Phytochemical analysis, antioxidant, and antitumor activity of Ligustrum ovalifolium leaves grown in Jordan: an in vitro and in vivo study

Asma Ismail Mahmod; Muna Oqal; Ali Muharrif Khalid; Fatma U. Afifi; Wamidh H. Talib

doi:10.3897/pharmacia.71.e111517

Abstract

Ligustrum ovalifolium (family Oleaceae) is a flowering plant with reported anti-inflammatory, antioxidant, hypotensive, and hypoglycemic activities. The present study investigated the phytochemical components and biological activities of L. ovalifolium leaves. Results showed that ethyl acetate extract has the highest potential to reduce cell growth in HeLa, T47D, and MDA-MB-231 cell lines with IC₅₀ values of 0.047, 0.07, and 0.072 mg/mL, respectively. Based on the LD₅₀ value, the tumor-bearing mice were IP injected with 12.5 mg/kg of ethyl acetate extract. Tumor growth was significantly reduced (-43.1%) compared to the control group. According to the LC-MS analysis, Apigenin-7-O-glucoside was the flavonoid with the highest percentage value in L. ovalifolium leaves. DPPH assay exhibited antioxidant activity of ethanol and water extracts with the percentage of scavenging around 91% at a concentration of 200 µg/mL. As well, the assessment of liver and kidney functions of the experimented animals showed no toxicity effect compared to the control result. All things considered, the outcomes of this study revealed an antitumor potential of L. ovalifolium leaves extracts, hence this activity may arise from the presence of different potent flavonoids and the antioxidant potential. Nevertheless, further investigations are needed to determine the targets and signaling pathways that are affected by L. ovalifolium leaves extracts.

Keywords

L. ovalifolium leaves, antitumor activity, LC-MS analysis, MTT assay, Apigenin-7-O-glucoside

Introduction

For centuries, plants have been a rich source for the discovery of potent pharmacologically active compounds, used in the treatment of different diseases (Khan et al. 2019, 2022). The biologically active principles of the plants, the so-called secondary metabolites are known to exert antioxidant, anti-inflammatory, antitumor, cardio-protective, antimicrobial, and several other activities (Leitzmann 2016). Accordingly, plants and herbs are promising sources for developing new remedies, in particular for the treatment of serious or complicated diseases such as cancer (Mohammed 2019). Cancer is considered one of the main causes of death worldwide (Cabasag et al. 2022; Kocarnik et al. 2022; Morgan et al. 2023). Statistically, more than 19.3 million new cancer incidences and about 10 million cancer deaths have been recorded in 2020 (Sung et al. 2021). Despite the remarkable advances in cancer therapy still, there are many obstacles needed to be solved for comprehensive success in cancer treatment (Khan et al. 2022). On the other hand, different phytochemicals such as flavonoids, phenolic compounds, terpenoids, phytosterols alkaloids, sulfides, and others have shown potential antitumor properties (Avato et al. 2017; Joshi et al. 2017; Talib et al. 2020; Mahmod et al. 2022; Talib et al. 2022b). In this context, numerous extracts from herbs have been investigated for their possible antioxidant and antiproliferative properties to inhibit tumor cell growth using accepted in vitro and in vivo models. Promising results for many plant species are reported (Shu et al. 2010; Mahmod and Talib 2021; Al Kury et al. 2022). Certain plant secondary metabolites have been reported to prevent cancer cell proliferation, invasion, and metastasis as well as to reduce chemo-resistance by increasing tumor cell sensitivity to the treatment (Mitra and Bhattacharya 2020; Talib et al. 2022a, 2022b).

Ligustrum ovalifolium Hassk., known by the common names “Korean privet”, “California privet”, “garden privet”, or “oval-leaved privet”, belongs to the olive family Oleaceae (Wang et al. 2009; Moldovan et al. 2018). It is a semi-evergreen shrub with thick, fleshy leaves and dark purple to black fruits (Hasskarl 1844; Yamada et al. 2014). It is widely distributed in East Asia and is usually cultivated as an ornamental plant in many other countries, including Jordan (Moldovan et al. 2018). Few studies reported the chemical compositions and biological activities of L. ovalifolium. Machida et al. isolated acylated triterpenoids from L. ovalifolium flowers (Machida et al. 1997). Secoiridoid glucosides with hypotensive activities were identified in L. ovalifolium leaves, grown in Egypt (Hosny et al. 2009). Also, other researchers discovered anti-inflammatory, and antioxidant, effects using. ovalifolium extracts (Hosny et al. 2009; Kim et al. 2011, 2012). Since this species with certain pharmacological activities very widely cultivated in Jordan, the present study was designed to investigate L. ovalifolium leaf extracts phytochemically, and biologically. The findings of the HPLC-MS screening, antioxidant and in vivo and in vitro evaluation of the antiproliferative efficacy will be detailed in the current study. Additionally, total phenol and total flavonoid concentrations were determined.

Materials and methods

Plant collection

The fresh leaves of the L. ovalifolium plant were collected from the campus of the Applied Science Private University (ASU) (Amman, Jordan) in March-April 2022. The taxonomic identity of the plant was authenticated by Prof. Fatma Afifi using herbarium samples and descriptive references. Herbarium samples are deposited in the Department of Pharmaceutical Chemistry and Pharmacognosy (ASU) (FMJ-OLE1).

Extracts preparation

Fresh leaves of L. ovalifolium were soaked separately in three solvents with different polarities (ethyl acetate, 70% ethanol, and water) and heated until boiling with continuous stirring (1:10 w/v). The extracts were covered and kept overnight at room temperature. After filtration, the solvents were evaporated using a rotary evaporator until dry. The obtained dried crude extracts were kept at -20 °C until use.

Determination of total phenol content

Total phenol content was determined according to Folin-Ciocalteu (F-C) method (Folin and Ciocalteu 1927; Aboalhaija et al. 2022; Mahmod and Talib 2023). To calculate the concentration of total phenol content, a calibration curve standard of gallic acid (400–3.125 µg.mL was used. The result was expressed in terms of mg of gallic acid equivalent per gram of dry extract (mg GAE/g). Methanol was utilized as a blank and each reading was taken in triplicate.

Determination of total flavonoid content

Total flavonoid content was investigated using the Aluminum chloride (AlCl₃) method as described earlier (Hossain and Rahman 2011; Mahmod and Talib 2023). The result was expressed in terms of mg of rutin equivalent per gram of dry extract (mg RE/g). Each reading was taken in triplicate.

Phytochemical analysis of ethyl acetate extract using LC-MS analysis

To prepare the sample, ethyl acetate extract was dissolved in 2 mL dimethyl sulfoxide (DMSO) completing the volume up to 50 mL with acetonitrile solvent. At 4000 rpm, the sample was centrifuged for 2 min, followed by moving 1 mL to the autosampler (injection volume was 3 µL). The analysis was performed using Burker Daltonik (Berman, Germany) impact II ESI-Q-TOF system equipped with the Burker Dalotonik Elute UPLC system (Bremen, Germany) as described by Al-Mterin et al.(Al-Mterin et al. 2021).

Antioxidant activity (DPPH assay)

The antioxidant potential of ethyl acetate, ethanol, and aqueous extracts of L. ovalifolium leaves was investigated according to the radical scavenging activity of the stable synthetic free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) as demonstrated in the literature (Blois 1958). In brief, a mixture was prepared by adding 3 mL of (0.1 mM) methanol solution of DPPH to 2 mL of plant extracts solution using different concentrations (200–1.65 µg/mL). After 30 min of incubation, the absorbance (Ax) was measured using a spectrophotometer (BEL PHOTONIC, Italy) at 517 nm. The solution of DPPH dissolved in methanol represents the blank sample (A°). IC₅₀ value was determined by plotting the percentage of inhibition against extract concentration. The percentage of inhibition was estimated based on the following equation 1:

%I = (A° – Ax) / A° × 100 (1)

In vitro evaluation of antiproliferative activity

Cell lines and cell culture conditions

To investigate the antiproliferative activity of L. ovalifolium extracts, six cancer cell lines were used. Human breast cancer cells (T47D and MDA-MB-231), human colon cancer (Caco-2), human prostate cancer (PC3), human cervical cancer (HeLa), and non-cancerous fibroblast cells were provided from the University of Jordan. Mouse mammary sarcoma cells (EMP6/P) were purchased from the European Collection of Cell Cultures (Salisbury, UK). The cells were cultured in a complete medium and incubated in proper conditions, including 37 °C, 5% CO₂, and 95% humidity. The type of culture media varied according to the cell line. In particular, T47D and PC3 cell lines were cultured in a completed RPMI 1640 medium (PAN-biotech, Germany), while MDA-MB-231, Caco-2, HeLa, and fibroblast were cultured in a complete DMEM medium (PAN-biotech, Germany). As well, a completed MEM medium (PAN-biotech, Germany) was used for EMT6/P culturing. All types of media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, UK), 1% penicillin-streptomycin (Sigma, USA), 1% L-glutamine (Sigma, USA), and 0.1% gentamycin (EuroClone, Italy).

Antiproliferative assay

The cytotoxicity of L. ovalifolium leaves extracts was detected using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as described in the literature (Mahmod and Talib 2021). In the present study, the cells were treated with L. ovalifolium extracts (5–0.039 mg/mL) and processed as mentioned by Mahmod and Talib (2021). The percentage of cell survival was calculated compared to the negative control (untreated cells) (Equation 2).

Percentage of cell viability (%) = (OD of treated cell/OD of control cell) × 100 (2)

The half-maximum inhibitory concentration (IC₅₀) of the treated cells was determined using SPSS (Statistical Package for the Social Science, Illinois version 24).

In vivo evaluation of the antitumor activity

Animals

The experiments with the animals were approved by the Research and Ethical Committee of Applied Science Private University (Approval Number: 2015-PHA-05). Thirty Balb/C female mice (4–6 weeks old, weight 21–25 g) were pathogen-free housed including convenient temperature (25 °C) and humidity of less than 60% in single cages, and had access to a standard pellet diet and water ad libitum before starting the experiments. The required conditions have been applied to keep the animals with ongoing air ventilation.

Acute toxicity of L. ovalifolium ethyl acetate extract

To determine the proper starting dose in the LD₅₀ estimation assay, a limit test was performed. A small group of female mice (n=2) was treated (IP injection) with L. ovalifolium ethyl acetate extract. After 24 hrs, mortality incidence was observed. In case the mice have tolerated the dose, a gradual increase in the concentration (the dose multiplied by 1.5) is applied; otherwise, the dose will be reduced by 0.7. The maximum non-lethal and minimum lethal doses demonstrated the lower and upper limits, which were used to achieve the LD₅₀ estimation assay (Akhila et al. 2007).

LD₅₀ estimation assay was carried out by treating (IP injection) three groups of mice (n=4) with three doses of ethyl acetate extract (100, 300, and 500 mg/kg). All the investigated doses were within the upper and lower range that was recognized in the limit test. The vital conditions of the treated mice were observed for 24 hrs. The arithmetical method of Karber was considered to estimate the LD₅₀ of L. ovalifolium leaves (Akhila et al. 2007).

Antitumor activity in an animal model

EMT6/P cells were collected and prepared to be inoculated in Balb/C mice (n=18) with a density of 1 × 10⁶ cells/0.1 mL via subcutaneous injection. After 10 days, the size of the growing tumors was measured using a digital caliper (Vogel, Germany). Tumor-bearing mice were divided into a control group (n=9) (with no treatment) and a treatment group (n=9) (treated with ethyl acetate extract). In particular, ethyl acetate extract was prepared for IP injection in a concentration of 12.5 mg/kg which is 10% of the estimated LD₅₀ value. The treatment stage was carried on for 10 days and during that tumor measurement was reported and serum samples were collected for further analysis. At the end of the experiment, the mice were sacrificed using cervical dislocation method and all the tumors were extracted and kept in 10% formalin. The volumes of the tumors were calculated according to the following equation (3) (Agrawal et al. 2004):

Volumes of the tumors = A × B² × 0.5 (3)

Whereas (A) = the length of the longest aspect of the tumor, (B) = the length of the perpendicular to A

Assessment of kidney and liver functions in the experimental animals

Creatinine was measured to evaluate nephrotoxicity. Alanine transaminase (ALT) and aspartate transaminase (AST) were evaluated to assess liver function. Quantification of these biomarkers was achieved by following the protocol of specific kits including ALAT (GPT) FS* (Cat. No. 1 2701 99 10 972, Holzheim, Germany), ASAT (GOT) FS* (Cat. No. 1 2601 99 10 021, Holzheim, Germany), and Creatinine FS* (Cat. No. 1 1711 99 10 021, Holzheim, Germany) using DiaSys Respons 920 analyzer (Holzheim, Germany).

Statistical analysis

Data was demonstrated using the mean ± SEM (Standard Error of Mean). The statistical significance between groups was detected utilizing SPSS student’s t-test. Variation between groups was approved significantly when the p-value is less than 0.05 (p < 0.05). IC₅₀ values were determined by applying non-linear regression in SPSS (Statistical Package for the Social Science, Chicago, Illinois version 24).

Results and discussion

Despite all technological advancements in cancer prevention and treatment, cancer is still one of the main causes of death around the world (Khan et al. 2022). Conventional treatment is associated with many side effects that may cause other critical disorders (Raina et al. 2014; Khan et al. 2022). Therefore, the search for new drugs for treatment with low cost and fewer adverse effects is the main goal in cancer research development (Raina et al. 2014; Mitra and Bhattacharya 2020). Natural plant compounds such as flavonoids, terpenoids, phenols, alkaloids, and others are rich sources of potential anticancer agents. Several chemotherapeutic agents, currently used in the treatment are derived from plants and their semisynthetic derivatives (Shaikh et al. 2016; Mitra and Bhattacharya 2020). According to the US National Cancer Institute (NCI), more than 35 thousand plant species had been evaluated. Vincristine, vinblastine, taxol, indicine-N-oxide, etoposide analogs, and many others are the results of these investigations (Shaikh et al. 2016). To benefit from nature, more research and experimentation are required using edible, medicinal, and ornamental plants (Raina et al. 2014). Although Ligustrum is an ornamental plant it has medicinal uses in traditional Chinese medicine. It is known to stimulate the immune system, improve osteoporosis, and enhance poor vision (Starr et al. 2003; Pang et al. 2015). L. ovalifolium, another representative of this genus, exerts various medicinal properties including anti-inflammatory, antioxidant, hypoglycemic, and hypotensive activities (Harsha et al. 2013). In the current study, LC-MS analysis, antioxidant, and antiproliferative assays were conducted to investigate the possible medicinal value of L. ovalifolium leaves extracts. The results revealed promising antioxidant and antiproliferative activity. Regarding extraction results, the percentage yield of the extracts was varied according to the solvent used in the extraction. Water extract exhibited the highest percentage followed by ethanol extract. (Table 1). Ethyl acetate extract showed the lowest percentage of yield.

Table 1.

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Extraction yield results of the three solvent extracts.

Source	Extraction solvent	% of dried extracts yield
Ligustrum ovalifolium leaves	Ethyl acetate	3.73%
	Ethanol	12.14%
	Water	17.24%

Based on MTT results, ethyl acetate inhibited tumor cell growth at a low concentration of around 40 to 70 µg/mL particularly in cervical and breast cancer cell lines (Table 2).

Table 2.

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IC₅₀ values of L. ovalifolium extracts against different cancer cell lines.

	T47D	MDA-MB-231	PC3	Caco-2	HeLa	EMT6/P	Fibroblast
	IC₅₀	IC₅₀	IC₅₀	IC₅₀	IC₅₀	IC₅₀	IC₅₀
	(mg/mL)±SEM	(mg/mL)±SEM	(mg/mL)±SEM	(mg/mL)±SEM	(mg/mL)±SEM	(mg/mL)±SEM	(mg/mL)±SEM
Ethyl acetate extract	0.07±0.08	0.072±0.03	0.17±0.02	0.16±0.06	0.047±0.08	0.21±0.05	>5
Ethanol extract	0.87±0.12	0.74±0.14	0.57±0.03	1.2±0.12	0.87±0.16	1.8±0.03	>5
Aqueous extract	>5	1.88±0.1	0.70±0.09	1.53±0.12	2.22±0.09	1.8±0.16	>5

As well, ethanol and aqueous extracts were able to reduce the percentage of survival in both PC3 and MDA-MB-231 cell lines (Fig. 1). On contrary, all the extracts showed less toxicity (IC₅₀ > 5 mg/mL) toward the fibroblast, the normal model cell line (Table 2). Using normal fibroblast cells the safety of these extracts was established.

Figure 1.

The antiproliferative activity of L. ovalifolium extracts against (A) T47D cell line (B) MDA-MB-231 cell line (C) HeLa cell line (D) Caco-2 cell line (E) PC3 cell line (F) EMT6/P cell line (G) Fibroblast cell line. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Based on the limit test result, the non-lethal dose of ethyl acetate extract IP injection was 100 mg/kg. By following the arithmetical method of Karber, the estimated LD₅₀ of ethyl acetate extract was 125 mg/kg (Table 3).

Table 3.

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Acute toxicity assay of L. ovalifolium ethyl acetate extract.

Groups (n=4)	Dose (mg/kg)	No. of mortality	Dose difference (a)	Mean mortality (b)	Probit (a × b)
1	100	0	0	0	0
2	300	2	200	1	200
3	500	3	200	2.5	500

In the animal model experiment, Ethyl acetate extract was selected for the in vivo assay since it exhibited the best antiproliferative efficacy in the in vitro assessment among the three extracts. After treating tumor-bearing mice (n=9) with L. ovalifolium ethyl acetate extract (12.5mg/kg), results revealed a -43.1% reduction in tumor size compared to the negative control (48.1%) (Table 4) (Figs 2, 3).

Figure 2.

A plot verifying the changes in average tumor size (mm³) vs time (days) of treatment with L. ovalifolium ethyl acetate extract in Balb/C mice inoculated with the EMT6/P cell line.

Figure 3.

L. ovalifolium ethyl acetate extract effect on tumor size and cure percentage. Compared to the control group, treating tumor-bearing mice with L. ovalifolium has reduced tumor size and improved the cure percentage. (n=9 per group).

Table 4.

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Antitumor effect of L. ovalifolium ethyl acetate extract in the animal model.

Treatment groups (n=9)	Initial tumor size (mm³) ± SEM	Final tumor size (mm³) ± SEM	% Change in tumor size	% of mice with no detectable tumor	Number of death	Average tumor weight (mg)
Control group	332.2±15	492.2±11	48.1	11.1	1	512.8
Ligustrum ovalifolium group	367.3±22	208.7±16	-43.1	55.1	0	228.2

In the present study, the antiproliferative efficacy of the ethylacetate extract of L. ovalifolium leaves is for the first time demonstrated while for some other species of the genus Ligustrum, cytotoxic effects against different cancer cells were reported. A recent study revealed the antiproliferative activity of different phytochemicals isolated from L. japonicum fruits (Kim et al. 2022). These compounds have suppressed cell growth and invasion by inhibiting MMP-2 and MMP-9 in HT 1080 fibrosarcoma (Kim et al. 2022). Also, ethanol extract of L. lucidum leaves decreased cell migration and invasion of hepatocellular carcinoma (Tian et al. 2019). Aqueous extract of L. robustum prevented tumor cell proliferation both, in vitro and in vivo by promoting apoptosis (Zuo et al. 2019). Moreover, methanol extract prepared from L. vulgare leaves and fruits showed cytotoxic effects against human leukemia cells (Zaric et al. 2021). The cytotoxic effect observed in the present study might be due to the high concentration of flavonoids, such as apigenin-7-O-glucosides (34%), luteolin (19%), baicalein (19%), and luteolin-7-O-glucosides (15%) detected in the ethylacetate extract using LC-MS (Table 5).

Table 5.

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LC-MS analysis of L. ovalifolium ethyl acetate extract.

NO	Compounds	RT (Retention time)	Formula	Relative % (ethyl acetate extract)
1	2,4-Dihydroxyacetophenone	5.41	C₈H₈O₃	0.130681
2	Luteolin 7-O-glucoside (Cynaroside)	5.88	C₂₁H₂₀O₁₁	15.84325
3	Isorhoifolin	6.54	C₂₇H₃₀O₁₄	7.621954
4	Apigenin-7-O-glucoside (Apigetrin)	6.77	C₂₁H₂₀O₁₀	34.6314
5	7-Glu Chrysoeriol (NMR)	7.11	C₂₂H₂₂O₁₁	2.079451
6	Luteolin	8.57	C₁₅H₁₀O₆	19.05762
7	Tiliroside	8.8	C₃₀H₂₆O₁₃	0.139487
8	Oct-1-en-3-yl Ara (1-6)Glu (NMR)	8.9	C₁₉H₃₄O₁₀	0.344667
9	naringenin	9.53	C₁₅H₁₂O₅	0.104087
10	Baicalein	9.86	C₁₅H₁₀O₅	19.7901
11	Madecassic acid	15.56	C₃₀H₄₈O₆	0.090174
12	Hederagenin	21.62	C₃₀H₄₈O₄	0.084362
13	Ursolic acid	27.19	C30H48O3	0.082776

Additionally, ethyl acetate extract revealed the highest phenol content (154.7 mg GAE/g), and flavonoids (100.3 mg RE/g) as well as a high value of IC₅₀ (33 µg/mL) according to DPPH assay (Table 6). Ethanol and aqueous extracts showed scavenging activity at a concentration of 200 µg/mL (Fig. 4).

Figure 4.

DPPH radical scavenging activity of L. ovalifolium extracts. Ascorbic acid is the positive control in this assay. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Table 6.

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Total phenol, flavonoid content, and antioxidant activity of L. ovalifolium.

L. ovalifolium leaves extracts	TFC* (mg RE/g)	TPC* (mg GAE/g)	DPPH assay IC₅₀ (µg/mL)
Ethyl acetate extract	100.3±0.4	154.7±0.4	33±9.2
Ethanol extract	41.5±0.1	115.2±1.1	46.7±11
Aqueous extract	22.6±0.3	87.9±0.1	52.2±8.6
Ascorbic acid			1.74±0.2

The natural flavonoid apigenin-7-O-glucoside with multiple biological activities has high stability and good solubility (Kowalski et al. 2005; Smiljkovic et al. 2017). Its cytotoxic effect was reported against colon cancer (Smiljkovic et al. 2017), and prostate and cervical cancers (Srivastava and Gupta 2009; Liu et al. 2020; Minda et al. 2020). Liu et al. have suggested that apigenin-7-O-glucoside mediated cell apoptosis by modulating PTEN/PI3K/AKT pathway and prevented cell migration in cervical cancer cells (Liu et al. 2020). A study has shown that apigenin-7-O-glucoside was more active than apigenin in inhibiting the cell growth of HeLa human cervical cancer cells (Minda et al. 2020). Baicalein is another flavonoid detected in L. ovalifolium leaves. This flavonoid has also various pharmacological activities such as antioxidant, anti-inflammatory, antiviral, antibacterial, and anticancer activities (Wang et al. 2014; Gao et al. 2016; Nik Salleh et al. 2020). Bacailein has been tested on many cancer cell lines and showed high potency as an antiproliferative agent (Gao et al. 2016). Zhang et al. suggested that baicalein inhibited cell growth in osteosarcoma through the upregulation of IncRNA-NEF, which suppressed the Wnt/β-catenin signaling pathway that led to prevent tumor growth in vitro and in vivo (Zhang et al. 2022). Moreover, baicalein exhibited an anti-metastatic effect against breast cancer cells via blocking STAT3 activity, MMP-2/9 expression, STAB1and Wnt/β-catenin pathway (Wang et al. 2010; Ma et al. 2016; Susmitha et al. 2020). Han et al. demonstrated the anticancer activity of baicalein by inhibiting AKT/NF-kB and STAT3 signaling pathways in cholangiocarcinoma cells (Han et al. 2021). On the other hand, luteolin, a natural flavone, has been reported to have antiproliferative activity against breast cancer (Lee et al. 2012), gastric cancer (Pu et al. 2018), cervical cancer (Chen et al. 2023), lung cancer, and other cancer cell lines (Imran et al. 2019; Ganai et al. 2021). Lee et al. (2012) experimented antitumor effect of luteolin on the MDA-MB-231 cell line, which showed suppression of AKT, PLK1, cyclin B¹, cyclin A, CDC2, CDK2, and Bcl-xL as well as induction of inducing p21 and Bax expression (Lee et al. 2012). Furthermore, luteolin-7-O-glucoside, a glycosyloxyflavone derived from luteolin, exhibited a potent cytotoxic effect on breast cancer cell lines (MCF-7 and MDA-MB-231 cells) (Goodarzi et al. 2020). It inhibited migration and invasion of oral cancer cells by downregulation of MMP-2 expression (Velmurugan et al. 2020) as well as induced apoptosis in human nasopharyngeal carcinoma (Ho et al. 2021).

There is a correlation between the antioxidant effect of plant extracts and their cytotoxic activity. Reactive oxygen species (ROS) can stimulate DNA damage which may lead to converting normal cells into cancerous ones by genetic mutation (Ziech et al. 2010). In fact, the unbalanced redox equilibrium mediates cancer development and progression (Jambunathan et al. 2014). Based on our results, L. ovalifolium extracts exhibited antioxidant activity compared to ascorbic acid. The positive correlation among total phenolic content, total flavonoids, and antioxidant activity of L. ovalifolium leaves was in agreement with what has been reported by Kim et al. (Kim et al. 2011).

To evaluate the effect of L. ovalifolium leaves treatment on the main functional biomarkers of the liver and kidney in mice; serum samples for both, treated and untreated groups of animals were analyzed. There are slight differences between the ethyl acetate group and the control group, which indicate the safety of the L. ovalifolium leaves extract with no toxic effects on kidney and liver functions (Table 7).

Table 7.

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L. ovalifolium effect on liver and kidney functions.

	Creatinine (mg/mL)±SEM	ALT (U/L)±SEM	AST (U/L)±SEM
L. ovalifolium ethyl acetate extract group	0.35±0.17	34.75±9	156±7
Control group	0.31±0.12	68.8±1.4	200±6.2
Healthy mice	0.17±0.05	61.3±9.8	150±11

Conclusion

The results of this study suggested that L. ovalifolium leaves extracts could be considered a promising medicinal plant with anticancer potential. Ethyl acetate extract of L. ovalifolium leaves exhibited high potency in preventing cell growth of various cancer types as well as reduced tumor size and enhanced quick recovery of mice bearing breast cancer. Besides, the phytochemical analysis revealed the richness of the active ethyl acetate extract in flavonoids, which are recognized by their antioxidant and antiproliferative activities. Nevertheless, further investigations are needed to determine the targets and signaling pathways that are affected by L. ovalifolium extracts.

References

Aboalhaija NH, Syaj H, Afifi F, Sunoqrot S, Al-Shalabi E, Talib W (2022) Chemical evaluation, in vitro and in vivo anticancer activity of Lavandula angustifolia Grown in Jordan. Molecules 27: 5910. https://doi.org/10.3390/molecules27185910

Agrawal N, Bettegowda C, Cheong I, Geschwind J-F, Drake CG, Hipkiss EL, Tatsumi M, Dang LH, Diaz Jr LA, Pomper M (2004) Bacteriolytic therapy can generate a potent immune response against experimental tumors. Proceedings of the National Academy of Sciences 101: 15172–15177. https://doi.org/10.1073/pnas.0406242101

Akhila JS, Shyamjith D, Alwar MC (2007) Acute toxicity studies and determination of median lethal dose. Current science, 917–920. https://www.jstor.org/stable/24099255

Al-Mterin MA, Aboalhaija NH, Abaza IF, Kailani MH, Zihlif MA, Afifi FU (2021) Chromatographic analysis (LC-MS and GC-MS), antioxidant activity, total phenol and total flavonoid determination of Ononis natrix L. grown in Jordan. Jordan Journal of Chemistry (JJC) 16: 31–39. https://doi.org/10.47014/16.1.4

Al Kury LT, Taha Z, Mahmod AI, Talib WH (2022) Xanthium spinosum L. Extracts inhibit breast cancer in mice by apoptosis induction and immune system modulation. Pharmaceuticals 15: 1504. https://doi.org/10.3390/ph15121504

Avato P, Migoni D, Argentieri M, Fanizzi FP, Tava A (2017) Activity of saponins from Medicago species against HeLa and MCF-7 cell lines and their capacity to potentiate cisplatin effect. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 17: 1508–1518. https://doi.org/10.2174/1871520617666170727152805

Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181: 1199–1200. https://doi.org/10.1038/1811199a0

Cabasag CJ, Fagan PJ, Ferlay J, Vignat J, Laversanne M, Liu L, van der Aa MA, Bray F, Soerjomataram I (2022) Ovarian cancer today and tomorrow: A global assessment by world region and Human Development Index using GLOBOCAN 2020. International Journal of Cancer 151: 1535–1541. https://doi.org/10.1002/ijc.34002

Chen Y-H, Wu J-X, Yang S-F, Hsiao Y-H (2023) Synergistic combination of luteolin and asiatic acid on cervical cancer in vitro and in vivo. Cancers 15: 548. https://doi.org/10.3390/cancers15020548

Folin O, Ciocalteu V (1927) On tyrosine and tryptophane determinations in proteins. Journal of Biological Chemistry 73: 627–650. https://doi.org/10.1016/S0021-9258(18)84277-6

Ganai SA, Sheikh FA, Baba ZA, Mir MA, Mantoo MA, Yatoo MA (2021) Anticancer activity of the plant flavonoid luteolin against preclinical models of various cancers and insights on different signalling mechanisms modulated. Phytotherapy Research 35: 3509–3532. https://doi.org/10.1002/ptr.7044

Gao Y, Snyder SA, Smith JN, Chen YC (2016) Anticancer properties of baicalein: a review. Medicinal Chemistry Research 25: 1515–1523. https://doi.org/10.1007/s00044-016-1607-x

Goodarzi S, Tabatabaei MJ, Mohammad Jafari R, Shemirani F, Tavakoli S, Mofasseri M, Tofighi Z (2020) Cuminum cyminum fruits as source of luteolin-7-O-glucoside, potent cytotoxic flavonoid against breast cancer cell lines. Natural Product Research 34: 1602–1606. https://doi.org/10.1080/14786419.2018.1519824

Han P, Shang J, Chen D-L, Li S-Y, Fan R, Li R-H, Li H-Q, Zhang S-Y, Shen D-Y (2021) Baicalein mediates anticancer effect on cholangiocarcinoma through co-targeting the AKT/NF-κB and STAT3 signaling pathway. Process Biochemistry 102: 304–314. https://doi.org/10.1016/j.procbio.2021.01.017

Harsha M, Ashish M, Pranav V, Megha J, Chokotia LS (2013) Review on pharmacological activities of Ligustrum ovalifolium. International Journal of Research and Development in Pharmacy and Life Sciences 2: 474–477.

Hasskarl JK (1844) Catalogus plantarum in horto botanico bogoriensi cultarum alter. Lands-Drukkerij, 391 pp. https://doi.org/10.5962/bhl.title.79159

Ho HY, Chen PJ, Lo YS, Lin CC, Chuang YC, Hsieh MJ, Chen MK (2021) Luteolin‐7‐O‐glucoside inhibits cell proliferation and modulates apoptosis through the AKT signaling pathway in human nasopharyngeal carcinoma. Environmental Toxicology 36: 2013–2024. https://doi.org/10.1002/tox.23319

Hosny M, Ragab EA, Mohammed AE-sI, Shaheen UY (2009) New secoiridoids from Ligustrum ovalifolium and their hypotensive activity. Pharmacognosy Research 1. https://www.phcogres.com/content/pharmacognosy-research-vol-1-issue-2-2009

Hossain MA, Rahman SMM (2011) Total phenolics, flavonoids and antioxidant activity of tropical fruit pineapple. Food Research International 44: 672–676. https://doi.org/10.1016/j.foodres.2010.11.036

Imran M, Rauf A, Abu-Izneid T, Nadeem M, Shariati MA, Khan IA, Imran A, Orhan IE, Rizwan M, Atif M (2019) Luteolin, a flavonoid, as an anticancer agent: A review. Biomedicine & Pharmacotherapy 112: 108612. https://doi.org/10.1016/j.biopha.2019.108612

Jambunathan S, Bangarusamy D, Padma PR, Sundaravadivelu S (2014) Cytotoxic activity of the methanolic extract of leaves and rhizomes of Curcuma amada Roxb against breast cancer cell lines. Asian Pacific Journal of Tropical Medicine 7: S405–S409. https://doi.org/10.1016/S1995-7645(14)60266-2

Joshi P, Vishwakarma RA, Bharate SB (2017) Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. European Journal of Medicinal Chemistry 138: 273–292. https://doi.org/10.1016/j.ejmech.2017.06.047

Khan AW, Farooq M, Haseeb M, Choi S (2022) Role of plant-derived active constituents in cancer treatment and their mechanisms of action. Cells 11: 1326. https://doi.org/10.3390/cells11081326

Khan AW, Khan A-u, Shah SMM, Ullah A, Faheem M, Saleem M (2019) An updated list of neuromedicinal plants of Pakistan, their uses, and phytochemistry. Evidence-Based Complementary and Alternative Medicine 2019: 6191505. https://doi.org/10.1155/2019/6191505

Kim H, Kong C-S, Seo Y (2022) Salidroside, 8 (E)-nuezhenide, and ligustroside from Ligustrum japonicum fructus inhibit expressions of MMP-2 and-9 in HT 1080 fibrosarcoma. International Journal of Molecular Sciences 23: 2660. https://doi.org/10.3390/ijms23052660

Kim Y-S, Lee S-J, Hwang J-W, Kim E-H, Park P-J, Jeong J-H (2011) Antioxidant activities of extracts from Ligustrum ovalifolium H. leaves. Journal of the Korean Society of Food Science and Nutrition 40: 1642–1647. https://doi.org/10.3746/jkfn.2011.40.12.1642

Kim Y-S, Lee S-J, Hwang J-W, Kim E-H, Park P-J, Jeong J-H (2012) Anti-inflammatory effects of extracts from Ligustrum ovalifolium H. leaves on RAW264. 7 macrophages. Journal of the Korean Society of Food Science and Nutrition 41: 1205–1210. https://doi.org/10.3746/jkfn.2012.41.9.1205

Kocarnik JM, Compton K, Dean FE, Fu W, Gaw BL, Harvey JD, Henrikson HJ, Lu D, Pennini A, Xu R (2022) Cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life years for 29 cancer groups from 2010 to 2019: a systematic analysis for the global burden of disease study 2019. JAMA oncology 8: 420–444. https://doi.org/10.1001/jamaoncol.2021.6987

Kowalski J, Samojedny A, Paul M, Pietsz G, Wilczok T (2005) Effect of apigenin, kaempferol and resveratrol on the expression of interleukin-1beta and tumor necrosis factor-alpha genes in J774. 2 macrophages. Pharmacological Reports: PR 57: 390–394. https://pubmed.ncbi.nlm.nih.gov/15985724/

Lee E-J, Oh S-Y, Sung M-K (2012) Luteolin exerts anti-tumor activity through the suppression of epidermal growth factor receptor-mediated pathway in MDA-MB-231 ER-negative breast cancer cells. Food and Chemical Toxicology 50: 4136–4143. https://doi.org/10.1016/j.fct.2012.08.025

Leitzmann C (2016) Characteristics and health benefits of phytochemicals. Complementary Medicine Research 23: 69–74. https://doi.org/10.1159/000444063

Liu M-M, Ma R-H, Ni Z-J, Thakur K, Cespedes-Acuña CL, Jiang L, Wei Z-J (2020) Apigenin 7-O-glucoside promotes cell apoptosis through the PTEN/PI3K/AKT pathway and inhibits cell migration in cervical cancer HeLa cells. Food and Chemical Toxicology 146: 111843. https://doi.org/10.1016/j.fct.2020.111843

Ma X, Yan W, Dai Z, Gao X, Ma Y, Xu Q, Jiang J, Zhang S (2016) Baicalein suppresses metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/β-catenin pathway. Drug Design, Development and Therapy, 1419–1441. https://doi.org/10.2147/DDDT.S102541

Machida K, Yamaguchi T, Kamiya Y, Kikuchi M (1997) Acylated triterpenoids from Ligustrum ovalifolium. Phytochemistry 46: 977–979. https://doi.org/10.1016/S0031-9422(97)00384-1

Mahmod A, Haif SK, Kamal A, Alataby IA, Talib WH (2022) Chemoprevention effect of the Mediterranean diet on colorectal cancer: preclinical studies and future prospects. Frontiers in Nutrition 9: 924192. https://doi.org/10.3389/fnut.2022.924192

Mahmod AI, Talib WH (2021) Anticancer activity of Mandragora autumnalis: An in vitro and in vivo study. Pharmacia 68: 827–835. https://doi.org/10.3897/pharmacia.68.e71695

Mahmod IA, Talib HW (2023) Chemical composition, antioxidant, antimicrobial, and immunomodulatory activity of Mandragora autumnalis Grown in Jordan. The Natural Products Journal 13: 72–83. https://doi.org/10.2174/2210315512666220602092915

Minda D, Avram S, Pavel IZ, Kis B, Ghitu A, Zupkó I, Dehelean C, Buda V, Diaconeasa Z, Scurtu A (2020) An in vitro evaluation of apigenin and apigenin-7-o-glucoside against hela human cervical cancer cell line. Revista de Chimie 71: 140–144. https://doi.org/10.37358/RC.20.2.7906

Mitra T, Bhattacharya R (2020) Phytochemicals modulate cancer aggressiveness: A review depicting the anticancer efficacy of dietary polyphenols and their combinations. Journal of Cellular Physiology 235: 7696–7708. https://doi.org/10.1002/jcp.29703

Mohammed AH (2019) Importance of medicinal plants. Research in Pharmacy and Health Sciences 5: 124–125.

Moldovan B, Sincari V, Perde-Schrepler M, David L (2018) Biosynthesis of silver nanoparticles using Ligustrum ovalifolium fruits and their cytotoxic effects. Nanomaterials 8: 627. https://doi.org/10.3390/nano8080627

Morgan E, Arnold M, Gini A, Lorenzoni V, Cabasag CJ, Laversanne M, Vignat J, Ferlay J, Murphy N, Bray F (2023) Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 72: 338–344. https://doi.org/10.1136/gutjnl-2022-327736

Nik Salleh NNH, Othman FA, Kamarudin NA, Tan SC (2020) The biological activities and therapeutic potentials of baicalein extracted from oroxylum indicum: a systematic review. Molecules 25: 5677 https://doi.org/10.3390/molecules25235677

Pang Z, Zhi-yan Z, Wang W, Ma Y, Feng-ju N, Zhang X, Han C (2015) The advances in research on the pharmacological effects of Fructus ligustri lucidi. BioMed Research International 2015: 281873. https://doi.org/10.1155/2015/281873

Pu Y, Zhang T, Wang J, Mao Z, Duan B, Long Y, Xue F, Liu D, Liu S, Gao Z (2018) Luteolin exerts an anticancer effect on gastric cancer cells through multiple signaling pathways and regulating miRNAs. Journal of Cancer 9: 3669. https://doi.org/10.7150%2Fjca.27183

Raina H, Soni G, Jauhari N, Sharma N, Bharadvaja N (2014) Phytochemical importance of medicinal plants as potential sources of anticancer agents. Turkish Journal of Botany 38: 1027–1035. https://doi.org/10.3906/bot-1405-93

Shaikh AM, Shrivastava B, Apte KG, Navale SD (2016) Medicinal plants as potential source of anticancer agents: a review. Journal of Pharmacognosy and Phytochemistry 5: 291–295.

Shu L, Cheung K-L, Khor TO, Chen C, Kong A-N (2010) Phytochemicals: cancer chemoprevention and suppression of tumor onset and metastasis. Cancer and Metastasis Reviews 29: 483–502. https://doi.org/10.1007/s10555-010-9239-y

Smiljkovic M, Stanisavljevic D, Stojkovic D, Petrovic I, Vicentic JM, Popovic J, Grdadolnik SG, Markovic D, Sankovic-Babice S, Glamoclija J (2017) Apigenin-7-O-glucoside versus apigenin: Insight into the modes of anticandidal and cytotoxic actions. EXCLI Journal 16: 795. https://doi.org/10.17179%2Fexcli2017-300

Srivastava JK, Gupta S (2009) Extraction, characterization, stability and biological activity of flavonoids isolated from chamomile flowers. Molecular and Cellular Pharmacology 1: 138. https://doi.org/10.4255/mcpharmacol.09.18

Starr F, Starr K, Loope L (2003) Ligustrum spp. Privet, Oleaceae Maui: United States Geological Survey-Biological Resources Division, Haleakala Field Station, 1–12.

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 71: 209–249. https://doi.org/10.3322/caac.21660

Susmitha GD, Miyazato K, Ogura K, Yokoyama S, Hayakawa Y (2020) Anti-metastatic effects of baicalein by targeting STAT3 activity in breast cancer cells. Biological and Pharmaceutical Bulletin 43: 1899–1905. https://doi.org/10.1248/bpb.b20-00571

Talib WH, Abuawad A, Thiab S, Alshweiat A, Mahmod AI (2022a) Flavonoid-based nanomedicines to target tumor microenvironment. OpenNano 8: 100081. https://doi.org/10.1016/j.onano.2022.100081

Talib WH, Alsalahat I, Daoud S, Abutayeh RF, Mahmod AI (2020) Plant-derived natural products in cancer research: extraction, mechanism of action, and drug formulation. Molecules 25: 5319. https://doi.org/10.3390/molecules25225319

Talib WH, Daoud S, Mahmod AI, Hamed RA, Awajan D, Abuarab SF, Odeh LH, Khater S, Al Kury LT (2022b) Plants as a source of anticancer agents: From bench to bedside. Molecules 27: 4818. https://doi.org/10.3390/molecules27154818

Tian G, Chen J, Luo Y, Yang J, Gao T, Shi J (2019) Ethanol extract of Ligustrum lucidum Ait. leaves suppressed hepatocellular carcinoma in vitro and in vivo. Cancer Cell International 19: 1–13. https://doi.org/10.1186/s12935-019-0960-5

Velmurugan BK, Lin J-T, Mahalakshmi B, Chuang Y-C, Lin C-C, Lo Y-S, Hsieh M-J, Chen M-K (2020) Luteolin-7-O-glucoside inhibits oral cancer cell migration and invasion by regulating matrix metalloproteinase-2 expression and extracellular signal-regulated kinase pathway. Biomolecules 10: 502. https://doi.org/10.3390/biom10040502

Wang L, Ling Y, Chen Y, Li C-L, Feng F, You Q-D, Lu N, Guo Q-L (2010) Flavonoid baicalein suppresses adhesion, migration and invasion of MDA-MB-231 human breast cancer cells. Cancer letters 297: 42–48. https://doi.org/10.1016/j.canlet.2010.04.022

Wang N, Ren D, Deng S, Yang X (2014) Differential effects of baicalein and its sulfated derivatives in inhibiting proliferation of human breast cancer MCF-7 cells. Chemico-Biological Interactions 221: 99–108. https://doi.org/10.1016/j.cbi.2014.08.003

Wang Z-h, Hsu C-c, Yin M-c (2009) Antioxidative characteristics of aqueous and ethanol extracts of glossy privet fruit. Food Chemistry 112: 914–918. https://doi.org/10.1016/j.foodchem.2008.06.078

Yamada T, Kodama K, Maki M (2014) Floral morphology and pollinator fauna characteristics of island and mainland populations of Ligustrum ovalifolium (Oleaceae). Botanical Journal of the Linnean Society 174: 489–501. https://doi.org/10.1111/boj.12092

Zaric M, Popovic S, Baskic D, Jovanovic D, Djurdjevic P, Zaric RZ, Canovic P, Zelen I (2021) Ligustrum vulgare leaves and fruit extracts induce apoptosis of human leukemia cells. Periodicum biologorum 123: 71–77. https://doi.org/10.18054/pb.v123i3-4.19206

Zhang F-w, Peng L-y, Shi C-J, Li J-c, Pang F-x, Fu W-m, Pan X-h, Zhang J-f (2022) Baicalein mediates the anti-tumor activity in Osteosarcoma through lncRNA-NEF driven Wnt/β-catenin signaling regulatory axis. Journal of Orthopaedic Translation 33: 132–141. https://doi.org/10.1016/j.jot.2021.12.001

Ziech D, Franco R, Georgakilas AG, Georgakila S, Malamou-Mitsi V, Schoneveld O, Pappa A, Panayiotidis MI (2010) The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chemico-Biological Interactions 188: 334–339. https://doi.org/10.1016/j.cbi.2010.07.010

Zuo H-j, Liu S, Yan C, Li L-m, Pei X-f (2019) In vitro and in vivo evaluation of antitumor activity of Ligustrum robustum, a Chinese herbal tea. Chinese Journal of Integrative Medicine 25: 425–430. https://doi.org/10.1007/s11655-018-2983-5

Supplementary material

Supplementary material 1

Ligustrum ovalifolium leaves ethyl acetate extract LCMS results

Asma Ismail Mahmod, Muna Oqal, Ali Muharrif Khalid, Fatma U. Afifi, Wamidh H. Talib

Data type: docx

Explanation note: Spectra of identified compunds in Ligustrum ovalifolium extraxts using LCMS

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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