extracts of M. oleifera reflected inhibitory potential of α-glucosidase (Muzammil et al. 2023). As mentioned by Spampinato et al (2020), α-glucosidase inhibition via phytochemical use is an effective strategy to management of hyperglycemia leading to promotion of wound healing in diabetic patients. The results in Table 5 indicate that M. oleifera extract obtained at 30 MPa exhibited excellent suppression of amylase and α-glucosidase activities particularly the α-glucosidase enzyme. Moreover, extraction at 35 MPa was better than at 15 MPa at all tested doses. The extract activity toward enzyme inhibition was confirmed via calculation of IC50, where less IC50 value of 12.97 µg/mL than IC50 (23.83 µg/mL and 53.46 µg/mL) was recorded using the extract at 25, 35, and 15 MPa, respectively compared to the acarbose IC50 (5.52 µg/mL) for amylase inhibition. The same observation was recorded for α-glucosidase inhibition with low IC50 (6.0 µg/mL), followed by 22.02 and 78.91 µg/mL using the extract at 25, 35, and 15 MPa, correspondingly compared to the acarbose IC50 (2.64 µg/mL). High inhibition of amylase and α-glucosidase activities at 30 MPa may be due to the existence of several compounds as identified by HPLC. Magaji et al. (2020) revealed that M. oleifera extract by different solvents possess an inhibitory potential to α-amylase and α-glucosidase but with different levels, where hexane and methanol leaves extract established proper inhibition of α-amylase with IC50 values of 9.40 and 8.217 mg/mL, respectively. Correspondingly, however, their inhibitory action was less than that of acarbose with IC50 value of 0.04 mg/mL, matching approximately with the current findings. Furthermore, root extract by hexane displayed more α-glucosidase inhibition with IC50 value of 0.38 mg/mL than that of acarbose with IC50 value of 0.88 mg/mL. Moringa oleifera extract was effective for several associated-diabetic factors, such as levels of insulin or blood glucose regulation, decreasing of insulin tissue resistance, and blood lipid level enhancement (Mthiyane et al. 2022). The activity of certain oxidative enzymes, such as α-glucosidase and α-amylase, has a major impact on glucose levels in blood and hyperglycemia. The metabolic processes that break down complex sugars into simple monomers such as glucose and its level of metabolism are aided by these enzymes (Al-Rajhi et al. 2023a&b).
Table 5. α-Amylase and α-Glucosidase Inhibitions by M. oleifera Extract
Table 6 demonstrates the results of healing properties of M. oleifera extract at different pressures through the scratch wound closure test. The values of wound closure %, rate of migration (RM), and % area difference (75.1 µm2, 16.6µm, and 786%, respectively) indicated the better efficacy of extract at 25 MPa than that at 35 MPa (Table 6). Generally, the extract of M. oleifera showed healing activity, where their extract at 15 MPa provided 59.1, 13.1, and 546 compared to values at control as of 53.9, 10.7, and 522 as wound closure %, RM, and area difference %, respectively. However, the anti-inflammatory of M. oleifera extract was not evaluated in the current study, but other studies documented this activity. In this context, lowering the inflammation of tissue may promote the wound healing progression. Moreover, the extract containing high contents of phenols, flavonoids, alkaloids, saponins, and tannins plays a stimulating role in lowering the oxidative stress, thereby improving the process and accelerating the wound healing time. In an earlier study, M. oleifera leaves extract stimulated the migration and proliferation of animal fibroblasts (Gothai et al. 2016). Agreeing to Singh et al. (2021) the formulated Moringa gum with a polyacramide hydrogel in wound dressing’s form promoted wound healing via fluid absorption, antioxidant action, and mucoadhesion. In a recent investigation, wound contraction and epithelization time were improved by utilization of M. oleifera extract, which was linked with the improved antioxidant enzyme potentials, capillary density, and formation of collagen in diabetic rats infected by MRSA (Al-Ghanayem et al. 2022).
Table 6. Healing Properties of M. oleifera Extract
CONCLUSIONS
- Supercritical fluid extraction with carbon dioxide (SFE-CO2) was shown to be an attractive green technique for the extraction of natural materials, which have probable uses in the pharmaceutical and food fields.
- The maximum extraction yield of Moringa oleifera (0.850 g/5 g), was obtained at 25 MPa.
- The most detected compounds of phenols and flavonoids, such as chlorogenic acid, gallic acid, rosmarinic acid, and coumaric acid, via HPLC were recorded at 25 MPa.
- Moreover, the extract at 25 MPa had the highest anti-yeast activity toward C. glabrata, C. tropicalis, and C. albicans with inhibition zones of 27 ± 0.1, 30 ± 0.2, and 30 ± 0.1 mm, respectively, compared to the activities of extracts at 15 or 35 MPa.
- Low values of IC50 (12.97 µg/mL and 6.0 µg/mL) were obtained from the extracted plant at 25 MPa for the inhibition α-amylase and α-glucosidase activities, respectively.
- From the wound healing properties test, it promotes the use of SFE-CO2 for M. oleifera extraction at 25 MPa.
FUNDING
This work was funded by the Deanship of Graduate Studies and Scientific Research at Jouf University under grant No. (DGSSR-2024-01-01121).
REFERENCES CITED
Abdelghany, T. M., Reham, Y., Bakri, M. M., Ganash, M., Basma, H. A., and Qanash, H. (2021). “Effect of Thevetia peruviana seeds extract for microbial pathogens and cancer control,” Int. J. Pharmacol. 17(8), 643-655. DOI: 10.3923/ijp.2021.643.655
Ahmadu, T., Ahmad, K., Ismail, S. I., Rashed, O., Asib, N., and Omar, D. (2020). “Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L.),” Brazilian Journal of Biology 81(4), 1007-1022. DOI: 10.1590/1519-6984.233173
Aisha, S. B., Kutama, A. S., Kabir, S., and Paul, A. T. (2016). “Phytochemical screening and antifungal activity of Moringa oleifera on some selected fungi in Dutse, Jigawa State,” Global Adv. Res. J. Agric. Sci. 5(6), 243-248.
Alawlaqi, M. M., Al-Rajhi, A. M. H., Abdelghany, T. M, Ganash, M., and Moawad, H. (2023). “Evaluation of biomedical applications for linseed extract: antimicrobial, antioxidant, anti-diabetic, and anti-inflammatory activities in vitro,” Journal of Functional Biomaterials 14(6), article 300. DOI: 10.3390/jfb14060300
Al-Ghanayem, A. A., Alhussaini, M. S., Asad, M., and Joseph, B. (2022). “Moringa oleifera leaf extract promotes healing of infected wounds in diabetic rats: Evidence of antimicrobial, antioxidant and proliferative properties,” Pharmaceuticals (Basel) 15(5), article 528. DOI: 10.3390/ph15050528.
Al-Rajhi, A. M. H., and Abdelghany, T. M. (2023a). “In vitro repress of breast cancer by bio-product of edible Pleurotus ostreatus loaded with chitosan nanoparticles,” Appl. Biol. Chem. 66, article 33. DOI: 10.1186/s13765-023-00788-0
Al-Rajhi, A. M. H., and Abdelghany, T. M. A. (2023b). “Nanoemulsions of some edible oils and their antimicrobial, antioxidant, and anti-hemolytic activities,” BioResources 18(1), 1465-1481. DOI: 10.15376/biores.18.1.1465-1481
Al-Rajhi, A. M. H., Bakri, M. M., Qanash, H., Alzahrani,H. Y., Halawani, H., Algaydi, M. A., and Abdelghany, T. M. (2023a). “Antimicrobial, antidiabetic, antioxidant, and anticoagulant activities of Cupressus sempervirens in vitro and in silico,” Molecules 28(21), article 7402. DOI: 10.3390/molecules28217402
Al-Rajhi, A. M. H., Qanash, H., Almashjary, M. N., Hazzazi, M. S., Felemban, H. R., and Abdelghany, T. M. (2023b). “Anti-Helicobacter pylori, antioxidant, antidiabetic, and anti-Alzheimer’s activities of laurel leaf extract treated by moist heat and molecular docking of its flavonoid constituent, naringenin, against acetylcholinesterase and butyrylcholinesterase,” Life 13(7), article 1512. DOI: 10.3390/life13071512.
Al-Rajhi, A. M. H., Qanash, H., Almuhayawi, M. S., Al Jaouni, S. K., Bakri, M. M., Ganash, M., Salama, H. M., Selim, S., and Abdelghany, T. M. (2022). “Molecular interaction studies and phytochemical characterization of Mentha pulegium L. constituents with multiple biological utilities as antioxidant, antimicrobial, anticancer and anti-hemolytic agents,” Molecules 27(15), article 4824. DOI: 10.3390/molecules27154824
Alsalamah, S. A., Alghonaim, M. I., Jusstaniah, M., and Abdelghany, T. M. (2023). “Anti-yeasts, antioxidant and healing properties of Henna pre-treated by moist heat and molecular docking of Its major constituents, chlorogenic and ellagic acids, with Candida albicans and Geotrichum candidum proteins,” Life 13(9), article 1839. DOI: 10.3390/life13091839
Anh, V. L., Parks S. E., Nguyen M. N., and Paul D. R. (2018). “Improving the vanillin-sulphuric acid method for quantifying total saponins,” Technologies 6(3), article 84. DOI: 10.3390/technologies6030084
Baldisserotto, A., Buso, P., Radice, M., Dissette, V., Lampronti, I., Gambari, R., Manfredini, S., and Vertuani, S. (2018). “Moringa oleifera leaf extracts as multifunctional ingredients for “natural and organic” sunscreens and photo protective preparations,” Molecules 23 (3), article 664. DOI: 10.3390/molecules23030664
Braham, F., Carvalho, D. O., Almeida, C. M. R., Zaidi, F., Magalhães, J. M. C. S., Guido, L. F., and Gonçalves, M. P. (2020). “Online HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from Moringa oleifera leaves,” South African Journal of Botany 129, 146-154. DOI: 10.1016/j.sajb.2019.04.001
Broadhurst, R. B., and Jones, W. T. (1978). “Analysis of condensed tannins using acidified vanillin,” Journal of the Science of Food and Agriculture 29(9), 788-794. DOI: 10.1002/jsfa.2740290908
Chemat, F., Abert, V. M., Ravi, H. K., Khadhraoui, B., Hilali, S., Perino, S., and Tixier, A.F. (2019). “Review of alternative solvents for green extraction of food and natural products: Panorama, principles, applications and prospects,” Molecules 24(16), article 3007. DOI: 10.3390/molecules24163007
Chen, X., Li, Z., Smith, S. A., Chen, M., Liu, H., Zhang, J., Tang, L., Li, J., Liu, Q., and Wu, X. (2022). “Optimization of supercritical CO2 extraction of Moringa oleifera seed oil using response surface methodological approach and its antioxidant activity,” Front. Nutr. 8, article 829146. DOI: 10.3389/fnut.2021.829146
Chiejina, N. V., and Onaebi, C. (2016). “Phytochemical constituents and antifungal properties of Chromolaena odorata L. and Moringa oleifera Lam on fungal rot of cucumber (Cucumis sativus L.) Fruit,” Asian Journal of Plant Sciences 15(1), 35-41. DOI: 10.3923/ajps.2016
da Silva, M., Trancoso, J., Tormen, L., Bombardelli, M. M., Corazza, M. L., and Bainy, E. M. (2022). “Extraction of compounds from Moringa oleifera leaves using supercritical CO2 plus ethanol as a cosolvent,” Journal of Food Process Engineering 45(3), article e13979. DOI: 10.1111/jfpe.13979
Gothai, S., Arulselvan, P., Tan, W.S., and Fakurazi, S. (2016). “Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts,” J. Intercult. Ethnopharmacol. 5(1), 1-6. DOI: 10.5455/jice.20160201055629.
Khalid, S., Arshad, M., Mahmood, S., and Lorenzo, J. M. (2023). “Extraction and quantification of Moringa oleifera leaf powder extracts by HPLC and FTIR. Food Anal. Methods 16(4), 787-797. DOI: 10.1007/s12161-023-02470-z
Magaji, U. F., Sacan, O., and Yanardag, R. (2020). “Alpha amylase, alpha glucosidase and glycation inhibitory activity of Moringa oleifera extracts,” South African Journal of Botany 128, 225-230. DOI: 10.1016/j.sajb.2019.11.024
Martinotti, S., and Ranzato, E. (2020). “Scratch wound healing assay,” Methods Mol. Biol. 2109, 225-229. DOI: 10.1007/7651_2019_259
Matshediso, P. G., Cukrowska, E., and Chimuka, L. (2015). “Development of pressurised hot water extraction (PHWE) for essential compounds from Moringa oleifera leaf extracts,” Food Chem. 172, 423-427. DOI: 10.1016/j.foodchem.2014.09.047
Mittal, M., Mittal, P., and Agarwal, A. C. (2007). “Pharmacognostical and phytochemical investigation of antidiabetic activity of Moringa oleifera lam leaf,” Ind. Pharm. 6(59), 70-72.
Moyo, B., Masika, P. J., and Muchenje, V. (2012). “Antimicrobial activities of Moringa oleifera Lam. leaf extracts,” African Journal of Biotechnology 11(11), 2797-2802 DOI:10.5897/AJB10.686
Mthiyane, F. T., Dludla, P. V., Ziqubu, K., Mthembu, S. X. H., Muvhulawa, N., Hlengwa, N., Nkambule, B. B., and Mazibuko-Mbeje, S. E. (2022). “A review on the antidiabetic properties of Moringa oleifera extracts: focusing on oxidative stress and inflammation as main therapeutic targets,” Front Pharmacol. 11(13), article 940572. DOI: 10.3389/fphar.2022.940572
Muhammad, A. A., Pauzi, N. A. S., Arulselvan, P., Abas, F., and Fakurazi, S. (2013). “In Vitro wound healing potential and identification of bioactive compounds from Moringa oleifera Lam.,” Biomed Res. 974580. DOI: 10.1155/2013/974580
Muhammad, H. I., Asmawi, M. Z., and Khan, N. A. K. (2016). “A review on promising phytochemical, nutritional and glycemic control studies on Moringa oleifera Lam in tropical and sub-tropical regions,” Asian Pacific Journal of Tropical Biomedicine 6(10), 896-902. DOI: 10.1016/j.apjtb.2016.08.006
Muzammil, S., Neves, C. J., Mumtaz, R., Rasul, I., Hayat, S., Khan, M. A., Khan, A. M., Ijaz, M. U., Lima, R. R., and Zubair, M. (2023). “Effects of drying temperature and solvents on in vitro diabetic wound healing potential of Moringa oleifera leaf extracts,” Molecules 28(2), article 710. DOI: 10.3390/molecules28020710
Ndong, M., Uehara, M., Katsumata, S.-I., and Suzuki, K. (2007). “Effects of oral administration of Moringa oleifera Lam on glucose tolerance in Goto-Kakizaki and Wistar rats,” Journal of Clinical Biochemistry and Nutrition 40(3), 229-233. DOI:10.3164/jcbn.40.229
Olayaki, L. A., Irekpita, J. E.,Yakubu, M. T., and Ojo, O. O. (2015). “Methanolic extract of Moringa oleifera leaves improves glucose tolerance, glycogen synthesis and lipid metabolism in alloxan-induced diabetic rats,” Journal of Basic and Clinical Physiology and Pharmacology 26(6), 585-593. DOI:10.1515/jbcpp-2014-0129
Patel, P., Patel, N., Dhara, P., Desai, S., and Dhananjay, B. M. (2014). “Phytochemical analysis and antifungal activity of Moringa oleifera,” International Journal of Pharmacy and Pharmaceutical Sciences 6(5), 144-147.
Pistia-Brueggeman, G., and Hollingsworth, R. I. (2001). “A Preparation and screening strategy for glycosidase inhibitors,” Tetrahedron 57(42), 8773–8778. DOI:10.1016/S0040-4020(01)00877-8
Qadir, R., Anwar, F., Bashir, K., Tahir, M. H., Alhumade, H., and Mehmood, T. (2022). “Variation in nutritional and antioxidant attributes of Moringa oleifera L. leaves at different maturity stages,” Front. Energy Res. 10, article 888355. DOI: 10.3389/fenrg.2022.888355
Qanash, H., Bazaid, A. S., Aldarhami, A., Alharbi, B., Almashjary, M. N., Hazzazi, M. S., Felemban, H. R., and Abdelghany, T. M. (2023). “Phytochemical characterization and efficacy of Artemisia judaica extract loaded chitosan nanoparticles as inhibitors of cancer proliferation and microbial growth,” Polymers 15, article 391. DOI: 10.3390/polym15020391
Qanash, H., Yahya, R., Bakri, M. M., Bazaid, A. S., Qanash, S., Shater, A. F., and Abdelghany, T. M. (2022). “Anticancer, antioxidant, antiviral and antimicrobial activities of Kei Apple (Dovyalis caffra) fruit. Sci. Rep. 12(1), article 5914. DOI: 10.1038/s41598-022-09993-1
Rani, N. Z. A., Husain, K., and Kumolosasi, E. (2018). “Moringa genus: A review of phytochemistry and pharmacology,” Front. Pharmacol. 9, 108. DOI: 10.3389/fphar.2018.00108
Rathi, B. S., Bodhankar, S. L., and Baheti, A. M. (2006). “Evaluation of aqueous leaves extract of Moringa oleifera Linn for wound healing in albino rats,” Indian Journal of Experimental Biology 44(11), 898-901.
Rodríguez-Pérez, C., Gilbert-López, B., Mendiola, J.A., Quirantes-Piné, R., Segura-Carretero, A., and Ibáñez, E. (2016a). “Optimization of microwave-assisted extraction and pressurized liquid extraction of phenolic compounds from Moringa oleifera leaves by multiresponse surface methodology,” Electrophoresis 37(13), 1938-1946. DOI: 10.1002/elps.201600071
Rodríguez-Pérez, C., Mendiola, J.A., Quirantes-Piné, R., Ibáñez, E., and Segura-Carretero, A. (2016b). “Green downstream processing using supercritical carbon dioxide, CO2-expanded ethanol and pressurized hot water extractions for recovering bioactive compounds from Moringa oleifera leaves,” The Journal of Supercritical Fluids 116, 90-100. DOI: 10.1016/j.supflu.2016.05.009
Sembiring, E. N., Elya, B., and Sauriasari, R. (2018). “Phytochemical screening, total flavonoid and total phenolic content, and antioxidant activity of different parts of Caesalpinia bonduc (L.) Roxb,” Pharmacolog. J. 10(1), 123-127.
Shamsa, F., Monsef, H., Ghamooshi, R., and Verdian-rizi, M. (2008). “Spectrophotometric determination of total alkaloids in some Iranian medicinal plants,” Thai J. Pharm. Sci. 32(1) 17-20. DOI: 10.56808/3027-7922.2196
Singh, B., Sharma, V., and Kumar, A. (2021). “Designing moringa gum-sterculia gum-polyacrylamide hydrogel wound dressings for drug delivery applications,” Carbohydr. Polym. Technol. Appl. 2, article 100062. DOI: 10.1016/j.carpta.2021.100062
Spampinato, S. F., Caruso, G. I., De Pasquale, R., Sortino, M. A., and Merlo, S. (2020). “The Treatment of impaired wound healing in diabetes: Looking among old drugs,” Pharmaceuticals (Basel) 13(4), article 60. DOI: 10.3390/ph13040060
Stephenson, K. K., and Fahey, J. W. (2004). “Development of tissue culture methods for the rescue and propagation of endangered Moringa Spp. germplasm,” Econ. Bot. 58 (Suppl 1), S116-S124. DOI: 10.1663/0013-0001(2004)58[S116:DOTCMF]2.0.CO;2
Verma, A. R., Vijayakumar, M., Mathela, C. S., and Rao, C. V. (2009). “In vitro and in vivo antioxidant properties of different fractions of Moringa oleifera leaves,” Food and Chemical Toxicology 47(9), 2196-2201. DOI: 10.1016/j.fct.2009.06.005
Wickramaratne, M. N., Punchihewa, J. C., and Wickramaratne, D. B. M. (2016). “In-vitro alpha amylase inhibitory activity of the leaf extracts of Adenanthera pavonina,” BMC Complement. Altern. Med. 16, article 466. DOI:10.1186/s12906-016-1452-y
Zhao, S., and Zhang, D. (2013). “Supercritical fluid extraction and characterisation of Moringa oleifera leaves oil,” Separation and Purification Technology 118, 497-502. DOI: 10.1016/j.seppur.2013.07.046
Zhong, J., Wang, Y., Yang, R., Liu, X., Yang, Q., and Qin, X. (2018). “The application of ultrasound and microwave to increase oil extraction from Moringa oleifera seeds,” Ind. Crops Prod. 120, 1-10. DOI: 10.1016/j.indcrop.2018.04.028
Zorić, M., Banožić, M., Aladić, K., Vladimir-Knežević, S., and Jokić, S. (2022). “Supercritical CO2 extracts in cosmetic industry: Current status and future perspectives,” Sustainable Chemistry and Pharmacy 27, article 100688. DOI: 10.1016/j.scp.2022.100688
Article submitted: April 15, 2024; Peer review completed: June 8, 2024; Revised version received and accepted: July 9, 2024; Published: July 16, 2024.
DOI: 10.15376/biores.19.3.5961-5977