In Vitro Anti-Cancer Effects of Artemisia sieberi on Human Breast Cancer Cells: A Systematic Review

Abstract

Artemisia sieberi (Asteraceae) is a grey, dwarf, dry woolly shrub, locally known as “Shih” in Arab countries, with significant medicinal properties due to its content of up to 160 active compounds with anti-cancer activity. A. sieberi has been investigated for its potential effects in various cancer types, including breast cancer. The aim of this systematic review is to examine the in vitro evidence on the potential activity of A. sieberi extracts against breast cancer, specifically in MCF-7 and MDA-MB-231 cells. A search of PubMed, ScienceDirect, Scopus, Web of Science, and Google Scholar electronic databases was conducted for studies published from inception to 2024, following PRISMA guidelines and the Cochrane Handbook for Systematic Reviews. The search strategy applied was ((Artemisia sieberi) AND (anti-breast cancer)). The Quality Assessment Tool (QUIN tool) was used to assess the risk of bias for in vitro studies. Six in vitro experimental studies were included. These studies suggested that A. sieberi extracts may exert anti-breast cancer effects via multiple mechanisms, including apoptosis induction, cell growth inhibition, and gene expression modulation. However, the anticancer potential of A. sieberi against breast cancer has been explored only at a preliminary in vitro level. Future research should evaluate different A. sieberi extracts across diverse breast cancer cell lines, particularly treatment-resistant types such as triple negative breast cancer (TNBC), and extend to in vivo and clinical investigation.

Introduction

Breast cancer is one of the leading malignancies affecting women worldwide. It ranks as the fifth most common cause of cancer-related death, with an estimated 2.3 million new cases diagnosed annually. ¹ Recent studies have indicated a growing incidence of breast cancer over the past three decades in almost all countries, with a particularly high incidence in developing countries.^2,3 Globally, breast cancer incidence among women aged 55 years and older increased between 2010–2019.^1,4 latest research indicates that the global number of breast cancer cases diagnosed annually will reach 2.7 million, with 0.87 million deaths by 2030. ⁵

Breast cancer is the most common cancer among women in Saudi Arabia. The age-standardized incidence and mortality rates were 27.3 and 7.5 per 100,000 population, respectively, in 2018. These rates remain lower than those reported in Western countries. ⁶ Other studies indicate an upward trend in the incidence of breast cancer in Saudi Arabia,^7,8 which may be related to socioeconomic changes, adoption of a western lifestyle, low physical activity, consumption of processed foods, tobacco use, and an aging population. ⁶

Breast cancer is divided into four major types: luminal A, luminal B, basal-like (also referred to as triple-negative breast cancer, TNBC), and human epidermal growth factor receptor (HER2).^9,10 TNBC is considered the most aggressive breast cancer type and accounts for approximately 15%–25% of the four previously mentioned types. As TNBC cells do not express the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2), treatment options are limited. These tumors are generally unresponsive to receptor-targeted therapies. ¹¹ Based on the histopathological examination, the treatment protocol is usually determined and may include surgery, ¹² chemotherapy, ¹³ radiation therapy, ¹⁴ hormonal therapy, ¹⁵ and biological therapy. ¹⁶ However, several previous studies have highlighted the potential adverse effects associated with each of these treatment modalities.^17,18

Evidence from literature has highlighted the potential role of plant-derived compounds in modulating several types of cancers, specifically breast cancer. The use of plant-derived compounds is generally associated with minimal adverse effects compared to conventional treatment methods and thus may represent a promising adjunct or alternative approach during cancer therapy.^11,19 Anti-cancer plant-derived compounds are reported to induce apoptosis in malignant cells through multiple molecular mechanisms. ¹⁸ Earlier reports indicated that breast cancer risk may be reduced by consuming a diet rich in flavonoid-containing vegetables and fruits, such as onions, garlic, tomatoes, peppers, oranges, and aromatic herbs. ²⁰ Furthermore, compounds including curcumin, capsaicin, cardamom, carnosol, cinnamaldehyde, and sinigrin found in some spices and herbs, have shown potential activity against breast cancer cells with comparatively fewer side effects than conventional chemotherapy agents. ²¹

Significantly, Artemisia sieberi (Asteraceae) is a gray, dwarf, dry woolly shrub that is locally known as “Shih” in Arab countries. ²² Artemisia plants are traditionally used in the treatment of several health conditions, including; intestinal disorders and helminth infections, common cold and associated cough, and skin wounds or injuries. ²³ The composition of Artemisia is believed to contribute to these therapeutic properties, as it contains various bioactive metabolites with reported anti-cancer, anti-inflammatory, anti-oxidation, and anti-microbial activities. These compounds include apigenin, flavonoids, flavones, antonin, luteolin, cyclic sesquiterpenes, davanone and bicyclic monoterpene glycosides.^24–26 Additionally, phytochemical analysis of A. sieberi leaves has revealed the presence of several important bioactive compounds, including artemisinin, gallic acid, quercetin, and tannic acid. These compounds are considered to have significant antioxidant activity and may exhibit anti-cancer and anti-microbial potential. ²⁷ The traditional use of A. sieberi for cancer treatment in Middle Eastern countries has been reported previously. The aerial parts of the plant are boiled in water, and the resulting preparation is traditionally consumed for a period of three weeks. ²⁸ Moreover, the potent anti-cancer activity of A. sieberi has been observed in various cancer types, including breast, prostate, ovarian, pancreatic, and lung cancers. ²⁹ A. sieberi extracts are reported to inhibit cancer cell growth through multiple mechanisms, including induction of apoptosis, ferroptosis via generation of reactive oxygen species (ROS), inhibition of angiogenesis, and cell cycle arrest. ²⁹ Recent pharmacological studies have indicated that A. sieberi alcoholic extracts exhibit in vitro anti-cancer activity. ²² The ethanolic extract of A. sieberi has demonstrated cytotoxic and anti-angiogenic effects against the HCT-116 colorectal adenocarcinoma cell line and human umbilical vein endothelial cells (HUVECs), respectively, highlighting the potential of A. sieberi in cancer prevention and as an adjunct in cancer therapy. ³⁰

Other species of the genus Artemisia, including A. annua and A. absinthium, have been shown in vitro and in vivo studies to exhibit significant cancer-preventive and anticancer properties. A recent Iranian study reported that a green-synthesized nanocomposite prepared using A. annua extract exhibited stronger anti-breast cancer activity than its chemically synthesized counterpart, both in vitro (4T1 cells) and in vivo (mouse model), highlighting its potential as a promising therapeutic candidate. ³¹ Another study from Saudi Arabia demonstrated that the methanolic extract of A. annua reduces oxidative stress and induces apoptosis in colon cancer, confirming its preventive and therapeutic efficacy. ³² Additionally, a Greek study reported that A. absinthium extract induced 99% cytotoxicity in HSC-3 tongue carcinoma cells while remaining safe for healthy cells, further supporting its potential as an anticancer agent. ³³

This systematic review fills the knowledge gaps regarding the anti-cancer potential of A. sieberi that have not been adequately addressed, as previous studies on the anti-cancer properties of its organic extracts were mostly limited to preliminary cell viability assays, such as (3-[4,5-dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide) (MTT) assay without detailed investigation of the underlying mechanisms of action. ²⁸ Furthermore, the existing literature is geographically concentrated, with five studies conducted in Saudi Arabia and one in Iran, suggesting that research on A. sieberi is largely focused on the Middle East. Nevertheless, the cytotoxic activity of A. sieberi extracts against TNBC (MDA-MB-231) cells has been insufficiently investigated. The main aim of this systematic review is to evaluate the existing in vitro evidence regarding the anticancer activity of A. sieberi extracts against breast cancer, specifically in MCF-7 and MDA-MB-231 cells. Therefore, the findings of this systematic review may provide insights into the potential role of A. sieberi as a complementary source for future breast cancer research.

Materials and Methods

Initially, a non-systematic search of relevant journals, electronic databases, and the PROSPERO website was conducted to identify any existing systematic reviews specifically addressing Artemisia sieberi in the context of breast cancer. This review was registered on PROSPERO (ID CRD420251050353). However, in recent years, no systematic reviews have specifically focused on Artemisia sieberi as an anti-breast cancer agent. The available literature has mainly discussed other Artemisia species and their association with various types of cancer. The literature lacks a comprehensive review evaluating the anti-cancer potential of Artemisia sieberi against breast cancer. Therefore, this current systematic review was developed following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). ³⁴ The study team consisted of researchers with experience in pharmacology and molecular biology, as well as a researcher with specialized expertise in systematic reviews and database searching.

Eligibility Criteria

Inclusion criteria included articles published in peer-reviewed scientific journals written in English up to December 2024. Eligible studies included experimental research investigating the anti-cancer activity of Artemisia sieberi against breast cancer. Exclusion criteria encompassed studies involving species other than Artemisia sieberi, unpublished academic theses such as master's or PhD dissertations, systematic reviews (including meta-analyses and scoping reviews), case reports, case-control studies, feasibility studies or pilot studies, and articles published in languages other than English. In addition, books, conference abstracts or proceedings, non-refereed articles, and non-refereed publications were also excluded.

Search Strategy

An electronic search was conducted on PubMed, Scopus, Science Direct, Web of Science, and Google Scholar. An appropriate search strategy was applied to each database. The search strategy included the use of the following keywords with Boolean operators: ((Artemisia sieberi) AND (anti breast cancer)). All selected studies were published from inception to December 2024. The following filters were applied: language (English) and document type (original research/academic journals).

Selection and Data-Collection Process

All studies were imported into EndNote version 21 for reference management. The screening process was conducted in two stages: In the first stage, three independent reviewers screened the titles and abstracts to assess their relevance. In the second stage, the same reviewers evaluated the full text of studies that met the initial inclusion criteria. Following screening, data were extracted in accordance with the objectives of this systematic review. Any disagreements or uncertainties arising during the screening or data extraction stages were resolved through periodic discussion meetings attended by all authors.

Data Extraction

The form of data collection included the following variables, which were extracted from each eligible study: first author, publication year, and the country. The study design was classified as in vitro. The type of cancer cell lines and any normal cell lines used were recorded. The part of the plant used, details of the type of Artemisia sieberi extract or isolated compound, and the extraction method were also collected. The dose (concentration) tested, period of treatment, and assays used (eg, MTT and FACS) were recorded. The main outcomes measured (eg, cytotoxicity, apoptosis) and mechanisms of action (if mentioned) were documented. In addition, the main findings and conclusions from each study were summarized (see Supplemental material table 1).

Quality Assessment

The quality and risk of bias of included in vitro studies were assessed using the QUIN (Quality and Risk of Bias tool for In Vitro studies), which was specifically developed for in vitro research. The QUIN consists of 12 criteria to evaluate methodological aspects, including study objectives, justification of sample size, sample selection technique, details of comparator groups, clearly described methodology, operator information, randomization, blinding, validity of outcome measures, outcome assessor information, appropriate statistical analysis, and complete reporting of results. Each criterion was scored as 0 (not applicable), 1 (not adequately specified), or 2 (adequately reported), and the total score for each study was calculated. The scores were then converted to percentages to determine the overall risk of bias and categorized as low risk (> 70%), moderate risk (50-70%), or high risk (< 50%) (Sheth et al, 2024). Details of the QUIN tool are provided in Supplemental Material Table 2.

Synthesis Methods

Descriptive data were extracted using a data collection tool that was specifically developed to achieve the aim of this review (Supplemental Material Table 3).

Results

Study Selection

A total of 685 articles were initially identified through database searches: 640 from Google Scholar, 37 from ScienceDirect, 4 from Scopus, 3 from Web of Science, and 1 from PubMed. After removing 150 duplicate records, 535 articles remained for title and abstract screening. After a more detailed screening process, 235 articles were selected for further verification. Subsequently, 57 articles were assessed for eligibility. Finally, although a broad search was performed, only six studies met the inclusion criteria, all published between 2015 and 2024, highlighting the limited available evidence in this area (Figure 1).

Figure 1.

PRISMA flow diagram.

Description and Characteristics of the Included Studies

All six selected studies were conducted in KSA except one study ³⁵ which was conducted in Tehran, Iran. All of the included studies were in vitro experimental studies performed on cancer cell lines, including breast cancer lines MCF-7 and MDA-MB-231. Five out of six studies investigated the MCF-7 breast cancer cell line,^{22,28,36–38} and examined the effects of different Artemisia sieberi extracts on breast cancer cells. Only one study reported the green synthesis of silver chloride nanoparticles using Artemisia sieberi extract (AgCINPs-ASE) ³⁵ and evaluated their in vitro activity against the MDA-MB-231 human breast cancer cell line, as illustrated in Table 1.

Table 1.

Summary of the Included Studies on Artemisia sieberi and Breast Cancer.

ID Studies	1	2	3	4	5	6
First author/ Year of Publication	Irani, et al, 2024 35	Maodaa, et al, 2024 38	Break, et al, 2023 28	Nasr, et al, 2020 22	El-Wassimy, et al, 2019 37	Abutaha, et al, 2015 36
Country	Tehran, Iran	Al Badiya – Tabuk, KSA	Hail, KSA	Al-Ghat, KSA	North area, KSA	Wadi Hanifa, Riyadh, KSA
Model Type	In Vitro	In Vitro	In Vitro	In Vitro	In Vitro	In Vitro
Breast Cancer Cell Line(s)	MDA-MB-231	MCF-7	MCF-7	MCF-7	MCF-7	MCF-7
Plant Part Used	Aerial	Leaves	Aerial In November, at flowering stage.	Aerial Leaves and stems, during winter season.	Aerial In flowering stage.	Aerial
Type of Extract / Compound	Aqueous crude extract	Methanol (70% methanol and 30% distilled water)	Essential oil	Methanol	Aqueous Methanol 80%	Unrefined Methanol Extract, Dichloromethane, Hexan, and Ethyl Acetate.
Extraction Method	- Boiling, with deionized water. - Filtration, - Synthesis of nanoparticles by mixing with silver nitrate and KCl salt.	- Air-drying, - Grinding, - Maceration in 70% methanol, - Percolation, - Filtration, and - Lyophilization	Hydrodistillation	Maceration	- Extracted twice with aqueous methanol (80%) at room temperature, - Evaporated	Methanol extraction and further purification by liquid-liquid extraction
Dose/Concentration Tested	- For cell viability: 0–100 μg/mL - IC50: 2.203 μg/mL	- For cell viability: 15.625–500 g/mL - IC50: 253.9 g/mL	- For cell viability: 0–300 µg/mL - IC50: 38.7 μg/ml	- For cell viability: 0–200 μg/mL - IC50: 86.2 μg/mL	- For cell viability: 3.9–500 µg/mL - IC50 for compound 1: 146 µg/mL, and IC50 for compound 2: 13.6 µg/mL.	- Unrefined methanolic extract: 50-500 µg/mL - LC50 333.01 µg/mL - Dichloromethane fraction: 10-200 µg/mL - IC50 139.39 µg/mL
Treatment Duration	- 24 h for viability assay and caspase activity.	- 48 h for cytotoxicity assay.	- For cell viability and western blotting 72 h. - For cell cycle analysis and apoptosis assay 48 h.	- For antioxidant 0.5 h. - For cytotoxicity 48 h	- 24 h for cell viability assay.	- 24 h for cytotoxicity assay, and Tali analysis.
Assays Used	MTT assay, qRT-PCR, Nitric oxide assay, Hoechst 33258 dye and AO/EB staining, Flow cytometry, Novex V R assay	MTT test assay, FT-IR, TPC, TFC.	SRB assay, migration assay, flow cytometry, western blotting, and GC/MS.	MTT assay, DPPH, and ABTS methods	MTT and trypan blue dye assay, NMR, MS, HPLC, and TLC methods.	MTT assay, hoechst 33342 dye, Tali image-based cytometer with annexin-V/PI staining, wound healing assay, and HPLC.
Mechanism of Action	Induction of apoptosis through upregulation of caspase-8 and 9 proteins. - Triggering early and late apoptosis stages. - Increased production of nitric oxide (NO). - Upregulation of the LncRNA GAS5 gene. - Cell cycle arrest in the SubG1 phase due to DNA damage."	Not reported	- Inhibition of cell migration - Induction of S-phase arrest - Caspase-independent apoptosis - Downregulation of total ERK and its downstream target LC3 - Synergistic effects of major compounds (davanone, 1,8-cineole, caryophyllene diepoxide) through PI3 K/Akt/MAPK and p53 pathways"	- Antioxidant activity exerted by phenolic and flavonoid compounds. - Anticancer activity of artemisinin and other sesquiterpene lactones.	Not reported	- The dichloromethane fraction of A. sieberia causes cell membrane integrity damage and initiates an apoptotic response in cancer cells, leading to apoptosis. - The exact molecular mechanisms or pathways are not detailed in the paper.
Key Findings	- AgClNPs-ASE showed high efficacy against breast cancer cells. - Induced apoptosis through upregulation of caspase-8 and 9 proteins. - Triggered apoptosis with early (1.93%) and late (58.86%) apoptosis stages. - Enhanced cell cycle arrest in sub-G1 phase. - Increased NO production in cancer cells. - Upregulated LncRNA GAS5 gene expression. - Demonstrated potent anticancer activity with an IC50 concentration of 2.203 µg/mL."	- The study identified 15 different functional groups in A. sieberi leaf extract (ASLE) using FT-IR. - ASLE contains high levels of phenolics (235.5 ± 2.7 mg/g DW) and flavonoids (47.89 ± 0.3 mg/g DW). - ASLE showed significant cytotoxicity against cancer cell line MCF-7, with LC50 values of 253.9 ± 4.4μg/mL	- ASEO exhibited highest cytotoxic activity against MCF-7 with an IC50 value of 38.7 μg/ml. - It Inhibited migration of MCF-7 cells. - It induced S-phase cell-cycle arrest and caspase-independent apoptosis-like cell death in MCF-7 cells. - It downregulated ERK and its downstream target LC3. - Major components include cis-crysanthenyl acetate, davanone, 1,8-cineole, and caryophyllene diepoxide.	- A. sieberi had higher phenol and flavonoid contents, correlating with higher antioxidant activity. - Both species of Artemisia studies, including Artemisia sieberi exhibited anti-proliferation activity in a dose dependent manner, and they are promising sources for antioxidant, anticancer agents.	- Isolation and structure elucidation of two compounds from A. sieberi: 3α,8βdihydroxygermacr-4(15),9-dien-6α,7β,11αH,12,6-olide (1) and 3'-hydroxy-genkwanin (2). - Cytotoxic effects of these compounds against human cancer cell line: MCF-7 (breast).	- The unrefined methanol extract of Artemisia siberi showed moderate anticancer activity against MCF-7 cells with an LC50 value of 333.01 µg/mL. - The dichloromethane fraction exhibited higher cytotoxic activity with an IC50 value of 139.39 µg/mL against MCF-7 cells.

Quality Assessment

The six included studies were assessed using the Quality Assessment Tool for in vitro studies (QUIN). ³⁹ Of the twelve criteria, three (operator details (6), randomization (7), and blinding of outcome assessor (10)) were excluded due to irrelevance. Based on the remaining criteria, three studies^22,28,35 were classified as having a low risk of bias (>70%), and the other three studies^36–38 were classified as having a moderate risk of bias (=66.7%). Most of the studies clearly reported the objectives, outcome measures, and statistical analysis, although several lacked sufficient details on controls and sample size justification (Table 2).

Table 2.

QUIN-Based Quality Assessment of the Included in Vitro Studies.

Studies	1	2	3	4	5	6
	Irani, et al, 2024 35	Maodaa, et al, 2024 38	Break, et al, 2023 28	Nasr, et al, 2020 22	El-Wassimy, et al, 2019 37	Abutaha, et al, 2015 36
1. Objective Clearly Defined	2	1	2	2	1	2
2. Sample Size Justified	1	1	1	2	1	1
3. Sample Selection Technique	1	2	2	2	2	1
4. Comparator Group Details	1	1	1	1	1	0
5. Methodology Clearly Described	2	1	2	2	1	2
6. Operator Details Provided	Exclude
7. Randomization	Exclude
8. Outcome Measurement Method	2	2	2	2	2	2
9. Outcome Assessor Information	1	0	1	0	0	0
10. Blinding of Outcome Assessor	Exclude
11. Appropriate Statistical Analysis	2	2	2	2	2	2
12. Complete Reporting of Results	2	2	2	2	2	2
Total Score	14	12	15	15	12	12
Risk of Bias (%)	77.8	66.7	83.3	83.3	66.7	66.7
Overall Risk of Bias Level	Low	Moderate	Low	Low	Moderate	Moderate

The Investigated Extracts of A. sieberi

All of the selected studies used the aerial parts of A. sieberi for extraction, except one, ³⁸ which used leaves only. Two of the six studies evaluated methanolic extracts on cancer cell lines.^22,38 Maodaa et al (2024) prepared a 70% methanolic extract from aerial parts (leaves) using air-drying, grinding, maceration, percolation, filtration, and lyophilization, whereas Nasr et al (2020) used simple maceration.

Among the included studies, other extracts were tested as follows: Irani et al (2024) prepared an aqueous extract by boiling aerial parts in deionized water, ³⁵ followed by green synthesis of silver chloride nanoparticles using silver nitrate and KCl. Break et al (2023) obtained an essential oil via hydrodistillation. ²⁸ El-Wassimy et al (2019) utilized an 80% aqueous methanolic extract prepared by dual extraction at room temperature followed by evaporation. ³⁷ Abutaha et al (2015) investigated crude extract obtained using liquid-liquid extraction with methanol, dichloromethane, hexane, and ethyl acetate. ³⁶

Most of the included studies evaluated the dose-response relationship. For instance, Irani et al (2024) tested concentrations ranging from 0 to 100 µg/mL and reported a potent IC₅₀ of 2.203 µg/mL. ³⁵ Maodaa et al (2024) examined concentrations between 15.625 and 500 µg/mL, with an IC₅₀ of 253.9 µg/mL, ³⁸ Break et al (2023) investigated concentrations up to 300 µg/mL, reported an IC₅₀ of 38.7 µg/mL, and applied 1× and 2× IC₅₀ values for further assays, ²⁸ Nasr et al (2020) assessed antioxidant potency at 10–1000 µg/mL and cytotoxicity at 0 to 200 µg/mL. ²² El-Wassimy et al (2019) reported two IC₅₀ values: 146 µg/mL and 13.6 µg/mL, from different isolated compounds. Abutaha et al (2015) tested unrefined methanolic extract at 50–500 µg/mL and the dichloromethane fraction at 10–200 µg/mL. ³⁶

Regarding the treatment duration, three of six included studies exposed cells to the investigated extracts for 24 h.^35–37 In two other studies, Maodaa et al (2024) applied a 48-h exposure, while Break et al (2023) used 72 h for viability assay and western blotting, and 48 h for cell cycle and apoptosis analysis. Each study employed different extraction methods (Table 1).

Phytochemical Composition of A. sieberi

Four of the six included studies identified some of the chemical compounds in the investigated extracts that may contribute to their biological activities, while the remaining two studies focused on assessing cytotoxicity rather than characterizing the phytochemical composition of the extracts.

The first study by Maodaa et al (2024) applied the FT-IR analysis to determine the composition of A. sieberi leaves extract (ASLE). The results revealed the presence of 15 distinct functional groups, indicating a complex phytochemical profile. However, it should be noted that FT-IR analysis provides information on functional groups only and is not sufficient to fully characterize the phytochemical composition. Therefore, the authors complemented this analysis with quantitative assays, which showed that ASLE is rich in phenolic compounds, with a concentration of 235.5 ± 2.7 mg/g dry weight (DW), and flavonoids, measured at 47.89 ± 0.3 mg/g DW. ³⁸

The second study, Break et al (2023), investigated the phytochemical composition of the extract, and the results identified several active components in A. sieberi essential oil. GC/MS analysis was performed to determine the major constituents, including cis-crysanthenyl acetate (48.56%), davanone (10.28%), 1, 8-cineole (6.81%) and caryophyllene diepoxide (5.34%), which may contribute to the bioactivity of the essential oil. ²⁸ Selected compound structures are shown in (Table 3).

Table 3.

Chemical Classification of Compounds in Artemisia sieberi by Extraction Type.

Compounds				Extraction type	Study ID / Citation
				Essential Oil	3 Break, et al, 2023 28
cis-Chrysanthenyl acetate	Davanone	1,8-Cineole	Caryophyllene diepoxide
p-Cymen-8-ol	α-Sinensal	Palustrol	cis-Jasmone
β-Myrcene	(+)-β-Costol	Ascaridole
				Aqueous Methanol 80%	5 El-Wassimy, et al, 2019 37
3α,8β-Dihydroxygermacr- 4(15),9-dien-6α,7β,11αH,12,6-olide	3'-Hydroxy-genkwanin

The third study, Nasr et al (2020), demonstrated that A. sieberi extract contained high levels of phenols and flavonoids, with total phenolics measured at 122.1 mg GAE/g and total flavonoids at 36.5 mg QE/g of powder. These high contents were associated with notable antioxidant activity as assessed by MTT, DPPH, and ABTS assays. ²²

The fourth study, El-Wassimy et al (2019), employed NMR, MS, HPLC, and TLC techniques to isolate and elucidate the structures of two compounds from A. sieberi extract: 3α,8βdihydroxygermacr-4(15),9-dien-6α,7β,11αH,12,6-olide (1) and 3'-hydroxy-genkwanin (2) ³⁷ (Table 3).These techniques allowed accurate identification of individual bioactive compounds, offering a more detailed and reliable characterization of the extract compared to general spectrophotometric or FT-IR methods.

Molecular Mechanisms Associated with Anti-Cancer Activity of A. sieberi

Almost all the included studies demonstrated the molecular mechanisms associated with anti-cancer activities of tested extracts, except for Maodaa et al (2024) and El-Wassimy et al (2019), who did not report specific mechanisms related to anti-cancer activity.^37,38 Several anticancer effects of A. sieberi extracts against breast cancer cell lines, particularly MCF-7 and MDA-MB-231, were reported. Irani et al (2024) demonstrated that A. sieberi AgClNPs-ASE induced apoptosis through multiple mechanisms, including upregulation of caspase-8 and −9, enhanced nitric oxide production, upregulation of the LncRNA GAS5 gene, and sub-G1 cell cycle arrest associated with DNA damage. ³⁵ Nasr et al (2020) suggested that the methanolic extract of A. sieberi promoted early and late apoptosis and exerted strong antioxidant activity, which may be linked to its high phenolic and flavonoid content. ²² Break et al (2023) reported that the essential oil of A. sieberi inhibited cell migration, caused S-phase cell cycle arrest, and induced caspase-independent apoptosis via downregulation of ERK and LC3, and modulation of PI3K/Akt/MAPK and p53 signaling pathways, potentially mediated by compounds such as davanone, 1,8-cineole, and caryophyllene diepoxide. ²⁸ Regarding the results of Abutaha et al (2015), the authors reported that the dichloromethane fraction of A. sieberi disrupted cell membrane integrity and induced apoptosis in MCF-7 cells. However, the study did not provide details of the underlying molecular pathways. ³⁶ The summary of the proposed mechanisms of A. sieberi extracts, as described in the included studies, is illustrated in Figure 2.

Figure 2.

Summarize the mechanisms of Artemisia sieberi extracts in inducing apoptosis and cytotoxic effects in breast cancer as mentioned in included studies.

Discussion

A. sieberi is considered a rich source of several clinically important chemical compounds and secondary metabolites, including terpenoids, phenolic compounds such as flavonoids, phenolic acids, quinones, coumarins, lignans, stilbenes, and tannins, as well as alkaloids, amines, betalains, and carotenoids. The presence of these compounds suggests that A. sieberi may have potential for medical applications, as it exhibits promising antioxidant, anticancer, and cytotoxic activities in vitro.^40,41

A study conducted by Break et al (2023) revealed some of the active ingredients in A. sieberi essential oils. In this study, GC/MS analysis reported that A. sieberi essential oil consists of cis-crysanthenyl acetate, davanone, 1, 8-cineole, and caryophyllene diepoxide, and they suggested that these compounds may contribute to the bioactivity of A. sieberi essential oil (Mohaddese et al, 2015). Many of these detected chemical compounds have been reported for their anti-cancer and antioxidant activities. Davanone is a member of oxolanes that have also been detected in several Artemisia species, ⁴² and this group is known for its potential anticancer and antioxidant properties. ⁴³ Regarding the 1,8-cineole, its presence in Artemisia has been previously reported, ⁴⁴ and its extensive pharmacological properties have been associated with the antioxidant activities of plant essential oils. ⁴⁵ Moreover, caryophyllene diepoxide is also one of the compounds of Artemisia essential oils^46,47 and has been reported for its potential antioxidant activities in animal models. ⁴⁸ All these previously published results support the findings of Break et al (2023) regarding the composition and potential biological properties of A. sieberi essential oil.

Break et al (2023) also reported that the essential oil extract exhibited its highest cytotoxic activity against MCF-7 cells, and oil-treated MCF-7 cells showed down-regulation of total ERK proteins and its target LC3, indicating potential inhibition of the ERK signaling pathway and suggesting reduced cancer cell growth. ²⁸ The ERK pathway is one of the most studied signal transduction pathways. ⁴⁹ In humans, feedback loops modulate the ERK signal transduction pathway during normal physiological and developmental processes to maintain cell reproduction, survival, and homeostasis. ⁵⁰ It was previously reported that certain genetic alterations in the ERK cascade effector molecules and/or regulatory components may lead to dysregulation of the ERK cascade, and its upregulation may contribute to the development of human cancers.^49,51 While Break et al (2023) observed downregulation of total ERK protein in MCF-7 cells, highlighting potential inhibition at the protein level, the ERK/MAPK pathway as a whole plays a key role in TNBC progression by promoting cell proliferation, survival, and metastasis^52,53 Small-nucleolar RNA host gene 14 (SNHG14) plays a functional role in TNBC, and analysis of its regulatory profile showed that its effects are linked to targets and pathways involving the MAPK cascade, including ERK. Experimental data demonstrated that silencing SNHG14 reduced the proliferation, migration, and invasion of TNBC cells in vitro through modulation of the ERK/MAPK signaling pathway and that its inhibition can suppress tumor aggressiveness. ⁵³ The other study shows that the ERK pathway enhances the invasive ability of TNBC cells by inducing expression of MM-1, a regulatory protein that promotes extracellular matrix degradation. Thus, ERK inhibition may offer a potential approach to limit metastasis in this cancer subtype. ⁵⁴ At a broader level, previous reports indicate that nearly 40% of human cancers are associated with an abnormal elevation of ERKp. ⁵⁵ Tumors harboring genetic alterations in ERK effectors often exhibit more complex clinical characteristics compared with those lacking such alterations, suggesting that these modifications may hold prognostic significance. ⁵⁶ For more than two decades, inhibition of the ERKp signaling pathway has been emphasized in cancer research and targeted therapy studies.^49,56 The potential effectiveness of the ERK pathway inhibition in cancer treatment arises from the pathway's Janus-faced immune regulatory effects. Briefly, ERKp inhibition can activate the associated T-helper cells and cytotoxic T-cells, potentially triggering an immune response against cancerous cells. ⁵⁷ Moreover, it may also activate dendritic cells, which play a central role in inhibiting immune response by acting as antigen-presenting cells.^49,57

Other types of A. sieberi extracts have been investigated in Abutaha et al (2015) study. This study examined the anti-cancer effects of aerial parts crude methanol, dichloromethane, hexane, and ethyl acetate extracts and demonstrated that dichloromethane and methanolic extracts showed anti-proliferative properties on MCF7 cell lines, while hexane and ethyl acetate fraction showed no anti-proliferative activity. ³⁶ According to available data, more future investigations are recommended to improve the knowledge regarding A. sieberi dichloromethane and methanolic extracts activity against breast cancer cells, with particular emphasis on the hard-to-treat types, including TNBC.

Additionally, Maodaa et al (2024) and Nasr et al (2020) utilized the methanol extract of A. sieberi in their analysis and reported its cytotoxicity against the tested cancer cells.^22,38 It was previously demonstrated that A. sieberi methanol extract is rich in several functional groups, including OH, CH, C=O, and aromatic skeletal stretches. ⁴⁰ Importantly, some studies did not perform a comprehensive phytochemical analysis of A. sieberi extracts, which limits the attribution of the observed cytotoxic effects to specific bioactive compounds. Despite these limitations, the anticancer potential of A. sieberi methanol extracts has been demonstrated in several cancer types, including breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and lung cancer. ²⁹ Active substances in A. sieberi were reported for their ability to inhibit cancer cell growth by targeting various molecules and signaling pathways. The molecular mechanisms underlying the anticancer activity of A. sieberi include apoptosis induction, ferroptosis by generation of ROS, angiogenesis inhibition, and cell cycle arrest. ²⁹

Nasr et al (2020) study revealed that A. sieberi showed cytotoxic effects against tested cancer cell lines, but it was less than the effects exerted by A. Judaica. However, A. sieberi indicated higher antioxidant activity than A. Judaica. The anticancer activity of A. sieberi was defined by its artemisinin (ART) and other sesquiterpene lactones composition, while the antioxidant activity was explained by its high phenolic and flavonoid compound content. ²²

In a previous study, Tin et al (2012) demonstrated that artemisinin, a sesquiterpene lactone originally isolated from Artemisia annua and identified in A. sieberi, exhibits anti-proliferative activity against human breast cancer cells. The study showed that artemisinin induced cell cycle arrest by downregulating the expression of the E2F1 transcription factor, which mediates the inhibition of cell proliferation. ⁵⁸ The activity of artemisinin as an anti-cancer agent has been reported in several previous studies, with different mechanisms of action proposed. Ma et al (2021) revealed that artemisinin caused a dose-dependent cell cycle arrest. ²⁹ Artemisinin induces G1-phase arrest mainly through the downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression, leading to the repression of cell proliferation, as well as through the upregulation of the tumor suppressor protein p16. ⁵⁹ In addition, G2/M-phase arrest associated with autophagy induction has also been observed in breast cancer cells treated with artemisinin. ²⁹

In addition, El-Wassimy et al (2019) tested the sesquiterpene lactone and flavonoid for their cytotoxic effects against MCF-7 cells, and they found that these compounds exhibited potential cytotoxic activity, although the study did not report the mechanism of action or the statistical significance of the results. ³⁷ Several previously published studies have explored the potential anticancer activities of sesquiterpene lactones. These compounds have demonstrated efficacy in inducing G1/G0 phase cell cycle arrest and apoptosis in various cancer models. ⁶⁰ Regarding flavonoids, they are known for their wide variety of potential anticancer effects, as they may modulate ROS-scavenging enzyme activities, participate in cell cycle arrest, induce apoptosis and autophagy, and suppress cancer cell proliferation and invasiveness. ⁶¹

The lack of selective toxicity remains one of the major limitations of many cancer therapeutic agents. Therefore, the development of efficient delivery systems could improve the targeted delivery of anti-tumoral agents, potentially minimizing side effects on normal cells and enhancing treatment efficiency. ⁶² In fact, approximately 50% of all internationally approved anti-cancer drugs are derived from natural products, their metabolites, or synthetic derivatives inspired by naturally occurring micro- or macromolecules. However, achieving selective toxicity remains essential to maximize efficacy against cancer cells while minimizing toxicity toward normal tissues. ⁶³ Nanotechnology has enabled the development of various cancer therapeutic delivery systems aimed at achieving better treatment outcomes with fewer side effects. The use of nanoparticles as drug carriers may enhance the selectivity of drug delivery to cancer cells through several mechanisms, including improving drug solubility and stability, prolonging circulation half-life, reducing adverse effects on normal cells, and concentrating drugs at the tumor site.^63,64

Among the included studies, Irani et al (2024) investigated the combined use of natural plant extracts (A. sieberi oil extracts) and nanotechnology (silver chloride nanoparticles) to evaluate the potential anticancer properties of these formulations. Their results showed that silver chloride nanoparticles-A. sieberi extract (AgClNPs-ASE) treatment led to an increase in nitric oxide (NO) production in cancer cells and upregulated the expression of the long non-coding RNA GAS5 (lncRNA GAS5), which is transcribed from the Growth Arrest Specific 5 Gene (GAS5). Moreover, AgClNPs-ASE induced apoptosis in cancer cells under in vitro conditions. ³⁵ GAS5 is a type of IncRNA that plays a tumor-suppressive role in the development of TNBC. One proposed mechanism is that GAS5 inhibits cell proliferation and invasion by competing for binding with miR-196a-5p, thereby reducing the effect of this microRNA on cancer cell growth. These findings support the potential of GAS5 as a prognostic biomarker for TNBC progression.^65,66 In cellular experiments, overexpression of GAS5 clearly reduced cell proliferation and increased apoptosis in TNBC, confirming that GAS5 contributes to suppressing the aggressive growth of cancer cells. ⁶⁶ These findings are indicating that increasing GAS5 can be an effective strategy to control TNBC, either alone or in combination with innovative treatments such as natural compounds or nanotechnology. Moreover, the results indicate that decreased levels of GAS5 in TNBC cells are associated with chemoresistance and a reduced capacity for apoptosis, while overexpression of GAS5 enhances the sensitivity of the cells to chemotherapy, that support the potential role of GAS5 as a tumor suppressor in TNBC^65,67 It has been previously reported that abnormal methylation of the GAS5 gene is associated with its downregulation in cervical cancer and that the upregulation of lncRNA GAS5 may suppress the proliferation and metastasis of cervical cancer cells. ⁶⁸ Increased NO concentrations have also been reported to contribute to cytotoxic effects and apoptosis induction in cancer cells. ⁶⁹

The QUIN tool showed that some studies were low-risk, such as Break et al (2023), Irani et al (2024), and Nasr et al (2020), which makes their results reliable, while moderate-risk studies, such as Maodaa et al (2024), El-Wassimy et al (2019), and Abutaha et al (2015), should be interpreted with caution due to methodological limitations.

While our review found limited studies for A. sieberi (all in vitro, conducted in Saudi Arabia and Iran), a study was conducted in Korea that evaluated the combined effect of polyphenols extracted from Korean A. annua L (pKAL) with the drug Docetaxel (DTX) on cancer cells. The results showed that pKAL significantly enhances the anticancer effect of DTX in colorectal cancer HCT116 cells with wild-type p53 through specific molecular changes, suggesting its potential to improve the efficacy of DTX-containing chemotherapy in tumors with functional p53. ⁷⁰ Other reviews of A. annua have included studies from a broader geographical scope. For example, Chinese research provided the historical foundation and the initial discovery of artemisinin, while additional laboratory and clinical studies were conducted in Western countries (Europe and the United States). ⁷¹ This broad geographical and institutional representation highlights the global interest in Artemisia species and clearly contrasts with the limited evidence available on A. sieberi. The geographic confinement of A. sieberi studies to Saudi Arabia and Iran limits the generalizability of current findings. Broader research across diverse regions is needed to verify these observations, expand the evidence base, and confirm their applicability.

Despite the promising findings, this study has several limitations that should be acknowledged. The generalizability of the results to animal or clinical models remains limited, and long-term effects or potential interactions within the tumor microenvironment were not addressed. These factors indicate that the current evidence, while valuable, is insufficient for direct clinical application. Therefore, further in vivo studies and applied research, particularly in triple-negative breast cancer (TNBC), are needed to validate these findings and explore their therapeutic potential.

Gap of Knowledge and Future Directions

According to the search conducted and the review of previously published articles, it can be noticed that the anticancer potential of A. sieberi against breast cancer remains insufficiently explored, and most existing studies have focused primarily on preliminary cytotoxicity assessment using assays such as cell viability assays like MTT, with limited understanding of the molecular pathways and mechanisms of action, although some studies, such as Braek et al (2023), have provided initial insights into these mechanisms. Notably, only one study has indirectly investigated the effectiveness of A. sieberi extract as an anticancer agent in TNBC through the synthesis of silver chloride nanoparticles, reporting encouraging results. These results suggest that broader and more rigorous investigations are needed, including studies on different types of A. sieberi extracts and various breast cancer subtypes, particularly TNBC, which is difficult to treat. A major limitation of some current studies is the lack of detailed phytochemical analysis of A. sieberi extracts, making it difficult to link the observed anticancer effects to specific compounds. Therefore, future studies should include comprehensive chemical profiling to identify the active constituents, improve methodological rigor, and ensure adequate sample sizes and appropriate comparator groups. In addition, in vivo validation is recommended to confirm the in vitro findings, and further investigation into molecular pathways and tumor microenvironment effects is warranted.

Conclusion

The potent anti-cancer activity of A. sieberi has been reported in multiple cancer types, including breast, prostate, ovarian, pancreatic, and lung cancers. A. sieberi extracts may inhibit cancer cell growth through several mechanisms, including induction of apoptosis, cell cycle arrest, and modulation of key signaling pathways. However, the anticancer potential of A. sieberi against breast cancer remains insufficiently studied, and the properties of its organic extracts have only been explored at a preliminary level. Therefore, further research is needed to investigate the effects of different A. sieberi extracts on various breast cancer cell lines, particularly the more difficult-to-treat subtypes such as triple negative breast cancer (TNBC).

Supplemental Material

sj-docx-1-chp-10.1177_2515690X261418415 - Supplemental material for In Vitro Anti-Cancer Effects of Artemisia sieberi on Human Breast Cancer Cells: A Systematic Review

Supplemental material, sj-docx-1-chp-10.1177_2515690X261418415 for In Vitro Anti-Cancer Effects of Artemisia sieberi on Human Breast Cancer Cells: A Systematic Review by Fatimah G. Albani, Entissar S. Alsuhaibani and Sahar S. Alghamdi in Journal of Evidence-Based Integrative Medicine