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BY 4.0 license Open Access Published by De Gruyter Open Access July 18, 2022

Phenolic contents, anticancer, antioxidant, and antimicrobial capacities of MeOH extract from the aerial parts of Trema orientalis plant

  • Sami Asir Al-Robai , Sami A. Zabin EMAIL logo , Abdelazim Ali Ahmed , Haidar Abdalgadir Mohamed , Abdullah A. A. Alghamdi and Aimun A. E. Ahmed
From the journal Open Chemistry


Medicinal plants contain phytochemical components of pharmaceutical importance, and Trema orientalis MeOH extracts are believed to have potential antioxidant and cytotoxic properties. This investigation explores the phenolic, antioxidant, and anticancer property of the methanol extracts of aerial parts of T. orientalis. The total polyphenol content (TPC) and the total flavonoid contents (TFC) were determined following standard methods. In vitro antioxidant property was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Cytotoxicity experiments were performed against eight cell lines and one fibroblast cell using the methylthiazolyldiphenyl-tetrazolium bromide assay. The antimicrobial activity assay was performed using the agar-diffusion method. Individual phenolic acids identified by GC/MS were examined in silico to estimate their drug likeness based on their structures. TPC and TFC were the highest in the leaf extract, with the strongest radical scavenging activity against ABTS (84.43%) and DPPH (79.60%) radicals. The highest cytotoxicity activity was exhibited by leaf (IC50 = 2.256 ± 0.85 μg/mL) and twig (2.704 ± 1.31 μg/mL) extracts against the HCT116 cell line, followed by bark (3.653 ± 0.05 μg/mL) and leaf (3.725 ± 0.30 μg/mL) extracts against the HT29 cell line. Clonogenicity resulted in a clear decrease of colony formation by HCT116 cells, suggesting a dose-dependent mode. In silico investigation suggested that phenolic acids detected have non-drug-like properties. Extracts showed antimicrobial inactivity.

1 Introduction

Since the time unknown, medicinal plants have been used to treat different infections. The World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population [1,2,3,4]. Medicinal plants are considered the prime source of chemical components of remedial importance and have numerous pharmacological activities, in addition to their role in traditional medicine and developing and discovery of new medicines [5,6]. One of the important medicinal plants is Trema Orientalis, which is utilized to treat numerous ailments by traditional practitioners [7].

T. orientalis, which is known as “charcoal tree,” is a species of fast-growing flowering and evergreen heavy branching plant belonging to the family Ulmaceae. The plant is known in native Saudi Arabia as “Shabarek” or “Shabrak.” It is characterized by high α-cellulose content, and for this reason, its wood is widely used by the paper industry [4]. It is considered a medicinal plant as its parts have been used for a long time to treat various diseases as antimicrobial, anti-malaria, anti-inflammatory, anti-arthritic, analgesic, and anti-worm agents [7,9,10,11]. The leaves are reported to be a general antidote to poisons, and the stem bark extract is used to control dysentery and is reported to reduce blood sugar levels [12,13,14]. The pharmaceutical effects may be due to the presence of biologically active constituents such as flavonoids, terpenoids, and saponins [11]. The methanol extract of T. orientalis leaves was reported to have a reasonable level of ability to alleviate the risk of Cd toxicity in rats [11].

This tree is found and distributed worldwide and can grow in different soil types and climate zones [8,15,16]. Saudi Arabia is one of the countries where the T. orientalis tree is grown widely. We have shown in our previous study that T. orientalis is one of the common plant species grown in the Al-Baha region located in the south-western part of Saudi Arabia [17]. The Al-Baha region is characterized by a dry and semiarid climate and has mountains with an elevation of 1,500–2,450 m above sea level, in addition to valleys and plains.

Some studies have reported on the botanical specification, phytochemical components, traditional usage, and pharmacological activity of T. orientalis from different locations in the world [7,11]. Salprima et al. [18] studied the antioxidant activity of the methanol extract of T. orientalis leaves collected from Seluma, Bengkulu Province in Indonesia and showed that the tested material exhibited an antiradical activity of 69.73% against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. To the best of our knowledge, we have not found any other study on the antioxidative or anticancer activity of the methanol extract of the aerial parts of the T. orientalis plant that is grown naturally in the Al-Baha region, Saudi Arabia.

In this investigation, we took into consideration the medicinal importance of the T. orientalis plant and the location variance, climate, type of soil, and other factors that are likely affecting the morphological characteristics and physical and chemical properties of the same species, thus affecting the pharmaceutical properties. Accordingly, this study is carried out with the aim of assessing the phenolic constituents and anticancer and antioxidant properties of the methanol extract of the aerial parts of T. orientalis. Additionally, we have performed an in silico study for the detected individual phenolic acid constituents in the plant parts for drug likeness.-

2 Materials and methods

2.1 Chemical standards and reagents

The Folin and Ciocalteu’s phenol reagent, gallic acid monohydrate (>98% pure), sodium carbonate, sodium nitrite, aluminum chloride, sodium hydroxide, DPPH (97%), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (>98%), methylthiazolyldiphenyl-tetrazolium bromide (MTT) (98%), N,O-bis(trimethylsilyl (TMS))trifluoroacetamide (BSTFA) (≥99%), potassium persulfate, and other solvents (analytical grade) were purchased from Sigma-Aldrich. All solutions used were freshly prepared before the experiments.

2.2 Plant materials

The aerial parts leaf, bark, twig and fruit samples from T. orientalis tree were collected during the month of October, 2020, from different localities around Al-Baha region, southwestern of Saudi Arabia, following guidelines for the collection of plant species [19]. The plant identification was carried out following the botanical criteria of Bock and Norris, 2016, and further was authenticated by a plant taxonomy expert Dr. Haidar Abd Algadir, Department of Biology, Albaha University and was archived with a voucher specimen herbarium number BUH-67 at the herbarium of the Faculty of Clinical Pharmacy, Albaha University, Albaha, Saudi Arabia. After cleaning and air-drying under shade, the collected samples were gently ground to coarse powder using an electric grinder (–2 mm mesh size) and stored properly for later use.

2.3 Total phenol, flavonoid, and antioxidant activity assessment

2.3.1 Preparation of extracts

The obtained coarse powder (10 g) of each plant part was mixed with 1,000 mL of MeOH solvent and undergone shaking at 120 rpm for 48 h. The obtained material (738–820 mL) for each plant part was filtered utilizing Whatman No.4 filter paper, concentrated to dryness using a rotatory evaporator, and then stored at 4°C for further usage.

2.3.2 Total polyphenolics contents

The total polyphenolics contents (TPC) were estimated in T. orientalis extract samples following the Folin–Ciocalteu assay technique with slight adjustment [20]. The individual extract (0.2 mL, 1 mg/mL) was added to 4.4 mL of deionized water and 0.4 mL of the Folin–Ciocalteu phenol reagent was added to the mixture, and the contents were mixed thoroughly. Double distilled water was utilized as control and for dilution purpose. The mixture was kept standing at room temperature (26°C) for 10 minutes, then a solution of Na2CO3 (20%) was added, and the whole mass was mixed properly and incubated away from the sunlight at a normal room temperature. With the help of UV-visible spectrophotometer (PD-303UV, Apel, Saitama, Japan), the absorbance was accurately measured at a wavelength of 765 nm. Each reading was repeated three times, and results were recorded as mean values with standard deviation (SD). Gallic acid was used as standard in order to compute the total phenolic content by standard curve (Y = 4.42x + 0.0102; R² = 0.9982). The TPC of the samples were shown as milligrams of gallic acid equivalent (GAE) per gram of the dry weight (DW) of the extract (i.e., mg GAE/g DW).

2.3.3 Total flavonoid content

Total flavonoid content (TFC) was measured using the reported aluminum chloride colorimetric assay [20]. Each extract (0.2 mL, 1 mg/mL) was added to a 10 mL volumetric flask containing 4 mL distilled water. To the flask, 0.3 mL of 5% sodium nitrite (NaNO2) was added, and after 5 min interval, 0.3 mL of 10% aluminum chloride (AlCl3) was added. Then, 2 mL of 1 M sodium hydroxide (NaOH) solution was added after 6 min. Finally, distilled water was added to complete the volume by up to 10 mL. After mixing the solution properly, the absorbance was recorded against a prepared reagent at a wavelength of 510 nm. TFC was calculated from the quercetin standard curve (Y = 4.826x + 0.0241; R² = 0.9992) and expressed as mg quercetin equivalent (QE)/g dry plant material.

2.3.4 Assessment of antioxidant property

The antioxidant property of the obtained MeOH extract was assessed in vitro following DPPH and ABTS assays [21]. These are the most commonly used methods, which are spectroscopic techniques based on quenching of the stabled colored free radical DPPH* or the radical cation ABTS*+ and caused a change in DPPH or ABTS+ absorbance after the addition of the test material which is often used as an index of the antioxidant property of the examined material. Ascorbic acid, propyl gallate, and Trolox compounds were used as effective antioxidant agents for comparison. Each assay is described in detail below. DPPH radical scavenging activity assay

The free radical DPPH is scavenged by the presence of naturally occurring antioxidant chemicals that may present in the plant extracts through the proton donation mechanism forming a reduced DPPH species. These species can be detected by measuring the decrease in the absorbance intensity of DPPH at 517 nm [22]. This decrease in the absorbance is considered the index of their antioxidant potential.

The antioxidant activity of the MeOH extracts of the T. orientalis plant parts was determined using the stable free radical DPPH according to the reported procedure with minor modification [23,24]. Briefly, 1 mm of each extract or standard with 0.5 mg/mL concentration was mixed with 2 mL of 0.01 mM methanol solution of DPPH. An equal amount of methanol and DPPH served as the experimental control. The mixtures were shaken vigorously and allowed to stand at room temperature for 5 min. The absorbance was measured for different samples and standards at 517 nm. The lower absorbance value of the sample marks the higher activity property of free radical scavenging. The ability of the antioxidant to scavenge the free DPPH radical is estimated as a percentage of discoloration of the DPPH in methanol solution. The percentage of DPPH scavenging (i.e., the antioxidant property) of the extracts was determined according to the following equation:

% of DPPH scavenging = Absorbance ( control ) Absorbance ( sample ) Absorbance ( control ) × 100 .

Each experiment was performed in triplicate, and the results recorded were the average of three separate measurements ± SD. ABTS*+ radical scavenging efficacy assessment

The ABTS+ radical scavenging efficacy evaluation was conducted following the reported procedure with some modifications [25]. In order to produce ABTS, free radical cations (ABTS•+) and 7 mM ABTS stock solution with 2.45 mM potassium persulfate (K2S2O8) solution were mixed, and the mixture was incubated for 15 h at room temperature (24 ± 2°C) in dark conditions. Methanol was used for dilution and absorbance was measured at 734 nm. For absorbance measurement, 1 mL of fresh ABTS*+ solution was incubated in the dark at 30°C with 3 mL (0.5 mg/mL) of the sample or standard for 30 min The mathematical expression used for computing ABTS*+ scavenging property is:

% of ABTS + scavenging = Absorbance ( control ) Absorbance ( sample ) Absorbance ( control ) × 100 .

Each experiment was performed in triplicate and the results recorded are the average of three separate measurements ± SD.

2.4 Determination of main individual phenolic acids-GC/MS analysis

In order to identify the phenolic acid constituents of each plant part, we have performed gas chromatography-mass spectrometry GC/MS analysis. Analyte separation, detection, and identification were performed by GC/MS on an Agilent (Palo Alto, CA) 6,890 N gas chromatograph equipped with an Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm film thickness) and 5,973 N mass selective detector. The oven temperature was ramped from 60°C (2 min) to 320°C (20 min) at a rate of 6°C/min. POP concentrations in filter extracts were determined in the selected-ion monitoring (SIM) mode using both electron impact and negative chemical ionization (NCI) mass spectrometry.

For analysis purpose, 500 mg of individual plant specimen was weighed accurately in a 60 mL tube and 5 mL MeOH was added to each sample and vortex for 1 min. Extraction was carried out by the sonication process using an ultrasonic bath system (Bransonic-CPXH digital ultrasonic bath, 40 kHz frequency, USA) for 30 min. Under the stream of nitrogen, the obtained extracts were concentrated to 1 mL. The cleaned samples were used for GC/MS analysis using the universal Agilent ALS method.

The derivatization step was performed before GC-MS analysis because phenolic compounds are non-volatile and were obtained as methanol extract, in which CH3OH is a polar protic solvent. For derivatization, the methanol crude extract of the plant materials (20 µL) of each sample was pipetted into a 2 mL amber vial and dried by evaporation under a mild stream of pure nitrogen at room temperature (24°C). Then, it was mixed with 30 µL of the derivatization reagent BSTFA, which is suitable for the derivatization of –OH groups of the phenolic compounds contained in the extract [26]. The mixture in the vial was capped, vortex, and kept for incubation at 70°C for 3 h. Samples were re-dried under a mild stream of N2 until dryness in order to get rid of the BSTFA excess, and Subsequently, re-dissolved in 200 µL hexane solvent. Derivatized specimens were analyzed by GC/MS applying SIM mode against calibration curves made from 10 individual phenolic standards.

2.5 In vitro cytotoxicity assay

For in vitro cytotoxicity assessment, the methanol extracts were prepared using the maceration method [22]. The powdered samples (10 g each) were extracted three times by maceration in 3 × 100 mL of MeOH solvent. Methanol was selected as the polar solvent in order to raise the level of the extraction yield by dissolving the polar compounds and minimizing the activity of oxidase enzymes. The solvent contains 1 g polyvinyl-pyrrolidone for detannification purposes. Extraction was performed for 48 h in the first time and then for 32 and 24 h and sequentially at room temperature (25 ± 3°C) in the dark, with manual shaking every 12 h for 5 min. The isolated extracts were filtered twice through bottle-top filter (Stericup 500 mL/1,000 mL, 0.45 µm PVDF filter membrane, Millipore, USA). Then, solvent was evaporated and the obtained concentrated extract of each part was stored separately in a refrigerator at 4°C for further use and analysis.

In vitro cytotoxicity of the MeOH extracts obtained from the aerial parts of T. orientalis plant as well as doxorubicin (0.01–10 µg/mL) as a drug reference were evaluated using the MTT colorimetric assay [25]. Each of the eight selected cell lines bought from ATCC, USA: MCF7 (human breast adenocarcinoma), A2780 (human ovary adenocarcinoma), HT29 (human colon adenocarcinoma), HepG2 (human liver cancer cell line), TK10 (human kidney renal cell adenocarcinoma), PC3 (human prostate cancer cell line), MDA231 (human breast cancer cell line), HCT116 (human colorectal carcinoma cell line, and MRC5 (normal human fetal lung fibroblast) were cultured in 96-well (3 × 103 cells/well) and incubated at 37°C overnight with 5% CO2, 95% air and 100% relative humidity. RPMI-1640 culture media (10% FBS, l-glutamine and 1% antibiotic–antimycotic) was used for all cells except MRC5, which was maintained in Eagles minimum essential medium (10% FBS). Every extract was set at a final concentration of: 0, 0.05, 0.5, 5, 25, 50 μg/mL in DMSO (0.1%) as media. Every experiment for every concentration was repeated thrice. Plates were incubated for 72 h in the presence of the extracts or its absence, then MTT solution was added to each well and plates were incubated for more 3 h. to allow reaction of MTT by mitochondrial dehydrogenases. Excess of MTT supernatant was aspirated and 100 μL of DMSO solvent was added to each well in order to dissolve formazan crystals formed.

The absorbance was recorded on a multi-plate reader (BioTek, Synergy HTX, USA) at 595 nm. The strength of the extract that causes growth inhibition was estimated by monitoring the concentration of the extract that gives rise to 50% cell growth inhibition (IC50) in comparison with that of the control cell growth (100%). This was done by comparing the amount of formazan dye compound (purple color) that produced by treated cells with that produced by untreated control cells. GraphPad Prism version 5.00 for Windows was employed for analysis.

2.6 Clonogenic cell survival assay

The clonogenic cell survival assay investigates the capability of single cancer cells to produce colonies and was conducted according to what was reported before [27]. Using two identical six-well plates, we have seeded colorectal human cell lines HCT116 in 2 mL of media at low density (5 × 102). Plates were left overnight at a temperature of 37°C for incubation and to permit attachment. Cells were processed with vehicle control and leaf extract (2.5, 5, and 7.5 µg/mL) and left for 72 h. Thereafter, the medium with each concentration was thrown out and replaced by 2 mL of fresh medium. The plates were inspected at every 2 days interval using a microscope. The cells of at least 50 in number were considered a colony after 14 days. After that, the medium was sucked out, and cells were washed with cold buffer solution phosphate-buffered saline (PBS), and then the cells were fixed using cold methanol for 5 min at room temperature. A staining mixture of 0.5% v/v methylene blue in methanol:H2O (1:1 v/v) was used to stain the cells for 15 min. The produced colonies were washed thoroughly using water, and PBS solution and plates were dried prior to colonies’ final counting.

2.7 In silico theoretical investigation

The individual phenolic acid constituents that were identified in the plant material extracts were theoretically examined in silico. The physicochemical properties such as lipophilicity, number of rotatable bonds, and topological polar surface area (TPSA) were computed in order to understand the transport characteristics of the tested compounds [28]. The absorption, distribution, metabolism, and excretion (ADME) pharmacokinetic processes that show how a chemical is processed in the body were investigated in silico for the targeted phenolic acid compounds using the Swiss ADME web interface (http// [29]. In addition, we have employed Lipinski, Ghose, Veber, Egan, and Muegge rules to predict the probable bioactivity of the tested phenolic acid compounds [29].

2.8 In vitro experimentation for antimicrobial property assessment

The microbial susceptibility of the four aerial parts (leaves, bark, twigs, and fruits) of the T. orientalis plant as well as tetracycline (70 µg/mL) as an antibiotic reference was assessed for their antimicrobial property against five microbial strains. The microbial strains used were two Gram-negative bacteria (Escherichia coli [ATCC35218] and Klebsiella pneumoniae [ATCC700603]), and two Gram-positive bacteria (Staphylococcus aureus [ATCC 25923] and Enterococcus faecalis [ATCC 29212]), in addition to Candida albicans (ATCC10231), the common pathogenic yeast as the fungal strain throughout all antimicrobial experiments. The assessment experiments were carried out in vitro at the Blood Bank Centre, Bisha City, Saudi Arabia. The agar disk-diffusion evaluation technique followed was in accordance with the Clinical and Laboratory Standards Institute methodology [29]. The growth medium for bacterial strains was Muller Hinton agar (MHA), and for fungal strains, Sabouraud dextrose agar (SDA) nutrient was utilized. Stock solutions of the tested plant parts’ methanol extracts were prepared by dissolving 1,000 µg of the extract of each part in 2.0 mL methanol solvent and used for experimental tests. Sterile filter paper discs (Whatman No.1, 6 mm in diameter) were placed on the surface of MHA and SDA and soaked in the targeted extracts. The reference antibacterial Augmentin drug was used for comparison. Bacterial strains were incubated for 24 h at 35°C and fungal strains for 48 h at 37°C. After the incubation period, we had measured the zones of complete inhibition (in mm) around every tested extract of each plant part that marks the property of the examined extract to harm or damage the bacterial or fungal strains used.

2.9 Statistical analysis

For statistical calculations and analysis, we used Graph Pad prism software version 0.5. The one-way ANOVA (analysis of variance), Dunnett’s post hoc test, and Tukey’s multiple comparison test were used to compute the statistical significance, and p ≤ 0.05 value was considered significant.

3 Results and discussion

The aerial parts of T. orientalis, leaves, bark, twigs, and fruits, were collected, treated, and finally extracted using methanol as the solvent. The total phenolic, total flavonoid, and individual phenolic acid contents of each part were estimated, and the crude extract of each part was explored for antioxidant and cytotoxicity properties.

3.1 TPC, TFC, and antioxidant activity

Polyhydroxylated phytochemicals such as phenolic acids and flavonoids are secondary metabolites that are highly abundant in plants. They are considered beneficial to human health as an important category of strong natural antioxidants because they can eliminate free radicals and provide some protection against the development of cancer [30,31]. Polyphenols are prime antioxidant active compounds that accompany bioactive molecules in plant materials. Therefore, to predict the antioxidant activity of natural products, it is very important to determine the total phenolic content in the selected plant material under investigation.

The total phenols found in the aerial parts (leaves, bark, twigs, and fruits) of the T. orientalis plant were estimated using a standard gallic acid curve. The resulting R 2 value indicates an accuracy of 99.82%. Significant differences (p ≤ 0.05) were observed among the studied plant parts in their total polyphenol contents (Table 1). Leaves exhibited a higher total phenolic value (20.65 GAE/g DW), followed by bark (7.54 GAE/g DW), and twigs (6.85 GAE/g DW), and the lowest was in fruits (3.84 GAE/g DW).

Table 1

Total phenolics, total flavonoid, and antioxidant activity in different aerial parts of T. orientalis

Plant part/standard Total phenolics (mg GAE/g DW) Total flavonoid (mg QE/g DW) DPPH (%) ABTS (%)
Leaves 20.65 ± 0.09a 6.73 ± 0.14a 79.60 ± 0.71a*** 84.43 ± 0.71aNS
Bark 7.54 ± 0.06b 1.88 ± 0.061b 22.06 ± 0.53c*** 58.54 ± 0.29c ***
Twigs 6.85 ± 0.08c 1.38 ± 0.10c 26.63 ± 0.50b*** 67.87 ± 0.56b***
Fruits 3.84 ± 0.10d 0.370 ± 0.05d 4.080 ± 0.19d*** 44.10 ± 0.91d***
Ascorbic acid 95.74 ± 0.96 92.86 ± 1.18
Propyl gallate 88.08 ± 1.35 82.30 ± 1.03
Trolox 84.91 ± 0.61 78.72 ± 0.51

Note: Results are means ± SD (n = 3); means within the same column which have no similar letters are significantly different (Tukey’s multiple comparison test, p ≤ 0.05). DPPH and ABTS values were compared with that of Trolox as a control using one-way ANOVA + post hoc test [p ≤ 0.001 (***); (NS) not significant statistically].

Flavonoids are also an important diverse class of plant compounds having a polyphenolic structure and are characterized by antioxidative, antimutagenic, and anticarcinogenic properties. For this reason, it is very important to determine the TFC in the selected T. orientalis tree material under investigation.

The linear regression equation is used to determine total flavonoid levels in the MeOH extracts of different parts of the T. orientalis plant. There were clearly significant differences in the level of total flavonoids among the investigated plant parts (Table 1). Similar to the total phenol contents, the level of total flavonoids was in the order leaves > bark > twigs > fruits with values of 6.73, 1.88, 1.38, and 0.37 mg QE/g DW, respectively.

The percent radical scavenging activity (% RSA) values in the case of DPPH and ABTS also varied significantly (p ≤ 0.05) among the examined extracts (Table 1). It is clear from the obtained values that the order in both assays was leaves > bark > twigs > fruits. Leaves showed the strongest activity against the ABTS radical (84.43%) and the DPPH radical (79.60%). The antioxidant activity of our examined material was compared with the standard known effective free radical scavengers ascorbic acid, propyl gallate, and Trolox as a positive control at the same conditions and concentrations as the examined materials. It was observed that the MeOH extract of the leaves has higher scavenging activity (84.43%) against ABTS than propyl gallate and Trolox (82.30 and 78.72%, respectively) and slightly less than ascorbic acid (92.86%) (Table 1). The percent radical scavenging activity against DPPH of the tested materials was less active compared to that of the standard used compounds, and the leaf extract showed a value (79.60%) close to that of Trolox (84.91%) against DPPH.

The DPPH radical scavenging activity of Trolox was significantly different (p ≤ 0.001) when compared with that of the four tested extracts. No significant difference was observed between the leaf extract and Trolox as the standard in their ability to scavenge the ABTS free radical in the reaction mixture.

By comparing with the results reported by Salprima et al., whose study reported an antiradical activity of 69.73% for the methanol extract of the T. orientalis leaves grown in Indonesia against DPPH free radicals [18], in our study, we obtained a higher antiradical activity of the methanol extract of the leaves against DPPH free radicals with a value of 79.60%.

The antioxidant activity is directly proportional to the level of polyphenols and flavonoids present in the tested materials, and hence, these observations explain the relationship between the polyphenols and flavonoid content and the antioxidant activity. The strong antioxidant property observed in the leaf extract followed by the bark extract was in agreement with that being leaves have the highest level of polyphenols and flavonoid content that consequently affects the degree of antioxidant activity. Hence, the leaves were more effective against the free radicals and exhibited the highest antioxidant property. This highest antioxidant activity is probably due to the presence of more abundant hydroxyl groups [30]. However, the fruits were the least, containing minimum polyphenol and flavonoid phytochemicals. It is believed that the antioxidant activity of polyphenols and flavonoids is mainly attributed to the phenolic OH groups that participate in redox reactions and the possibility of hydrogen transfer to free radicals causing neutralization of the free radicals responsible for the oxidative stress that causes dangerous diseases. Consequently, the more transfer of hydrogen atoms increases the % RSA [32].

3.2 GC/MS analysis for determination of main individual phenolic acids

For correlation between the chemical composition and biological activity, it is important to determine the main individual phenolic acids that may present in the obtained extracts. There is little information available in the literature regarding the phenolic composition of T. orientalis parts. GC/MS analysis was performed in order to determine the amount of individual phenolic acid compounds in different aerial parts (leaves, bark, twigs, and fruits) of the T. orientalis plant. The results obtained are presented in Table 2. GC/MS analysis revealed the presence of 10 different phenolic acid compounds in the tested parts of the plant. p-Hydroxybenzoic acid, which is a popular antioxidant of low toxicity, was dominant, with the highest level of 52 µg/g in leaves, followed by 25 µg/g in twigs. t-cinnamic acid has 29 µg/g in leaves, followed by sinapic acid and caffeic acid at 25 and 22 µg/g, respectively. These compounds are well-known antioxidants and have strong free radical scavenging properties with low toxicity [33,34,35].

Table 2

Main individual phenolic acids (µg/g) in different parts of T. orientalis

Molecule # Compound Leaves Bark Twigs Fruits
Molecule 1 t-Cinamic acid 29 8 10 10
Molecule 2 p-Hydroxybenzoic acid 52 15 25 17
Molecule 3 Vanillic acid 9 9 17 8
Molecule 4 3,4-Dihydroxybenzoic acid 8 10 17 8
Molecule 5 Syringic acid 10 12 19 9
Molecule 6 p-Coumaric acid 15 16 23 19
Molecule 7 Gallic acid 13 12 16 16
Molecule 8 Ferulic acid 16 15 17 15
Molecule 9 Caffeic acid 22 20 22 21
Molecule 10 Sinnapic acid 25 22 24 22

Some researchers have reported that syringic acid as a phenolic compound has an inhibiting effect on colorectal tumors in rats and it is proved and recommended as a potential therapeutic applicant for human ovarian cancer [36,37]. Caffeic and gallic acids were reported to induce morphological changes in breast cancer cells and were suggested as future antitumor agents [38]. It is also reported that p-coumaric acid has an effect against lung cancer and colon carcinoma developed at the adhesion and migration steps [39]. Moreover, it can inhibit the proliferation of human and mouse melanoma cells by inducing cell cycle arrest and inducing apoptosis [39].

The increased in vitro DPPH and ABTS radicals scavenging activity of leaves extract may be related to a significant amount of p-hydroxybenzoic acid, t-cinnamic acid, sinapic acid, and caffeic acid and hence a greater number of hydroxy groups which increases the reaction with the free radicals [30].

3.3 In vitro anticancer property

Eight human cancer cell lines and one normal fibroblast cell were utilized to assess the anticancer properties of the crude methanol extract of the different aerial parts of the T. orientalis plant under investigation. The anticancer effect of the methanol extract was estimated using an MTT assay and a morphological study. The IC50 values ± SD of the examined methanol extracts are presented in Table 3. The examined MeOH extracts were observed to show good anticancer properties against the selected cell lines. The obtained IC50 values for the leaf extract against A2780, HT29, HepG2, TK10, MDA231, and HCT116 cancer cell lines were significantly (p ≤ 0.001) high when compared with those of the normal cell line. On the other hand, the cytotoxic results of the leaf extract against the other two cancer cell lines, MCF7 and PC3, were not significantly different when compared with those of the fibroblast cell line. The cytotoxicity results of the bark extract against MCF7, HepG2, TK10, MDA231, and HCT116 cancer cell lines were not significantly different from the cytotoxicity result against the MRC5 cell line. Significant differences (p ≤ 0.05) exist among the tested extracts in their cytotoxic activity against MCF7, A2780, and MDA231 cancer cell lines.

Table 3

Inhibition concentration activity of extracts from aerial parts of T. orientalis against eight cancer cell lines and one normal fibroblast (MTT 72 h, IC50 ± SD μg/mL)

Plant part cell line Leaves Bark Twigs Fruits Doxorubicin
MRC5 34.75 ± 0.53 10.99 ± 1.02 55.97 ± 3.22 32.71 ± 1.05 0.74 ± 0.06
MCF7 33.59 ± 0.72bNS 9.671 ± 0.25aNS 38.31 ± 0.34c*** 13.81 ± 4.38a*** 0.01 ± 00
A2780 21.26 ± 1.36c*** 4.310 ± 1.46a*** 18.50 ± 0.57b*** 27.29 ± 1.16d** 0.02 ± 0.01
HT29 3.725 ± 0.30a*** 3.653 ± 0.05a*** 13.61 ± 2.57b*** 25.72 ± 0.62c*** 0.38 ± 0.03
HepG2 12.48 ± 0.74ab*** 10.26 ± 0.37aNS 17.02 ± 1.50c*** 13.30 ± 0.68b*** 1.16 ± 0.09
TK10 15.75 ± 1.08b*** 9.970 ± 0.23aNS 51.23 ± 2.76c* 14.41 ± 1.16b*** 0.71 ± 0.04
PC3 33.58 ± 0.81bNS 8.847 ± 0.40a* 55.43 ± 3.36cNS 12.16 ± 1.24a*** 0.60 ± 0.07
MDA231 6.232 ± 0.80a*** 9.983 ± 0.77bNS 13.75 ± 1.10c*** 8.791 ± 1.25ab*** 0.01 ± 0.00
HCT116 2.256 ± 0.85a*** 10.16 ± 1.36bNS 2.704 ± 1.31a *** 14.13 ± 1.11c*** 0.47 ± 0.05

Notes: Results are presented as mean with SD (±); n = 3. IC50 values were compared with the IC50 value of MRC5 normal cell in the same row using one-way ANOVA + Dunnett’s multiple comparison test, where p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), (NS), not significant statistically. Means followed by different letters in the same column are significantly different according to Tukey’s means test at p ≤ 0.05.

The modified guidelines from those of the US National Cancer Institute (NCI) for the cytotoxic activity of the crude extract, which are established by Srisawat et al., are as follows: IC50 < 20 µg/mL = highly active, IC50 21–200 µg/mL = moderately active, IC50 < 201–500 µg/mL = weakly active, and IC50 > 501 µg/mL = inactive [40]. Based on these criteria, our investigated extracts can be categorized as highly and moderately active extracts in terms of their activity on the tested cell lines. The bark extract was highly active (IC50 < 20 µg/mL) against all tested cancer cell lines.

3.4 Clonogenicity of leaf extract

It was observed in the cytotoxicity experiment that the MeOH leaves’ extract exhibited the highest cytotoxicity property against colorectal carcinoma HCT116 cell lines with a value of (2.256 ± 0.85 μg/mL) (Table 3). Therefore, the colonogenic survival assay was carried out in order to assure the growth inhibitory effectiveness of the leaves methanol extract against the HCT116 cells. The assay results obtained, as shown in Figure 1, revealed that the control cells (not treated with the MeOH leaf extract, i.e., at 0 μg/mL) formed colonies, which were nearly uniformly distributed in the plate. While the data showed that the cells treated with 2.5 and 5.0 μg/mL methanol leaf extract were reduced by nearly 30% and 50%, respectively. Finally, the data showed that the colony proliferative and formation number of HCT116 cells was significantly decreased (nearly by 98%) on using 7.5 μg/mL of leaves’ extract after an incubation period of 14 days and exhibited the strongest colonogenic activity. Moreover, the assay reveals that the cell growth inhibition effectiveness is a dose-dependent condition.

Figure 1 
                  Clonogenicity of HCT116 cells following 72 h treatment in a six-well plate with vehicle control, 2.5, 5, and 7.5 µg/mL. Experiments were repeated three times. Statistical differences compared to untreated control cells were assessed by one-way Anova with the Tukey’s post hoc multiple comparison test (p < 0.100 [*], p < 0.010 [**], p < 0.001 [***] was taken as significant).
Figure 1

Clonogenicity of HCT116 cells following 72 h treatment in a six-well plate with vehicle control, 2.5, 5, and 7.5 µg/mL. Experiments were repeated three times. Statistical differences compared to untreated control cells were assessed by one-way Anova with the Tukey’s post hoc multiple comparison test (p < 0.100 [*], p < 0.010 [**], p < 0.001 [***] was taken as significant).

3.5 In silico studies on the identified individual phenolic acid compounds

Considerable investigations have indicated that natural polyphenols have pharmaceutical activity, and the structure of these compounds has been utilized as a scaffold for developing and discovering new drugs [41]. In silico investigations are important and have a major role in discovering and developing new drugs, because in silico studies reduce the time, material and facilities factors required for discovering new materials for biological applications. This type of investigation can estimate the required pharmacokinetic parameters for ADME. Moreover, it can be supported through in silico assessment and estimating the physiochemical criteria.

3.6 Physicochemical properties

The calculation and measurement of physiochemical properties can assist in setting the priority of the suggested chemicals for inspection as possible successful medicines and give the permission for making tentative decisions in drug discovery [42]. The natural product phenolic acids detected in the investigated T. orientalis plant were investigated in silico to understand the compound transport properties. The results presented in Table 4 for in silico absorption percentage calculations indicated that the absorption percentage was in the range of 75.92–96.13% among the phenolic compounds, where compound 1 (t-cinnamic acid) was observed to have the highest absorption percentage of 96.13%. Furthermore, the scored TPSA values for the tested phenolic acid compounds were lower than the set limit of 140 Å2 polar surface area, suggesting that these chemical ingredients had appreciable permeability into the cellular plasma membrane [38]. The obtained coefficient of solubility (log S) values for all targeted phenolic acids were in the range of –2.37 to –1.64 (Table 4), which are more than the 4.00 value determined by the estimated solubility (ESOL) method on Swiss ADME for many known drugs available in the market [43]. These observations suggest that these compounds have high solubility.

Table 4

Physicochemical properties of the phenolic acid compounds

Molecule Fraction Csp3a No. of rotatable bonds No. of H-bond acceptors No. of H-bond donors Molar reactivity TPSAb M log P c ESOL log S d ESOL solubility (mg/mL) In silico % absorption
Molecule 1 0 2 2 1 43.11 37.3 1.9 –2.37 6.29 × 10−1 96.13
Molecule 2 0 1 3 2 35.42 57.53 0.99 –2.07 1.18 × 10+00 89.16
Molecule 3 0.12 2 4 2 41.92 66.76 0.74 –2.02 1.60 × 10+00 85.97
Molecule 4 0 1 4 3 37.45 77.76 0.4 –1.86 2.14 × 10+00 82.18
Molecule 5 0.22 3 5 2 48.41 75.99 0.49 –1.84 2.84 × 10+00 82.78
Molecule 6 0 2 3 2 45.13 57.53 1.28 –2.02 1.58 × 10+00 89.16
Molecule 7 0 1 5 4 39.47 97.99 –0.16 –1.64 3.90 × 10+00 75.92
Molecule 8 0.1 3 4 2 51.63 66.76 1 –2.11 1.49 × 10+00 85.97
Molecule 9 0 2 4 3 47.16 77.76 0.7 –1.89 2.32 × 10+00 82.18
Molecule 10 0.18 4 5 2 58.12 75.99 0.73 –2.16 1.54 × 10+00 82.78

Note: a = the ratio of sp3 hybridized carbons over the total carbon count of the molecule; b = TPSA (0 A2); c = lipophilicity; d = water solubility.

3.7 ADME properties and drug-likeness properties

In this research, pharmacokinetics is used to examine the time course of chemical ADME properties and decide the destiny of administration of these phenolic acids to the living organisms. Table 5 shows the results obtained from the in silico pharmacokinetic predictions for tested natural phenolic acid products. The obtained results revealed excellent absorption possibility from the intestine after oral administration as the gastrointestinal absorption values were high. It was very interesting that compounds 1, 2, 6, and 8 were able to penetrate through the blood–brain barrier (BBB), while compounds 3, 4, 5, 7, 9, and 10 were unable to penetrate the BBB. Also, the drug-likeness (log p) parameter (the logarithm of the partition coefficient of octanol/water) that employed to foretell the possible solubility of a likely oral drug with a value between −0.4 and 5.6 [29]. In this investigation, log K p estimated values for all tested compounds were between −6.02 and −5.69, which are below −0.4, suggesting weak lipophilicity and inability to break through the lipid bilayers of the cells and pointing out that the tested phenolic acid ingredients have poor drug-likeness. Furthermore, we applied the following rules of Lipinski, Ghose, Veber, Egan, and Muegge to foresee whether the tested phenolic acid ingredients are likely to be bioactive and evaluate qualitatively the possibility of these chemicals to be oral drug nominees. Moreover, these rules were used to differentiate between the drug-like and non-drug analogues [24]. Table 6 presents the results of the number of violations of these rules. All rules were satisfied by compounds 3, 5, 6, 8, 9, and 10, and these compounds exhibited zero violation against all of the rules except one that was shown against the Muegge rule. In contrast, compounds 1, 2, 4, and 7 have fulfilled all of the rules and showed zero violations against all rules except the Ghose rule where they showed two and three violations and one violation against the Muegge rule, where they showed one violation. All compounds in the screening processes exhibited good bioavailability with a score of 0.56, which is a sign that all of these phenolic compounds can reach the circulation system readily.

Table 5

Pharmacokinetic/ADME properties of the phenolic acid compounds

Molecule GI absorptiona BBB permeantb P-gp substratec CYP1A2 inhibitord CYP2C19 inhibitore CYP2C9 inhibitorf CYP2D6 inhibitorg CYP3A4 inhibitorh log Kp i (cm/s)
Molecule 1 High Yes No No No No No No –5.69
Molecule 2 High Yes No No No No No No –6.02
Molecule 3 High No No No No No No No –6.31
Molecule 4 High No No No No No No Yes –6.42
Molecule 5 High No No No No No No No –6.77
Molecule 6 High Yes No No No No No No –6.26
Molecule 7 High No No No No No No Yes –6.84
Molecule 8 High Yes No No No No No No –6.41
Molecule 9 High No No No No No No No –6.58
Molecule 10 High No No No No No No No –6.63

Note: a = gastro intestinal absorption; b = BBB permeant; c = P-glycoprotein substrate; d = CYP1A2: cytochrome P450 family 1 subfamily A member 2 (PDBHI4); e = CYP2C19: cytochrome P450 family 2 subfamily C member 19 (PDB4GQS); f = CYP2C9: cytochrome P450 family 2 subfamily C member 9 (PDB1OG2); g = CYP2D6: cytochrome P450 family 2 subfamily D member 6 (PDB5TFT); h = CYP3A4: cytochrome P450 family 3 subfamily A member 4 (PDB4K9T); i = Skin permeation in cm/s.

Table 6

Drug likeness predictions of the target compounds

Molecule Lipinski #violations Ghose #violations Veber #violations Egan #violations Muegge #violations Bioavailability score
Molecule 1 0 2 0 0 1 0.56
Molecule 2 0 3 0 0 1 0.56
Molecule 3 0 0 0 0 1 0.56
Molecule 4 0 3 0 0 1 0.56
Molecule 5 0 0 0 0 1 0.56
Molecule 6 0 0 0 0 1 0.56
Molecule 7 0 2 0 0 1 0.56
Molecule 8 0 0 0 0 1 0.56
Molecule 9 0 0 0 0 1 0.56
Molecule 10 0 0 0 0 0 0.56

The prediction of the interaction of the tested compounds with cytochrome CYP450 major isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) was performed in order to check which compound would cause significant pharmacokinetic interactions that lead to a toxic effect through inhibition of these isoenzymes. The CYP450 inhibition prediction results (Table 5) indicated that all compounds have no inhibition to all isoenzymes except compounds 4 and 7, which showed inhibition possibility to the CYP3A4 isoform only. Furthermore, the likelihood of the compounds to be the substrate of P-glycoprotein (P-gp) recommended that the compounds have potentiality to be a non-substrate of P-gp. These results suggest that the tested phenolic ingredients are mostly in favor of non-drug-likeness.

3.8 In vitro antimicrobial potentials

The obtained methanol extracts of each aerial plant part were subjected to in vitro assessment for the potential antimicrobial efficacy following the agar disc diffusion technique. The zones of complete inhibition were monitored and recorded in millimeters (Table 7). It is observed that all the tested extracts of the plant parts were inactive against the tested clinical bacterial and fungal strains and the microbial strains showed high resistance. This was in agreement with the in silico theoretical investigations, which suggested that the tested phenolic acid compounds detected in the plant material were more in favor of non-drug likeness.

Table 7

Antimicrobial observations for the obtained methanol extracts

Methanol extract Zone of inhibition (mm)
Gram-positive bacteria Gram-negative bacteria Fungus
S. aureus E. faecalis E. coli K. pneumonia C. albicans
Leaves 0 0 0 0 10 (Partially sensitive)
Twigs 0 0 0 0 0
Bark 0 0 0 0 0
Fruits 0 0 0 0 0
NC 0 0 0 0 0
Tetracycline 32 30 31 30 Not tested

4 Conclusion

The investigation of TPC and TFC values and the antioxidant and anticancer properties of the methanol extracts of the aerial parts of the T. orientalis plant collected from the Al-Baha region, Saudi Arabia, was carried out in this study. Additionally, determination of individual phenolic acids present in the leaves, bark, twigs and fruits and an in silico ADME investigation for individual determined phenolic acids were done. The results obtained revealed that both polyphenol and flavonoid contents decreased in the order leaves > bark > twigs > fruits. The antioxidant assessment results indicated that the leaf extract showed the highest antioxidant activity, which is consistent with the higher levels of polyphenols and flavonoids that were present in the leaves. Moreover, the leaf extract showed higher scavenging activity against ABTS+ free radical compared with propyl gallate and Trolox as reference materials. GC/MS analysis revealed the presence of 10 phenolic acid constituents in the methanol extract of the areal parts. Cytotoxicity assay experiments revealed that the methanol extract of the different tested parts of the plant had excellent activity against most of the tested cell lines and the highest activity was exhibited by the leaf and twig extracts against the HCT1116 cell line, followed by bark and leaf extracts against the HT29 cell line. The results of in silico investigation suggested non-drug-likeness for the examined phenolic acids present in the extracts. The antimicrobial assessment tests showed inactivity of all examined methanol extracts of the aerial parts of the plant.

Overall, the present investigation pointed out that the extracts of the T. orientalis plant grown naturally in Al-Baha region, Saudi Arabia, may have reasonable polyphenolic and flavonoid contents that showed in vitro antioxidant and anticancer properties, and we recommend further in vivo investigation.

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The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number: MOE-BU-7-2020.

  1. Funding information: This study was funded by the Deputyship for Research & Innovation, Ministry of Education, Saudi Arabia, under Grant number MOE-BU-7-2020.

  2. Author contributions: Conceptualization, Sami A. Alrobai, Abdelazim A. Ahmed, and Haidar A. Mohamed; methodology, Sami A. Alrobai, Abdelazim A. Ahmed, Ayman A. Ahmed, and Haidar A. Mohamed; software, S. A. Zabin and Ayman A. Ahmed; validation, Abdelazim A. Ahmed., Abdullah A. Alghamdi, and Ayman A. Ahmed; formal analysis, Abdelazim A. Ahmed and Sami A. Zabin; investigation, Sami A. Alrobai, Haidar A. Mohamed, and Abdelazim A. Ahmed.; resources, Sami A. Alrobai and Abdullah A. Alghamdi; data curation, Abdelazim A. Ahmed and Sami A. Zabin; writing – original draft preparation, Abdelazim A. Ahmed and Sami A. Zabin; writing – review and editing, Abdelazim A. Ahmed and Sami A. Zabin; visualization, Sami A. Alrobai, Abdelazim A. Ahmed, Abdullah A. Alghamdi, and Haidar A. Mohamed; supervision, Sami A. Alrobai; project administration, Sami A. Alrobai; funding acquisition, Sami A. Alrobai.

  3. Conflict of interest: The authors declare no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.


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Received: 2022-05-18
Revised: 2022-06-13
Accepted: 2022-06-22
Published Online: 2022-07-18

© 2022 Sami Asir Al-Robai et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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