Dynamic extraction time’s effect on phytochemical characterization of Vitex agnus-castus dry biomass with healing properties and their activity against microorganisms and ovarian cancer

Abstract

Efficacies of plant metabolites are known to be dependent on their extraction methods. Yields and compositions of phytoconstituents in the extract were evaluated following supercritical fluid extraction (SFE) of Vitex agnus-castus leaves, static extraction times (SET) for 30 min, subsequently dynamic extraction time (DET) for 30 min (condition A) and SET for 0 min followed by DET for 60 min (condition B). The extract exposed to condition B gave an extraction yield of 0.169 g compared to 0.115 g for condition A. High-performance liquid chromatography analysis revealed compounds including cinnamic acid, kaempferol, ferulic acid, rutin, and caffeic acid, in high concentrations in the extract exposed to condition B. Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, and Candida albicans were more affected by the condition B with 32 ± 0.1, 20 ± 0.2, 32 ± 0.2, 35 ± 0.2, and 40 ± 0.1 mm inhibition zones, respectively. Less MIC and MBC were noticed of the exposed extract to condition B than to condition A against C. albicans and bacteria. The IC₅₀ of the extract exposed to condition B was high against ovarian tumor cells. Presently the efficacy of the exposed extract to condition B for wound healing process was documented.

Full Article

Dynamic Extraction Time’s Effect on Phytochemical Characterization of Vitex agnus-castus Dry Biomass with Healing Properties and their Activity Against Microorganisms and Ovarian Cancer

Samy Selim,^a,* Yasir S. Alruwaili,^a,b Emad Manni,^a Muhammad Atif,^a

Mohammed S. Almuhayawi,^c Mohammed H. Alruhaili,^c,d Mohammed A. Bazuhair,^e,f Eman M. Abdelkareem,^g Badriah Saleh Alammari,^h and Soad K. Al Jaouni ^i,*

Efficacies of plant metabolites are known to be dependent on their extraction methods. Yields and compositions of phytoconstituents in the extract were evaluated following supercritical fluid extraction (SFE) of Vitex agnus-castus leaves, static extraction times (SET) for 30 min, subsequently dynamic extraction time (DET) for 30 min (condition A) and SET for 0 min followed by DET for 60 min (condition B). The extract exposed to condition B gave an extraction yield of 0.169 g compared to 0.115 g for condition A. High-performance liquid chromatography analysis revealed compounds including cinnamic acid, kaempferol, ferulic acid, rutin, and caffeic acid, in high concentrations in the extract exposed to condition B. Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, and Candida albicans were more affected by the condition B with 32 ± 0.1, 20 ± 0.2, 32 ± 0.2, 35 ± 0.2, and 40 ± 0.1 mm inhibition zones, respectively. Less MIC and MBC were noticed of the exposed extract to condition B than to condition A against C. albicans and bacteria. The IC₅₀ of the extract exposed to condition B was high against ovarian tumor cells. Presently the efficacy of the exposed extract to condition B for wound healing process was documented.

DOI: 10.15376/biores.19.3.5793-5810

Keywords: Vitex agnus-castus; Antimicrobial; Supercritical fluid extraction; SKOV3; Wound healing

Contact information: a: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka 72388, Saudi Arabia; b: Sustainable Development Research and Innovation Center, Deanship of Graduate Studies and Scientific Research, Jouf University, Sakaka, Saudi Arabia; c: Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia; d: Special Infectious Agents Unit, King Fahad Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; e: Department of Clinical Pharmacology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; f: Centre of Research Excellence for Drug Research and Pharmaceutical Industries, King Abdulaziz University, Jeddah, Saudi Arabia; g: Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt; h: Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), P. O. Box: 90950, Riyadh 11623, Kingdom of Saudi Arabia; i: Department of Hematology/Oncology, Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia;

* Corresponding author: sabdulsalam@ju.edu.sa (S.S.), saljaouni@kau.edu.sa (S.K.A.)

GRAPHICAL ABSTRACT

INTRODUCTION

Natural products, particularly from plants, have gained great scientific consideration in current decades because of their various therapeutic possessions, including numerous biological activities (Abdelghany et al. 2014, 2016; Qanash et al. 2022; Alghonaim et al. 2023). Vitex agnus-castus, which belongs to the Lamiaceae family, has been broadly employed in traditional medicine. Within the Mediterranean regions such as Southern Europe, Western Asia, and North Africa, it grows along seacoasts and riverbanks as a wild plant. It is a tall shrub (3 to 6 m) or low tree. It appears in the shape of an erect shrub or a prostrate habit. The shoots of the young plant are tetrahedral with gray color. Its leaves consist of 3 to 7 leaflets, with white developed tomentum on its lower surface (Adamov et al. 2020). According to several investigations, V. agnus-castus has several utilizations in biological systems. Its antiseptic, digestive, diuretic, anti-anxiety, aphrodisiac, anti-estrus, emmenagogus, aperitif, analgesic, and antispasmodic effects have been used in traditional treatment. Moreover, V. agnus-castus is an effective, traditional plant used to minimize uterine cramps during menstruation regulation (Mari et al. 2012; Zahid et al. 2016). The Vitex agnus-castus fruit is frequently used for a range of reproductive illnesses in females, comprising of female hormonal imbalances and premenstrual syndrome (PMS), such as depression, mood swings, cramps, weight gain, and water retention linked with the disorder of premenstrual dysphoric, PMS, lactation problems, and menopause-related complaints besides low fertility (Shaw et al. 2018).

Antioxidant and antihyperglycemic activities, besides antibacterial properties of V. agnus-castus were reported by Berrani et al. (2021). Moreover, its seeds extract exhibited suppressive effect against bacteria, besides antioxidant, and anti-alzahimer activities (Kavaz et al. 2022). Al-Otibi et al. (2022) reported the inhibitory potential of V. agnus-castus toward numerous species of yeasts comprising Candida krusei, C. tropicalis, C. albicans, C. parapsilosis, C. dublinesis, C. famatai, and C. rhodotorula.

Many secondary constituents, such as iridoids, terpenoids, flavonoids, oils, as well as ketosteroids, are found in various organs of V. agnus-castus, including the flowering stems, fruits, and leaves based on phytochemical investigations (Chen et al. 2011). The majority of plant materials naturally contain these bioactive constituents, which are recognized to have intriguing biological properties including anti-inflammatory, cancer suppressive, antibacterial, antiviral, and antioxidant agents (Teugwa et al. 2013; Al-Rajhi et al. 2022a,b; Al-Rajhi et al. 2023a; Al-Rajhi and Abdelghany 2023a,b; Alsalamah et al. 2023). However, there have been few investigations into V. agnus-castus for its pharmaceutical uses. The presence of various bioactive chemical groups and compounds, such as terpenes, polyphenols, terpenoids, fatty acids, steroids, alcohols, aldehydes, and esters, were documented in V. agnus-castus extract via Fourier transform infrared and gas chromatography-mass spectrometry analyses (Al-Otibi et al. 2022).

Supercritical fluid extraction (SFE) is an excellent technique for the extraction of natural compounds because it allows the extraction of heat-susceptible compounds without causing any degradation and is also recognized as an environmentally friendly technology. Through adjusting the extraction temperature and pressure, SFE allows for the manipulation of the yield of extracted compound and selectivity (Jokić et al. 2017). Because carbon dioxide (CO₂) (solvent used in the supercritical fluid extraction) is nontoxic, and is easily accessible, affordable, and has low critical point requirements for both pressure and temperature, it has been the most widely used solvent (Bimakr et al. 2009). It is unlike the conventional approaches of extraction, which are commonly performed at high temperatures that can be responsible for the damage of appreciated ingredients. This method also has the advantage that the extraction can be performed under different conditions of temperature, pressure, and extraction time (Bimakr et al. 2011; Chamali et al. 2023). Currently, there are no data about the influence of static extraction time and dynamic extraction time via SFE on V. agnus-castus. Therefore, the present study focuses on the influence of extraction time on constituent’s analysis of V. agnus-castus L. leaves by HPLC. Additionally, this study investigates the antimicrobial, anticancer, and healing activity of maximum yield of V. agnus-castus extract.

EXPERIMENTAL

Supercritical Fluid Extraction

According to the description of Žitek et al. (2020), the SFE leaves extraction of V. agnus-castus (collected from market in Egypt and identified by Prof. Tarek Mohamed, Botany and Microbiology department) was carried out in an ISCO-Sitec modified SFX 220 supercritical fluid extraction system. In this study, 6.0 g of V. agnus-castus dried powder were subjected to SEF at two conditions including static extraction time (SET) for 30 min, followed by dynamic extraction time (DET) for 30 min at constant pressure (206.84 bar) and temperature (50 ℃) (sample code A). Another sample of V. agnus-castus dried powder was extracted at SET for 0 min, followed by DET for 60 min at constant pressure (206.84 bar) and temperature (50 ℃) (sample code B). In every run, the supercritical CO₂ consumption and the solvent flow rate remained constant (Hassim et al. 2020).

HPLC Analysis

The extract was subjected to HPLC analysis (Waters 2695 Alliance, Waters Inc., Milford, CT, USA) for determining the phenolic and flavonoids contents, which was furnished utilizing an ultraviolet-visible (UV-Vis) DAD. A Waters SunfireTM C18 reverse-phase chromatography column (dimensions: 250 mm length, 4.6 mm width, and 5 μm particle size) was employed to perform the separation. The extract solution and mixture of standard compounds were introduced into the apparatus using an autoinjector. A variety of gradient and isocratic mobile phases were tested at various column temperatures and flow rates to determine an effective separation technique for the standards. The gradient approach was ultimately selected after a sequence of initial investigations. A combination of acetonitrile as mobile phase A and phosphoric acid as mobile phase B was used. The phosphoric acid was prepared by adding 85% orthophosphoric acid dropwise to HPLC grade water until pH = 2. The concentration gradient was changed in the following ways during the method’s 60-minute total runtime: A) 5% A + 95% B at first; b) 35% A + 65% B for 15 min; c) 35% A + 65% B for 20 min; d) 40% A + 60% B for 30 min; e) 40% A + 60% B for 35 min; f) 50% A + 50% B for 40 min; g) 70% A + 30% B for 52 min; and h) 5% A + 95% B for 60 min. There was a static flow rate (0.5 mL/min) and temperature (5 °C). Following the examination of the UV-Vis spectra of separate standards, three wavelengths—minimum 210, median 280, and maximum 360 nm—were selected for HPLC examination in this study.

Antimicrobial Screening

The antibacterial and antifungal potential of V. agnus-castus extract under different conditions of SFE (static and dynamic extraction times) were examined by the agar well diffusion technique as designated by Qanash et al. (2023b) against the microorganisms: Mucor circinelloid (AUMMC 11656), Candida albicans (ATCC 10221), Staphylococcus aureus (ATCC 6538), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 90274), and Enterococcus faecalis (ATCC 29212). Dimethyl sulfoxide (DMSO) was utilized as a solvent for the extract, and then tested as antimicrobial agent. One mL of freshly cultured bacteria/fungi was placed into the midpoint of a sterile petri plates. After cooling, liquefied Mueller-Hinton/potato dextrose for bacteria/fungi was added to the Petri plate comprising the inoculum and mixed thoroughly. In the solidified agar, sterile cork borers of 6 mm diameter were applied to some wells in the agar plates containing the microbial inoculum. Subsequently, 100 µL (extract) was added to the corresponding well. The Petri plates were cooled for 30 min to thoroughly diffuse the extracts in the layer of agar, and then incubated for 1/4 days at 37/30 °C for bacteria/fungi. Measuring the inhibition zone was recorded at the end of incubation period. A 10% of DMSO was used as a negative control while nystatin (500 µg/mL) and ampicillin (1000 µg/mL) were employed against fungi and bacteria, respectively, as positive controls (Abdelghany et al. 2019). Minimum inhibitory concentration (MIC) was assayed as follows: the bacterial and fungal inoculum activation and preparation were performed using Mueller Hinton broth for 24 h at 37 °C and potato dextrose broth for 48 h at 30 °C, respectively. The microbial culture was diluted using the appropriate broth to modify the inoculum dose to an optical density of 0.5 McFarland standards. Subsequently, 100 μL of the inoculum was added to each well of a 96-well microtiter plate. Various doses of the extract were then introduced to the wells through serial dilution. The wells containing only media + extract (negative controls) were used, while the wells with microbial inoculum without extract were employed as positive control to estimate the maximum growth. Absorbance of plates was documented at 0 h of inoculum time and again after 24 h at a wavelength of 570 nm. Finally, the MIC value was estimated employing log it analysis. Fungal growth was assessed using the MIC method, where 100 μL of fungal inoculum (adjusted to 0.5 McF of 1.5 × 10⁸ CFU/mL) was spread on a petri dish containing sabouraud dextrose agar medium. The extract was diluted in a DMSO solution (0.1%) to obtain different concentrations ranging from 7.8 to 1000 μg/mL. Subsequently, 10 μL of each dose was applied to a 6 mm agar well, and the fungal culture was then incubated for 4 days at 30 °C. To determine the minimum bactericidal concentration (MBC), certain dilution of examined microbes in MH broth at a concentration of 1 × 10⁶ CFU/mL. The V. agnus-castus extract was then diluted at 100% of the MIC and added to 96 microtiter plates in equal volumes (1:1 dilutions). Each concentration of the extract was injected with equal volumes of the examined microbes. Controls (positive and negative) were included in some wells to ensure proper growth during the incubation period. The MBC was determined by observing the dilution that showed a defined decrease in CFU/mL, along with at least two more determined test product dilutions (French 2006). The MBC/MIC index was used to determine whether the V. agnus-castus extract had a static or cidal effect. If the ratio of MBC/MIC value was less than or equal to 4, the extract was considered to have a cidal value (Al-Rajhi et al. 2023b).

Anticancer Activity

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was utilized to measure the V. agnus-castus extract cytotoxicity on human ovarian tumor cell line (SKOV3). In summary, a 96-well plate containing 200 μL of DMEM and 2 × 10⁴ cells/well was seeded with the SKOV3, and the cells were cultured for 12 h. Subsequent treatment, the cells were preserved at 37 °C for 48 h and 5% of CO₂ while being exposed to varying concentrations of V. agnus-castus extract (31.25 up to 1000 μg/mL). Following incubation, 5 mg/mL MTT reagent (20 μL) was added to the cells, and subsequently incubated for 2 h in a CO₂ incubator after the removal of spent medium. After solubilizing the crystals of formazan in 100 μL of DMSO, the wavelength at 570 nm was measured via a microplate reader. The cells that were only treated by DMEM were regarded as 100% viable negative controls (Qanash et al. 2023a). Using the next formula No:1, the cell viability (%) was determined:

Wound Healing via Cell Scratch Test

Using an earlier described method for in vitro cell migration studies on L929 cells, the wound healing capabilities of V. agnus-castus extract was evaluated. In a nutshell, 6-well plates were seeded by 2 × 10⁴ cells/mL, and then cultured for an entire night. After that, the cells were cleaned using DPBS (Delbucco’s Phosphate Buffered Saline) and a sterile 200 μL tip was utilized to make a scratch. The tested cells were washed with DPBS to get rid of the detached cells and cellular debris. After applying 100 μg/mL of V. agnus-castus extract, the cells were incubated for 24 h. Negative control cells were untreated. Images captured with an inverted microscope to show cell migration and changes in the morphological profile (Alsalamah et al. 2023). Three duplicates of each experiment were run. Analysis was done on the width of the scratch and the wound closure at various time periods (0, 24, and 48 h). The next analysis was computed using Eqs. 2, 3, and 4:

RESULTS AND DISCUSSION

According to Khaw et al. (2017), the SFE was selected as the extraction technique because it guarantees rapid and effective extraction that does not need purification steps and does not include the use of unsafe organic solvents. The extracts of V. agnus-castus were subjected to HPLC analysis and biological activities, as presented (Fig. 1). The yield of extraction was higher (0.169 g) at 60 min of DET than the yield of extraction (0.115 g) of the sample exposed to DET (30 min) (Table 1). Via SFE, Vitex negundo L. leaves were extracted at different conditions of operation including temperature (40 to 65 °C), and pressure (20 to 30 MPa) at constant time (60 min), where the yield of the extract was increased with increasing of temperature up to 50 °C (Mohd et al. 2014). Regarding the pressure, the yield of the extract increased at the pressure range from 20 to 30 MPa and at temperature up to 55 °C. Previously, olive leaves were exposed to static condition (for 1 min) of SFE followed by dynamic extraction (5 min up to 140 min), and the quantity extracted yield was 6.7 mg/g and 8.0 mg/g at 20 and 140 min, respectively. Le Floch et al. (1998) and Bimakr et al. (2009) studied the impact of DET on the yields of spearmint leaves extract. At constant pressure of 100 bar, they estimated dynamic extraction at different times after the static extraction up to 30 min. The yield of extraction was improved as the dynamic time increased until 90 min, but it reached a maximum yield at 60 min and at 300 bar of pressure.

Fig. 1. Extraction of V. agnus-castus via SFE at two conditions including static extraction time (S) and dynamic extraction time (D), followed by HPLC analysis of flavonoid and phenolic contents with wound healing, antimicrobial, and anticancer activities of the extracts. Created with BioRender.com

After SFE extraction, the collected extract of V. agnus-castus was subjected to HPLC analysis (Figs. 2 and 3) to recognize the contents of phenols and flavonoids in the extract. In sample code B, all constituents of phenols and flavonoids were detected with high concentrations compared to its concentrations in sample code A except one compound namely gallic acid (Table 2). For instance, in the sample code B, the concentrations of cinnamic acid, kaempferol, ferulic acid, rutin, and caffeic acid were respectively 88.7, 1950, 198, 172, and 1210 µg/g, while in the sample code A it was 3.32, 286, 39.0, 35.2, and 302 µg/g with decreasing levels of 96.3%, 85.4%, 80.3%, 79.6%, and 75.1%, respectively. Moreover, hesperetin was detected only in the sample code A. Generally, gallic acid, querectin, chlorogenic acid represent the main detected compounds with high concentration in the extract. The authors’ result indicated that the DET was effective in releasing the active constituents in the extract. From recent investigation, vanillic acid represented the major content (22800 μg/L) in the ethanol extract of V. agnus-castus seeds besides other phenols, including luteolin, quercetin, fumaric acid, 4-hydroxybenzoic acid, caffeic acid, kaempferol, salicylic acid, butein, resveratrol, ellagic acid, catechin hydrate, phloridzin dehydrate, and naringenin (Kavaz et al. 2022). Berrani et al. (2021) reported the presence of 25 flavonoids and phenols via HPLC-DAD-QTOF-MS analysis with a notable variability among plant parts. Hesperidin, chlorogenic, luteolin, vanillic, 3-hydroxy-benzoic, and 3,4-dihydroxybenzoic were registered with high levels in V. agnus-castus. Regarding the effect of static and extraction dynamic time on the phenolic and flavonoid compounds in V. agnus-castus extract, previous reports indicated that the extraction yield of phenolic constituents is affected by pressure, time, temperature, and addition of co-solvents (Junior et al. 2010; Bimakr et al. 2011). Moreover, in the SFE mode (static or dynamic), the solvent flow rate (Pourmortazavi and Hajimirsadeghi 2007; Leal et al. 2008) affected the extraction yield of natural extracts.

Table 1. Extraction Yield of V. agnus-castus via SFE at Two Different Conditions (SET and DET) at Constant Temperature and Pressure

Table 2. Phenols and Flavonoid Compounds of V. agnus-castus Extracted via SFE in SET (Sample Code A) and DET (Sample Code B) Conditions

*RT: retention time, **Conc., Concentration

In the present investigation, the extraction via SFE focused on the effect of extraction time on the yield of the extract. Several studies were reported on other plants, for instance, the best conditions were 60 °C, 60 min, and 200 bar for spearmint flavonoids extraction comparable to other conditions, primarily temperature (40 °C and 50 °C), extraction time (30 min and 90 min), and pressure (100 bar and 300 bar) via SFE (Bimakr et al. 2011). According to the result of Hassim et al. (2020), 60 min of SET was the best condition for total yield of the extract of Phyllanthus niruri. Influence of temperature and time of extraction was studied on phytochemical characterization, extraction yield, anti-xanthine oxidase, and antioxidant activities. Dynamic time (36 min) and a temperature (179 °C) were the optimum conditions for extraction and biological activities of Eucalyptus intertexta (Chamali et al. 2023).

Fig. 2. HPLC analysis of metabollites namely flavonids and phenols in V. agnus-castus extractred via SFE at static extraction time

Fig. 3. HPLC analysis of metabollites namely flavonids and phenols in V. agnus-castus extractred via SFE at dynamic extraction time

The antimicrobial properties of V. agnus-castus extracts under SET and DET against S. aureus, P. areginosa, K. pneumoniae, E. faecalis, C. albicans, and M. circinelloid were evaluated in this study (Table 3 and Fig. 4). The obtained findings revealed that the V. agnus-castus extracts are effectively suppressing the microbial growth with variable potency based on the conditions of the extraction process. As stated in the results, sample code B of the V. agnus-castus extract had high zones of inhibition 32 ± 0.1, 20 ± 0.2, 32 ± 0.2, 35 ± 0.2, and 40 ± 0.1 mm, whereas sample code A of the extract showed less zones of inhibition 30 ± 0.1, 16 ± 0.1, 25 ± 0.1, 33 ± 0.1, and 35 ± 0.1 mm against S. aureus, P. areginosa, K. pneumoniae, E. faecalis, and C. albicans, respectively. The extract failed to suppress the growth of M. circinelloid. This result may be due to the structure of cell wall that differed from the other tested microorganisms, or because of the incapacity of the extract to pass through the cell membrane. The antimicrobial activity of V. agnus-castus extract is attributed to their content of phenols and flavonoids, particularly in the sample of code B that could join to cell membrane proteins via hydrophobic and hydrogen bonding. Currently from HPLC analysis, the extract of V. agnus-castus contains high concentration of caffeic acid. In another report, Alfarrayeh et al. (2021) noted that leaves of V. agnus-castus contained a great concentration of caffeic acid. It was discovered that caffeic acid inhibited the growth of various strains of Candida by influencing their capacity to form biofilms and mature, ultimately leading to their mortality. Based on findings of Kavaz et al. (2022), Escherichia coli, Salmonella typhimurium, and S. aureus were inhibited by seed extract of V. agnus-castus. Morphological and ultrastructure alterations were observed Candida famata as a result of exposure to V. agnus-castus extract (Al-Otibi et al. 2022). The results in Table 3 showed that P. areginosa was less affected by the extract than other bacteria. This may be explained by the ability of this bacterium to form biofilm that represent one of the mechanisms of drug resistance. Moreover, the extracellular matrix of bacterial biofilm is commonly impermeable and may control the diffusion of antibacterial compounds via attaching to the antibacterial compound and obstructing target locations (Alsolami et al. 2023).

Table 3. Activity of V. agnus-castus extractred via SFE at SET (Sample Code A) DET (Sample Code B) against Different Microorganisms

* Ampicillin /Nystatin was applied as positive control

Sample code A of the V. agnus-castus extract possesses higher MIC and MBC values than the sample of code B against tested bacteria and C. albicans (Table 4). The highest MIC and MBC extract were associated with P. areginosa with MIC quantities of 250 and 62.5 μg/mL, MBC quantities of 1000 and 125 μg/mL, correspondingly. Gonçalves et al. (2017) mentioned that the extract of V. agnus-castus from ethanol had promising growth inhibition against Lactobacillus casei and Streptococcus mutans with an MIC value of 15.6 μg/mL and Streptococcus mitis with an MIC value of 31.2 μg/mL. Bouyahya et al. (2017) mentioned that the antibacterial potential of Vitex agnus-castus extracts is perhaps due to the main phenolic constituents such as chlorogenic acid and luteolin that display antibacterial activity. Moreover, analysis of the different parts of V. agnus castus showed the presence of 25 compounds associated to phenols, where the registered compounds with high levels were vanillic acid, chlorogenic acid, hesperidin, luteolin, 3-hydroxybenzoic acids and 3,4-dihydroxybenzoic. The growth of five species of bacteria was inhibited by the extracts with MIC values ranging from 7.81 and 31.2 µg/mL (Berrani et al. 2021).

Fig. 4. Activity of V. agnus-castus extracted via SFE at SET (A) DET (B) against different microorganisms. Negative control (NC) (10% of DMSO), positive control (PC) nystatin (500 µg/mL) and ampicillin (1000 µg/mL)

Table 4. Values of MIC and MBC Besides the MIC/MBC Index of V. agnus-castus Extracted via SFE at SET (Sample code A) and DET (Sample Code B) against Different Microorganisms

The extract showed anticancer activity against SK-OV3 cells line, but extraction condition of the sample code B reflected better toxicity than the sample code A (Table 5 and Fig. 5). No toxicity was observed at 31.2 and 62.5 µg/mL of the extract of sample code A. Moreover, less IC₅₀ value was recorded, 164.51 ± 1.2 µg/mL for the extract of the sample code B than the IC₅₀ value of 209.02 ± 4.11 µg/mL for the extract of the sample code A. As presented in Table 5, the cytotoxic effect was linearly associated with the dose of the extract. Previously, Ohyama et al. (2003) reported the DNA fragmentation and apoptosis of SKOV-3 cells treated by V. agnus-castus extract, which may be attributed to amplified intracellular oxidation. Another type of cancer cells, namely MCF-7 breast cells was suppressed by seeds extract of V. agnus-castus (Sultan and Aşkın 2013). From the detected flavonoids in the extract of V. agnus-castus, daidzein was detected in high concentration. Hua et al. (2018) found that daidzein stimulated the morphological alteration in SKOV3 cells and mitochondrial apoptosis with IC₅₀ value of 20 µM, while it reflected high IC₅₀ value (100 μM) against normal ovarian cells. Data from Hamza et al. (2019) recorded ameliorative effects of Vitex agnus-castus extract on the syndrome of polycystic ovary via the modulation of lipid and hormonal profile in addition to oxidative stress. Furthermore, the promising effects of these constituents are comparable to metformin. As the dose of the extract increased, particularly the extract of the sample code B, the cancer cells of SKOV3 became shrunken, rounder, and detached from the substratum, which are vital morphological alterations linked with apoptosis (Fig. 5). The several changes in the treated MCF-7 breast cells by V. agnus-castus extract were observed by Sultan and Aşkın (2013) including condensation of chromatin, cell shrinkage, nuclear fragmentation, and visualization of membrane-linked apoptotic bodies.

Table 5. Cytotoxicity of V. agnus-castus Extract via SFE at SET (Sample Code A) and DET (Sample Code B) against SK-OV3 Cells

Management of inflammation is a critical agent of wound-healing stages because extreme inflammation minimizes healing of wounds. The anti-inflammatory influence of V. doniana may be assistance for wound repair. The present investigation showed the presence of chlorogenic acid in the V. agnus-castus fruit extracts, and according to Rohrl et al. (2017), this acid reflected good antioxidant and minimize the inflammations of tissues. In the present investigation, sample code B of the V. agnus-castus extract was tested for healing of wounds in vitro (Fig. 6 and Table 6) because it contains high concentration of active compounds. From the obtained results, treatment by V. agnus-castus extracts reflected wound healing. The indicated signs of wound healing involving migration rate (13.4 um), wound closure % (75.5 µm²), and area difference (489,000 %) were tabulated (Table 6) as a result of exposure to V. agnus-castus extract.

Fig. 5. Morphological changes of SK-OV3 cells exposed to various doses (31.25 to 1000 µg/mL) of extracted V. agnus-castus via SFE at SET (A) DET (B). Photos were imagined prior to 24 h cultivation at magnification 40X

Table 6. Healing Activity of V. agnus-castus extracted via SFE at Dynamic Extraction Time (Sample Code B)

Fig. 6. Descriptions of scratch exam showed the influence of V. agnus-castus (VA) extracted via SFE at dynamic extraction time (Sample code B) compared to control on the area of wounds at 0 and 48 h

Untreated cells exhibited less values of migration rate (12.8 µm), wound closure % (72.2 µm²), and area difference (468,000%). The mechanism of V. agnus-castus extracts may improve the healing stages through reducing the inflammation and oxidative stress. Previously, Nyiligira et al. (2008) mentioned that several species of Vitex, including V. agnus-castus, V. piramidata, V. pubescens, and V. cienkowskii, have been registered to be applied in traditional treatments of an extensive variety of ailments, such as venereal diseases, depression, allergy, malaria, skin diseases, wounds, and inflammation.

CONCLUSIONS

1. In the present study the dynamic extraction time was positively efficient relative to extraction yield except for flavonoid and phenolic compounds.
2. Moreover, anticancer activity against SK-OV3 cells, healing process, and antimicrobial activity against different microorganisms represented with inhibition zones, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values of the V. agnus-castus extract was highest at the extraction dynamic time of 60 min.
3. This study helps to authenticate the traditional application of V. agnus-castus particularly when extracted by SFE under the effect of extraction time as well as encourages investigators to further research the separated compounds from V. agnus-castus.

ACKNOWLEDGEMENT

This work was funded by the Deanship of Graduate Studies and Scientific Research at Jouf University under grant No. DGSSR-2023-01-02002.

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Article submitted: April 28, 2024; Peer review completed: June 8, 2024; Revised version received and accepted: June 10, 2024; Published: July 12, 2024.

DOI: 10.15376/biores.19.3.5793-5810