Antioxidant activity and total phenolic compounds of Commiphora gileadensis extracts obtained by ultrasonic‐assisted extraction, with monitoring antiaging and cytotoxicity activities

Abstract Commiphora gileadensis (C. gileadensis) has been identified and linked with various health benefits and pharmaceutical potential for its phytochemical activities and chemical constituents. This study aimed to evaluate ultrasonic‐assisted extraction (USE) technique for total phenols content from C. gileadensis leaf compared to the hydrodistillation extraction (HDE). Our results showed that the USE operating conditions were identified as: MeOH·H2O solvent‐to‐fresh sample ratio of 80:20 (v/v); ultrasonic power/frequency at 150 W/20 kHz; and a temperature of 40 ± 1°C; subjected to acoustic waves intermittently for a calculated time (5 min) during the total programmed time of 12 min. The USE exhibited (118.71 ± 0.009 mg GAE/g DM) more amounts of all phenols than HDE (101.47 ± 0.005 mg GAE/g DM), and antioxidant (77.78 ± 0.73%, 75.27 ± 0.59% scavenging inhibition of DPPH), respectively. Anti‐aging and Cytotoxicity activities were investigated. The results of biological evaluations showed that the crude extracts of C. gileadensis significantly extended the replicative lifespan of K6001 yeast. In addition, in vitro cytotoxicity against the HepG2 cell line showed significant anticancer activity, and approximately 100 μg/mL is required to decrease viability compared with that of the control. This study is proven for a larger scale to extract and isolate compounds of C. gileadensis for potential utilization in the pharmaceutical industry. In conclusion, advanced methods afford an extract with high activity in the biological properties of the extract.


| INTRODUC TI ON
Recently, various botanical extracts have gained special attention as natural sources of antioxidants and antimicrobiotic agents due to the potential toxicity of synthetic antioxidants and antibiotic resistance (Ielo et al., 2022). Phytochemicals such as phenolic compounds are a large group of substances and are found in large quantities in vegetables, fruits, spices, and seeds because they are considered potential antioxidant agents, and their role in the pharmaceutical and food industry and the chemical prevention of disease has become an area of active research in many areas (Maqsood et al., 2020). It has been shown that oxidative reactions are involved in aging and the development of many diseases. Therefore, it has been suggested that antioxidant molecules may slow the aging process, disease progression, and extend lifespan (Umaru et al., 2019).
Scientific research has focused on plant extracts as natural sources of antioxidant compounds (Kyzioł et al., 2021;Pirzadeh et al., 2021). Several studies have demonstrated the potential applications of medical, pharmaceutical, food industries, and cosmetics of secondary plant metabolites (Klimek-szczykutowicz et al., 2020).
More than 60% of the overall time is spent on the sample preparation stage, so choosing the proper extraction method is very essential (Yahya et al., 2018). The extraction of bioactive compounds from plant sources can be improved with ultrasound technology assistance. The phenomenon of cavitation bubble collapse caused by acoustic and radiation shocks is responsible for the intensification of mass transfer, which facilitates the solvent to flow into plant tissues and thus increases the extraction efficiency of phenolic compounds (Kumar et al., 2021;Ojha et al., 2020).
Also, unlike conventional extraction methods like soaking and aqueous distillation, modern techniques can utilize the chemical and physical properties of the plant cell (such as osmotic pressure and water activity), which differ significantly when applied compared to conventional methods. Therefore, ultrasonic technology can increase the extraction yield, reduce chemical risks, and shorten the processing time (Švarc-Gajić et al., 2013;Wang et al., 2012).
In the process of phenolic compound extraction, conventional extraction techniques such as maceration, percolation, and Soxhlet have been found to be extensively applied due to the simplicity of their operation and the simple equipment that is needed. Usually, extraction is carried out using organic solvents, which require a large volume of solvent and a long extraction period. In contrast, water is utilized as a solvent in the hydrodistillation and decoction methods. However, long extraction times, high amounts of organic solvents, and high energy consumption could lead to low yields and degradation of the compounds (Cannavacciuolo et al., 2023;Jha & Sit, 2022).
Recent studies have revealed that ultrasound-assisted, supercritical fluid and microwave-assisted extraction techniques are environmentally friendly extraction methods (Alahmad et al., 2023).
Ultrasound-assisted extraction is one of the simplest and most costeffective technologies available for improving extraction efficiency, decreasing extraction time, and enhancing overall effectiveness of processing (Usman et al., 2022). The process of ultrasound-assisted extraction (USE) involves the formation of bubbles by a process called "acoustic cavitation," which is facilitated by ultrasonic waves' ability to cause expansion and compression cycles (Soltani Firouz et al., 2022). Cavitation forces create a localized pressure that ruptures cell walls in the matrix when these bubbles burst, releasing their contents (Jadhav et al., 2023). Therefore, an ultrasound-assisted technique may extract phenolic compounds quickly and easily.
Most plant sources, including those in the Burseraceae family, contain polyphenolic compounds or their derivatives. The Burseraceae family has promising potential for use as antioxidants and antimicrobials in the pharmaceutical and food industries (Afonso et al., 2018;da Silva et al., 2022;Emmanuelle Sika et al., 2021;Ifejirika et al., 2022).
Plant phenols are essential because they are low-molecular-mass antioxidants that can be obtained from food (Nadeem et al., 2022).
In vitro and in vivo, several plant phenols demonstrated antibacterial or antioxidant activity (Prior, 2003). The intensity of the antioxidant activity depends on many factors, such as the number of hydroxyl groups attached to the aromatic ring (Mercado-Mercado et al., 2020), and the number and locations of the double bonds in the molecule (Singla et al., 2019).
The objective of this study is to evaluate the antioxidant activity of three vital Yemeni plant species, including Commiphora gileadensis, Indigofera spinosa, and Salvadora persica (Table 1), and the total phenolic content (TPC) in the extracts of these plants by comparing the ultrasonic-assisted technique with the hydrodistillation method to choose the best plant with the highest of total phenolic content pharmaceutical industry. In conclusion, advanced methods afford an extract with high activity in the biological properties of the extract.

K E Y W O R D S
anti-aging, Commiphora gileadensis, extraction, HepG2 cells, hydrodistillation, ultrasonic assisted TA B L E 1 Description of collected plant species investigated in this study. and the highest antioxidant capacity for subsequent studies as study the anti-aging activity and cytotoxicity to better contribute to the promotion of the use of natural compounds as an important and safe.

| Extraction procedures
Two extraction techniques were employed for hydrodistillation extraction (HDE) and ultrasonic-assisted extraction (USE).

Equation
(1) was used to determine the extraction yield (%):

| Hydrodistillation extraction (HDE)
To study the antioxidant activities and TPC of the three selected medicinal plants, we used hydrodistillation extraction (HDE) as a conventional method to select the plants with high antioxidant and TPC. Upson et al. (2000) described the procedure followed with minor adjustments. Briefly, hydrodistillation was performed in a Clevenger-type apparatus. For each plant sample, 4 g was added in a 500 mL hydrodistillation apparatus flask and dissolved in 100 mL of MeOH·H 2 O at a ratio of 80:20 (v/v). All prepared samples were heated for 60 min at 60°C. The supernatants were centrifuged for 10 min at 5000 rpm and 4°C after being filtered using Whatman paper No. 1. Also, filtering was repeated using a Buchner funnel and Whatman No. 1 paper under a vacuum. The volume was filled up to 100 mL with deionized distilled water and kept at 4°C until the next analysis.

| Ultrasonic-assisted extraction (USE)
The ultrasonic power/frequency at 150 W/20 kHz was tuned for the USE operating conditions. In a coated glass container, with magnetic stirring, 100 mL of MeOH·H 2 O at a ratio of 80:20 (v/v) was used to dissolve 4 g of plant powder. The total programmed time was 12 min, and the mixture was treated to sonic waves intermittently for a considered time of 5 min. The temperature was maintained constant at 40 ± 1°C using a thermostatic external cold-water bath. After that, filtration and solvent removal were carried out as shown in item (Section 2.3.1).

| Determination of total phenolic content (TPC)
As Rashed et al. (2016) described, Singleton and Rossi's colorimetric oxidation reaction technique was used to determine the TPC of plant powder extract. The results were presented as mg gallic acid equivalents per gram of dry matter (mg GAE/g DM), using the standard curve of gallic acid (R 2 = .99; Figure 1).

| Determination of antioxidant activity using the DPPH
The antioxidant scavenging effect of extracts was assessed based on the DPPH assay (Rashed et al., 2016), according to Rashed et al. (1) Yield % = (Weight of dried extract ∕ Weight of sample) × 100 F I G U R E 1 Calibration curve for gallic acid (mg/g of dry extract) for three plants.
without modification. The results were expressed according to the following equation: where A is the optical density of the control sample and B is the optical density of the sample. TBHQ served as a positive model at 120 mg/L. There were three duplicates of each treatment.

| Lifespan assay of C. gileadensis
Based on Weng et al. (2010), the bioassay method was used to study the anti-aging activity. A yeast strain (K6001) was grown in YPGalactose or YPGlucose mediums. Galactose (3%), hipolypeptone (2%), and yeast extract (1%) were used to prepare YPGalactoses, while glucose was used to prepare YPGlucoses, which included 2% glucose instead of galactose. Agar plates were prepared by adding agar to the medium at a 2% concentration. To carry out bioassays, the yeast peptone galactose medium was used to cultivate the K6001 yeast strain for 24 h with shaking. After 24 h, centrifugation was performed on the yeast strain medium. The phosphate buffer solution (PBS) was used three times to rinse the diluted yeast pellets.
Around 4000 cells were injected into agar plates containing various substances after the cells had been diluted. The plates were kept at 28°C in an incubator for the next 2 days. After 48 h, the agar plate containing the yeast cells was examined under a microscope. Each of the 40 microcolonies randomly selected from each plate was then analyzed and counted the number of daughter cells.

| Cell viability assay of C. gileadensis
Measurement of cell viability was determined using the HepG2 cells.
In the assay for cell viability, 100 mL of DMEM was added to 96-well plates, where 1 × 10 4 cells per well were cultured overnight. C. gileadensis extract was added to cells in different dilutions (1, 10, and 100 μg/mL), and cultured at 37°C for 24 h with 5% CO 2 . Each well was incubated for 1 h at 37°C with 10 μL of CCK-8 reagent before readings were taken at 450 nm with an optical microplate reader (Enspire2300).

| Statistical analysis
All results were reported as a mean ± standard deviation after three replicates of each test. The SPSS 22.0 (SPSS Inc.) software program on Microsoft Windows 10 was used to conduct one-way analysis of variance (ANOVA) utilizing Duncan's new multiple-range test to determine statistically significant differences between means.

| Extraction yield (%)
Extract yield of C. gileadensis, I. spinosa, and S. persica leaves was prepared by hydrodistillation extraction and ultrasonic-assisted extraction methods. The extraction yield (mass of extract/dry matter) was employed as an index of the extraction methods' effects. Based on the product obtained after evaporation, the yield percentage of the extracts of the three Yemeni medicinal plants was determined (Table 2; Akkol et al., 2011).
The quantitative estimation of the percentage yield of various crude extracts of the plants is calculated as the weight (g) of crude extracts obtained from 4 g dried leaves. The results of the percentage yield are adequately described by Equation (1).
Among the hydrodistillation extracts of all three plants, the yield of C. gileadensis (20.25%) showed the highest yield percentage, and the lowest yield percentage was observed in the extract of I. spinosa (15.75%) among the three plants studied by hydrodistillation extraction. The yield of S. persica was 18.25%. While the ultrasonic extracts of C. gileadensis (11.00%) showed the highest yield percentage, the lowest yield percentage was observed in the extract of S. persica (6. 25%) among the three plants studied by ultrasonicassisted extraction. The yield of I. spinosa was 6.75% ( Table 2).
The extract yield of I. spinosa and S. persica with the conventional method was about 2.6 times higher than that of an advanced method for the same plant. This could be due to the more vital polarity of water, as well as the fact that at elevated extraction temperatures, and in the case of HDE, water has a similar dielectric constant to organic solvents such as methanol and acetonitrile. In the case of HDE, the extract yield increased with the time of extraction by the conventional method. The extract yield of C. gileadensis by ultrasonic-assisted method was lower than the maximum extract yield obtained by the conventional method may be due to the time of extraction.

| Effect on TPC of three plants
Polyphenols In recent years, the demand for modern extraction technologies that are ecologically friendly, more efficient, and faster than conventional methods has increased. Many studies have compared modern techniques for extracting medicinal plants, either among themselves or with conventional methods (Jovanovic et al., 2017).
Although modern techniques offer several benefits for extracting biomolecules from various plants, notably concerning extraction time, solvent consumption, and extraction yields, the best methods for extracting bioactive compounds need to be carefully evaluated (Fierascu et al., 2020;Herrero et al., 2015).
In this work, the TPC of the dried leaves of three plants (C. gileadensis, I. spinosa, and S. persica) in Yemeni's system of medicine has been investigated and determined as shown in As well as a rise in TPC, an increase in solubility of solute and solvent diffusivity may have resulted from an increase in temperature. Due to the sonication-generated acoustic cavitation bubbles increasing the extraction temperature and destroying the plant cell wall, more phenolic compounds were released into the solution matrix (Altemimi et al., 2016).
The process of extraction is the major difference between ultrasonic extraction and hydrodistillation. The ultrasonic extraction method utilizes high-frequency sound waves to generate cavitation in the solvent, which helps break down the plant material's cell walls to extract the bioactive compounds. This is due to ultrasonic extraction's ability to extract volatile and non-volatile compounds from the plant material (Dash et al., 2021). TPC extraction from C.

| Antioxidant activity using (DPPH) radical scavenging
Antioxidants are substances that can scavenge free radicals and prevent the oxidative damage they generate in cells. Lowering oxidative damage is very important for preventing cancer and other diseases (Mfotie Njoya, 2021).
In comparison to C. gileadensis, I. spinosa, and S. persica, extracts of C. gileadensis had stronger scavenging values, when tested at a concentration of 100 μg/mL, the extracts of C. gileadensis had antioxidant activity of 77.8%, while the extracts of the other two plants were less effective. One explanation for these results is that radical-scavenging capacity depends not just on the redox potentials of compounds but also on their hydrogen-donating ability (Lucarini et al., 1999). The high polyphenol content may be a contributing factor. Other investigations have found a similar link between antioxidant activity and phenolic concentration (Apak et al., 2007;Ricci et al., 2006).
Many functional groups, such as hydroxyl and carbonyl groups, have been linked to antioxidant activity in these extracts (Galati & O'Brien, 2004;Payet et al., 2005). The DPPH assay revealed that several extracts exhibited high levels of antioxidant activity. The efficiency of antioxidants is mostly dependent on the concentrations of the extracts. Their ability to free radical scavenger was ranked in the following ascending order: S. persica ˂ I. spinosa ˂ C. gileadensis.
Hydrodistillation extracts showed that extracts of C. gileadensis were fairly close inhibitors of DPPH scavenging to reference antioxidants such as TBHQ ( Table 3) Ultrasonic-assisted extraction has been shown to be an effective method for improving the yield of antioxidants from C. gileadensis compared to the HDE method. This is because the ultrasonic energy helps break down the plant material's cell walls, which increases the availability of antioxidants for extraction (Mounika et al., 2022).
However, the amount of antioxidants obtained through ultrasonicassisted extraction depends on extraction parameters such as ultrasonic energy duration, frequency, and intensity, as well as the type and amount of solvent used. (More & Arya, 2021). Extracting antioxidants from C. gileadensis using ultrasound-assisted extraction is a promising approach. Although the antioxidant yield of C. gileadensis leaves was increased through the use of ultrasound-assisted extraction, the mechanism may involve a combination of physical and chemical effects, including cell wall disruption, solvent permeation, temperature control, and enhancement of chemical reactions.
These effects enhance the mass transfer of the antioxidants from F I G U R E 2 Comparison of TPC obtained by HDE and USE. F I G U R E 3 Comparison of DPPH obtained by HDE with USE.
the plant material into the solvent, leading to a greater yield of antioxidants compared to HDE method (Dash et al., 2021;Khadhraoui et al., 2021).

| Anti-aging effect
Aging is a biological fact and an inevitable process that has its way of occurring beyond human control, which increases the prevalence of age-related illnesses.
The majority of anti-aging agents are derived from plants, such as resveratrol. The K6001 yeast strain was used to build a convenient bioassay system to evaluate the anti-aging activity of natural product extracts. The anti-aging activity of the samples was investigated by counting the number of daughter cells produced before the death of yeast mother cells. The data analysis was done through the statistical software of SPSS, 40 data of each sample were analyzed, the average yeast life of each group was calculated, and a one-to- one T-test was carried out between each group and the negative control group. The p-value determined the active size of the sample.
Resveratrol was employed as the positive control group, while untreated was used as the negative control group.
As shown in Figure 4, the result revealed that C. gileadensis compared with the negative control, the replicative lifetime of the K6001 yeast strain increased significantly. The average lifespan of the negative control was 7.55 ± 0.48. In comparison, the K6001 at 30 μg/mL of C. gileadensis (9.15 ± 0.62) increased significantly, whereas the C.

| In vitro cytotoxicity
In 2018, liver cancer had the third-highest cancer-related deaths; by 2020, it had increased to the second-highest mortality rate worldwide (Cao et al., 2021). Natural products are one of the important alternatives for drugs against cancers, including liver cancer. Many phenolic chemical prevention studies focus on liver cell response, as the liver is the primary site for xenobiotic metabolism (Arora et al., 2019;Nardo et al., 2021). The human hepatoma cell line (HepG2 cells) is a good model for studying the xenobiotic metabolism of microorganisms in vitro and liver toxicity because they maintain many specialized functions characteristic of normal human hepatocytes (Štampar et al., 2019). The HepG2 model has also been widely used in biochemical and nutritional studies where many compounds and conditions have been tested using minor intravariability (Ding et al., 2019).
The CCK-8 assay was used to investigate in vitro cytotoxicity against the HepG2 cell line at various concentrations of C. gileadensis extract (1, 50, and 100 μg/mL). After 24 h of treatment, the cell viability was assessed. Figure 5 shows the substantial anticancer effect of the C. gileadensis extract, with a concentration of 100 μg/mL required to reduce viability compared with that of the control, but C. F I G U R E 4 Effect of C. gileadensis (Cg) on the replicative lifespan of the K6001 yeast strain. Average lifespan of K6001 was as follows: control (7.85 ± 0.34); resveratrol at 10 μg/mL (9.225 ± 0.53*); and Cg at 10 μg/mL (7.9 ± 0.36) and at 30 μg/mL (9.125 ± 0.62*) (*p < .05, compared with the control).
In previous studies, aliphatic alcohol glycosides, triterpenoids, sesquiterpenoids, and several new cycloartane types were isolated from C. gileadensis, and the cytotoxic effect of C. gileadensis, especially against human prostate, liver, and cervical cancer cell lines, was

| CON CLUS ION
In sum, following the use of different extraction methods, the ultrasonic-assisted extraction employing an ultrasonic device with direct agitation resulted in the highest polyphenols with higher antioxidant activity compared to conventional extraction. Direct sonication with a probe system was more efficient than conventional extraction. The values of total phenol extracted with ultrasonicassisted extraction under the following conditions are as follows: MeOH·H 2 O solvent-to-fresh sample ratio of 80:20 (v/v); ultrasonic power/frequency at 150 W/20 kHz; and the temperature of 40 ± 1°C; with subjected to acoustic waves intermittently for a calculated time (5 min) during the total programmed time of 12 min; were higher than conventional extraction (60°C, 60 min) with a significant shortening of processing time. Furthermore, based on the findings of this study, C. gileadensis demonstrated promising bioactivity. They can thus be considered a potential plant-based polyphenol agent for anti-free radicals, anti-aging, and HepG2 protection. Further studies using more technical methods to elucidate the exact constituent (s) responsible for these benefits without side effects are required in order to approve and expand these findings.

ACK N OWLED G M ENTS
The authors thank the School of Pharmaceutical Science at Nanchang University for the administrative and technical assistance. The authors also appreciate Mrs. Sara Al-Mahrooq (Faculty of Languages, University of Aden) for the linguistic revision of this manuscript.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

E TH I C S S TATEM ENT
This article does not involve humans or animals.

CO N S E NT TO PA RTI CI PATE
All the co-authors participated in the preparation of this manuscript.