Potential Therapeutic and Medicinal Applications of Four Invasive Non-Native Plant Species: A PRISMA-Guided Systematic Review of PubMed Studies

Abstract

Invasive non-native plant species are of ecological concern globally, as they may negatively affect biodiversity, the economy, and human health. At the same time, invasive non-native plants comprise an underutilised biomass that contains valuable natural bioactive compounds, which could find various biomedical applications and potential medicinal uses. In this paper, we aimed to systematically review the published data surrounding four selected invasive non-native plant species in a medical and therapeutic context. The search was conducted using PubMed and PRISMA guidelines, and strict criteria were employed to provide a thorough framework for the study selection process. After rigorous screening of the 53 selected articles, we were able to summarise the main findings and current knowledge regarding the valorisation opportunities for the selected plants in a medical context and to identify research gaps and highlight further research opportunities. Finally, we concluded that the selected invasive non-native plant species may provide valuable services in the biomedical field if the focus of future research is concentrated on their potential applicability in clinical settings. Furthermore, the valorisation of invasive non-native plant species may prove to be a viable strategy for controlling their spread.

1. Introduction

1.1. The Impact of Invasive Non-Native Plant Species and Their Possible Valorisation

Invasive non-native plant species (INPS) are those plant species that are introduced into a new ecosystem to which they do not naturally belong, either through natural means (wind, water, animals, etc.) or through human activity (directly and indirectly). Some of the introduced plant species may become naturalized in their non-native ecosystems and thus cause no harm; however, others tend to spread uncontrollably, while at the same time causing damage to the local biodiversity (flora and fauna) and disturbances of the soil, potentially causing harm to humans, domestic animals, pets, and the economy. Among the human-assisted factors contributing to the spread of INPS are increased travel and tourism activities, increased global trading, climate change, and intentional or unintentional use of non-native plant species as decorative plants [1,2,3,4,5].

The impact of INPS spreads across multiple domains, and significant efforts are being made to eradicate them. Most countries adhere to action plans for controlling these plant species, which generally aim to reduce the social and economic harm that they cause. The action plans usually consist of educating and involving communities in spotting and reporting newly introduced species, planning out and implementing control management actions at the authority level (through chemical control, uprooting, or controlled fire), and by strictly regulating the handling of these plant species through local, nationwide, or union-level laws [6,7,8,9]. At the European Union (EU) level, the Invasive Alien Species Regulationspecifies a list of strictly regulated non-native species, some of which are considered of Union concern but are not strictly regulated. According to this law, all member states are required to take action to prevent the introduction of new non-native species in their territories, to take measures for early detection and quick eradication of these species, and to find feasible ways to manage the spread of already widely established non-native species in their territories. More importantly, species that are strictly regulated are prohibited from being kept, imported, sold, bred, grown, and/or released into the environment [10].

Conventional methods for the management of the spread of INPS are very costly, sometimes ineffective, and often result in wasted biomass [11,12,13]. Considering that resources in general are limited, management methods such as finding value in INPS by collecting and utilising them through the valorisation of plant biomass could potentially be a more viable spread control alternative [14,15]. This method includes, but is not limited to, the extraction and purification of bioactive compounds with medicinal, therapeutic, and cosmetic applications, transforming the plant mass into various materials (fibres, paper, etc.), livestock feed, bioenergy/biofuel, biochar, compost, and wood products, all of which could contribute to covering at least some parts of the cost of eradication, a concept that is known as the circular economy [16,17,18,19,20,21,22,23]. Most importantly, this approach contributes to a more sustainable, cost-friendly, and more efficient management of INPS.

Plants are recognized as a significant source of bioactive compounds that can be extracted and utilized in the medical, pharmaceutical, and cosmetic fields. Screening plants for bioactive compounds and testing them using various assays has led to essential drug discoveries, supplement formulation, and applications in diverse biological activities, such as antioxidant, anti-inflammatory, antimicrobial, and anticancer uses, to name a few [24,25,26]. Although significant progress has been made in elucidating the therapeutic potential of plant species and the mechanisms of action of their phytochemicals, substantial gaps in knowledge persist. What makes INPS especially interesting to study in this sense is the fact that they are highly adaptable organisms with the ability to spread and compete with the native flora and fauna, and the specific conditions of the ecosystems in which they are introduced is in part due to their chemical composition, which varies based on the location [27,28,29].

1.2. Argumentation of the Study

There is growing interest in the ecological management of INPS, but their biomedical potential remains underexplored. Although numerous studies have investigated the phytochemical profiles of different plant species, there is a lack of systematic reviews focused explicitly on the therapeutic applications of INPS. The existing scientific literature tends to highlight ecological control strategies or isolated pharmacological discoveries, without synthesising the broader biomedical relevance of these species. Thus, a comprehensive evaluation of INPS as sources of bioactive compounds with potential therapeutic value is needed, especially in the context of the circular economy and sustainable resource use.

To select the INPS to be included in the literature review, databases and studies focusing on the most abundant and problematic non-native plant species established in Romania were consulted. EU regulations on invasive alien species were also considered during the selection process. The first compiled database of invasive and potentially invasive plant species in Romania was published in 2022. After the authors of the aforementioned study performed a literature review, they identified 102 plant species of concern [30]. Following this, the first-ever nationwide field survey in Romania brought new information regarding the distribution of non-native plant species. It revealed that there are 396 species belonging to 77 families established in Romania, updating the initially published database. Of the invasive plants established and widespread on Romania’s territory, four are also of EU concern: Ailanthus altissima, Asclepias syriaca, Elodea nuttallii, and Impatiens glandulifera. The study also proposed a set of seven plants of interest for Romania: Ambrosia artemisiifolia, Ambrosia tenuifolia, Ambrosia trifida, Cyclachaena xanthiifolia, Phytolacca americana, Phytolacca acinosa, and Verbesina encelioides, from which three were considered to exert a significant impact and to require even more focused management plans and measures: Ambrosia artemisiifolia, Ambrosia trifida, and Phytolacca americana [31].

The spread of INPS in national parks and protected natural areas poses an even greater challenge, since in these areas, chemical control is strictly prohibited. Several studies have highlighted potentially damaging INPS that are spreading and causing biodiversity loss within these protected areas. Dumitrascu M et al. performed a survey of several protected areas in Romania, including the Danube Delta Biosphere Reserve, the Mureș Floodplain, and Comana Natural Park. They identified Amorpha fruticosa as being the INPS with the most negative impact on these protected ecosystems [32].

For the present study, we have selected the following INPS to be included, based on their impact, spread, and possible valorisation opportunities for therapeutic uses as means of managing their population numbers: Ailanthus altissima, Amorpha fruticosa, Asclepias syriaca, and Phytolacca americana.

1.3. Study Objectives

The primary objective of this systematic review is to critically evaluate the current scientific literature concerning the therapeutic and medicinal applications of four invasive non-native plant species: Ailanthus altissima, Amorpha fruticosa, Asclepias syriaca, and Phytolacca americana. The selected INPS, while ecologically problematic, possess underutilised biomass rich in bioactive compounds with potential biomedical value. By synthesising existing research, the study aims to identify and highlight the therapeutic potential of INPS, including their antioxidant and antimicrobial properties, as well as their cytotoxic and neuroprotective effects.

Additionally, the review proposes to uncover gaps in current knowledge and suggest future research directions that could support the sustainable valorisation of these invasive species. Our approach aims to contribute to the advancement of natural product-based therapeutics and to offer an innovative strategy for managing INPS through their biomedical exploitation, aligning with principles of a circular economy and ecological sustainability.

2. Methodology

2.1. Botanical Terminology

All four botanical names were cross-checked for accuracy with Plants of the World Online (https://powo.science.kew.org/, accessed on 16 June 2025), the International Plant Names Index (IPNI) (https://www.ipni.org/, accessed on 16 June 2025), and the World Flora Online (WFO) Plant List (https://wfoplantlist.org/, accessed on 16 June 2025) [33,34,35]. The native or non-native status of the plants in each study was checked using Plants of the World Online (https://powo.science.kew.org/, accessed on 16 June 2025) [33].

2.2. Data Collection

For this systematic review, we followed the PRISMA protocol and guidelines to effectively and accurately identify, screen, and include relevant articles for the chosen topic. Rayyan (Cambridge, MA, USA), an AI-powered web-based platform designed to simplify the process of conducting systematic reviews, was used for article screening [36].

Searches were conducted on PubMed, considering reviews focusing on the medicinal and therapeutic use of the selected plants. The keywords used were the botanical names of the plants included in the study, as follows: “Ailanthus altissima”, “Amorpha fruticosa”, “Asclepias syriaca”, and “Phytolacca americana”. The screening process was conducted independently by two reviewers. Discrepancies during the study selection process were resolved through discussion, and when consensus could not be reached, a third reviewer served as an arbitrator.

The quality of the included studies was assessed narratively, considering methodological aspects such as whether the study design was suitable for the stated objectives, the level of detail and reproducibility of methods, the traceability of the sample, how confounding factors were managed, and transparency in reporting the results. No formal validated instrument was used because the primary goal of the analysis was descriptive, aiming to synthesize and contextualize existing evidence without applying a standardized quality classification.

The inclusion criteria for the studies were the following:

1.

The cited research must utilise a traceable plant material or isolated phytocompound, with either a deposited voucher specimen or a clearly stated source.

2.

The cited articles must be written in English or have an English translation available.

3.

Only original research articles were considered; reviews, commentaries, and other non-original formats were excluded.

4.

The study must evaluate at least one type of biological activity, conducted either in vitro, in vivo, or both.

2.3. Data Analysis

The data obtained during the article screening process were manually recorded and managed using Microsoft Excel for Microsoft 365 (Version 2508). Visualisation of data and graphics was conducted manually using Microsoft Excel for Microsoft 365 (Version 2508).

3. Results

3.1. Literature Screening

The literature search was performed on 26 April 2025 using PubMed, yielding a total of 1100 articles as follows: for Ailanthus altissima, the search provided a total of 408 results; for Amorpha fruticosa, a total of 132 results; for Asclepias syriaca, a total of 173 results; and Phytolacca americana, a total of 387 results. No lower time limit for the publication date was applied, while the upper limit was 26 April 2025.

Following the initial literature search, duplicates (n = 14) were removed automatically by Rayyan, and 1086 articles were included in the initial screening process, which consisted of title screening for relevancy to the topic. After the title screening, 939 articles were excluded because they were unrelated to medicinal uses. Next, 147 reports were sought out for full-text retrieval, and a handful of them were not retrieved (n = 38); therefore, they were excluded from this review. Full-text screening was performed on 109 articles, and after rigorous screening of the data presented and applying the inclusion criteria, we established a final number of 53 articles to be included in the study. Thus, from the articles considered relevant to the topic of the study, 48.6% also met the inclusion criteria. The screening process is presented in more detail in Figure 1.

3.2. Studies Overview

The included studies were analysed rigorously, and relevant data regarding therapeutic and medicinal uses were extracted from them. A general overview of all the included studies can be found in

Table 1. Summary of included original research articles by plant species, biological activity, origin of plant material or compounds, and non-native status.

Author, Year	Plant/Compound	Activity	Origin	Non-Native (Yes/No)	Reference
Zhuo Z et al., 2015	Ailanthone	Cytotoxic	Jinan University, Guangzhou, China	N/A	[37]
Wei C et al., 2018	Ailanthone	Cytotoxic	Jinan University, Guangzhou, China	N/A	[38]
Han F et al., 2018	Ailanthone	Cytotoxic	Puruifa Science & Technology Development Co., Chengdu, China	N/A	[39]
Daga M et al., 2019	Ailanthone	Cytotoxic	Baoji Herbest, Bio-Tech Co., Ltd., Baoji, China	N/A	[40]
Cucci MA et al., 2020	Ailanthone	Cytotoxic	Baoji Herbest, Bio-Tech Co., Baoji, China	N/A	[41]
Ding H et al., 2021	Ailanthone	Cytotoxic	Shanghai Yiyan Biotechnology Co., Ltd., Shanghai, China	N/A	[42]
Wang S et al., 2022	Ailanthone	Cytotoxic	Jiangxi Herb Tiangong Technology, Nanchang, China	N/A	[43]
Zhang Y et al., 2022	Ailanthone	Cytotoxic	GlpBio Technology, Montclair, CA, USA	N/A	[44]
Wang Y et al., 2023	Ailanthone	Cytotoxic	MedChemExpress Ltd., Monmouth Junction, NJ, USA	N/A	[45]
Fang C et al., 2024	Ailanthone	Cytotoxic	Dasfbio Nanjing, Nanjing, China	N/A	[46]
Yang H et al., 2024	Ailanthone	Cytotoxic	Chengdu Alfa Biological Technology Co., Ltd., Chengdu, China	N/A	[47]
Liang J et al., 2025	Ailanthone	Cytotoxic	GlpBio Technology, Montclair, CA, USA	N/A	[48]
Li J et al., 2025	Ailanthone	Cytotoxic	ChemFaces, Wuhan, China	N/A	[49]
Tamura S et al., 2003	Ailanthus altissima	Antiviral	Taiwan	No	[50]
Okunade AL et al., 2003	Ailanthus altissima	Antimalarial	United States	Yes	[51]
Chang Y et al., 2003	Ailanthus altissima	Antiviral	South Korea	Yes	[52]
De Feo V et al., 2005	Ailanthus altissima	Cytotoxic	Italy	Yes	[53]
Jin MH et al., 2006	Ailanthus altissima	Anti-inflammatory	South Korea	Yes	[54]
Ammirante M et al., 2006	Ailanthus altissima	Cytotoxic	Italy	Yes	[55]
Wang Y et al., 2013	Ailanthus altissima	Cytotoxic	China	No	[56]
Kim HM et al., 2015	Ailanthus altissima	Anti-inflammatory	South Korea	Yes	[57]
Kim H et al., 2016	Ailanthus altissima	Anti-inflammatory	South Korea	Yes	[58]
He Q et al., 2018	Ailanthus altissima	Cytotoxic	China	No	[59]
Wang R et al., 2018	Ailanthus altissima	Cytotoxic	China	No	[60]
Yan ZY et al., 2018	Ailanthus altissima	Cytotoxic	China	No	[61]
Jeong M et al., 2018	Ailanthus altissima	Cytotoxic	South Korea	Yes	[62]
Rahman HMA et al., 2019	Ailanthus altissima	Hypotensive Anticoagulant Smooth muscle relaxant	Pakistan	Yes	[63]
Du YQ et al., 2019	Ailanthus altissima	Neuroprotective	China	No	[64]
Yan ZY et al., 2020	Ailanthus altissima	Cytotoxic	China	No	[65]
Mo Y et al., 2021	Ailanthus altissima	Antioxidant	Bozhou Baohua Pharmaceutical Co., Ltd. Bozhou, China	N/A	[66]
Kim SR et al., 2022	Ailanthus altissima	Anti-inflammatory	South Korea	Yes	[67]
Gao ZH et al., 2022	Ailanthus altissima	Cytotoxic	China	No	[68]
Muhammad Abdur Rahman H et al., 2023	Ailanthus altissima	Neuroprotective Antioxidant Anti-enzymatic	Pakistan	Yes	[69]
Boukhibar H et al., 2023	Ailanthus altissima	Antibacterial	Algeria Tunisia	Yes	[70]
Andonova T et al., 2023	Ailanthus altissima	Antioxidant DNA protective	Bulgaria	Yes	[71]
Song Q et al., 2024	Ailanthus altissima	Anti-enzymatic	China	No	[72]
Cocîrlea MD et al., 2024	Ailanthus altissima	Antioxidant Antibacterial	Romania	Yes	[73]
Cselőtey A et al., 2024	Ailanthus altissima	Antibacterial	Hungary	Yes	[74]
Weidner C et al., 2012	Amorfrutin	Antidiabetic	Analyticon Discovery	N/A	[75]
Kim YS et al., 2011	Amorpha fruticosa	Antibacterial	South Korea	Yes	[76]
Wu X et al., 2015	Amorpha fruticosa	Cytotoxic	China	Yes	[77]
Cui X et al., 2017	Amorpha fruticosa	Cytotoxic	China	Yes	[78]
Lee W et al., 2016	Amorpha fruticosa	Antidiabetic	South Korea	Yes	[79]
Jankovská D et al., 2024	Amorpha fruticosa	Anti-enzymatic	Czech Republic	Yes	[80]
Schlick J et al., 2000	GnRH-PAP	Cytotoxic	N/A	N/A	[81]
Ishag HZ et al., 2013	PAP	Antiviral	Huazhong University of Science and Technology, Wuhan, China	N/A	[82]
Mansouri S et al., 2009	PAP	Antiviral	N/A	N/A	[83]
Zhabokritsky A et al., 2014	PAP	Antiviral	N/A	N/A	[84]
Takahasi H et al., 2003	Phytolacca americana	Neurotrophic	Japan	Yes	[85]
Zheleva-Dimitrova Dzh et al., 2013	Phytolacca americana	Antioxidant Anti-enzymatic	Bulgaria	Yes	[86]
Patra JK et al., 2014	Phytolacca americana	Antibacterial	South Korea	Yes	[87]
Saleri FD et al., 2017	Phytolacca americana	Cytotoxic	China	Yes	[88]
Popovici LF et al., 2025	Phytolacca americana	Anxiolytic, Anti-enzymatic	Romania	Yes	[89]

N/A = not applicable; PAP = pokeweed antiviral protein; GnRH-PAP = pokeweed antiviral protein fused with gonadotropin-releasing hormone.

. The studies that passed the criteria evaluation and quality assessment for each studied plant are as follows: 38 studies on Ailanthus altissima, 6 studies on Amorpha fruticosa, and 9 studies on Phytolacca americana. Although the plant Asclepias syriaca was included in the initial literature search, none of the retrieved articles met the evaluation criteria because they were either irrelevant to the topic or did not report at least one type of activity assay that could be correlated to a therapeutic or medicinal use. Considering that understanding the potential use in these fields constituted the main objective of this study, the identified studies that did not meet this criterion were excluded. Among them, we have identified and included studies focusing on testing the properties of both crude extracts and isolated compounds, either obtained directly from the plant through isolation or by purchasing and directly testing the purified compound of the selected plant origin.

Data analysis was conducted to identify patterns and trends in publishing, as well as the distribution of biological activities that were assessed, as found in Figure 2. Most of the studies included, i.e., 47%, evaluated the cytotoxic potential of the selected INPS and of some isolated compounds obtained from them on various cell lines to serve as preliminary research for potentially developing new antitumor agents. The antimicrobial activity, including antibacterial and antiviral assays, is also a frequent topic of research among the selected studies (17% of the total studies), followed by antioxidant activity (8% of the total studies) and anti-inflammatory activity (7% of the total studies). Amongst other tested activities, we noticed more specific types of activities, such as various enzyme inhibition assays, neuroprotective effect assays, DNA protective assays, and cardiovascular effect assays, to name a few.

In the following sections, each study will be discussed in more detail with a focus on the therapeutic/medicinal use analysed, the potential applications in the medical field, the limitations and gaps identified, and the future directions of research for these specific topics.

3.3. INPS and Their Antimicrobial Potential

3.3.1. The Antibacterial Activity of INPS

There is no doubt that the search for new antibacterial agents is an essential and currently ongoing topic of research in the medical and pharmaceutical fields. Bacterial resistance to the conventionally used antibiotics is on the rise due to the high adaptability of bacteria and their overall evolutionary nature, the inappropriate prescribing and use of antibiotics (overuse and misuse), environmental contaminations, the use of large-spectrum antibiotics, and the under-utilisation of antibiograms in medical practice [90,91,92,93].

Plants have been used historically for their antibacterial properties [94], and screening the phytocomponents of plants is one of the strategies for discovering new therapeutic agents that could help combat the crisis of bacterial resistance [95,96,97]. For the chosen plants, we have identified a range of tested bacterial strains, including a clinical isolate. The growth inhibition activity was tested on crude extracts, chromatography-fractionated extracts from various plant parts, and on some isolated compounds. The findings are summarised in

Table 2. Overview of antibacterial activity reported in selected studies, including bacterial strains tested, type of extract or isolated compound, plant material and origin, experimental model (in vitro or in vivo), and reference. Only effective results are presented, ordered after the tested bacterial strain.

Bacterial Strain	Growth Inhibition Capability	Extract Type/Isolated Compound	Plant Material/Origin	In Vivo/In Vitro	Reference
Bacillus subtilis ATCC 6633	IZ = 8.00	Hydroethanolic crude extract of dried autumn leaves.	Ailanthus altissima, leaf, Romania	In vitro	[74]
Bacillus subtilis F1276	MIC = 0.07	13-HODE isolated from fractionated methanolic crude young stem bark extract.	Ailanthus altissima, bark, Hungary	In vitro	[74]
	MIC = 0.07	9-HODE isolated from fractionated methanolic crude young stem bark extract.
	MIC = 0.07	Juniperic acid isolated from fractionated methanolic outer trunk bark extract.
	MIC = 0.01	Canthin-6-one isolated from fractionated methanolic inner trunk bark extract.
Clostridium perfringens Neuraminidase (E.C. 3.2.1.18.)	IC50 = 4.15	Amoradicin isolated from the hexane–acetone fraction.	Amorpha fruticosa, root, South Korea	In vitro	[76]
	IC50 = 0.12	Amorisin isolated from the hexane–acetone subfraction.
	IC50 = 7.86	Isoamoritin isolated from the hexane–acetone subfraction.
	IC50 = 22.03	Amoricin from the hexane–acetone subfraction.
	IC50 = 12.94	Amorphigenin from the 80% methanolic subfraction.
	IC50 = 16.74	Dalbinol isolated from the hexane–acetone subfraction.
	IC50 = 8.34	6-ketodehydroamorphigenin isolated from the hexane–acetone subfraction.
Enterococcus faecalis ATCC 29212	IZ = 8.00	Hydroethanolic crude extract of frozen summer leaves.	Ailanthus altissima, leaf, Romania	In vitro	[73]
	IZ = 9.00	Hydroethanolic crude extract of frozen autumn leaves.
	IZ = 9.00	Hydroethanolic crude extract of dried summer leaves.
	IZ = 10.00	Hydroethanolic crude extract of dried autumn leaves.
Escherichia coli ATCC 25922	MIC = 31.25	Crude 80% methanolic extract, macerated.	Ailanthus altissima, leaf, Blida, Algeria	In vitro	[70]
	MIC = 8.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	MIC = 31.25		Ailanthus altissima, leaf, Bizerte, Tunisia
	MIC = 31.25		Ailanthus altissima, leaf, Sousse, Tunisia
	MBC = 250.00		Ailanthus altissima, leaf, Blida, Algeria
	MBC = 250.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	IZ = 9.00	Hydroethanolic crude extract of frozen summer leaves.	Ailanthus altissima, leaf, Romania	In vitro	[73]
	IZ = 10.00	Hydroethanolic crude extract of dried autumn leaves.
Porphyromonas gingivalis W83 ATCC BAA-1703	MIC = 0.20	Crude 80% methanolic	Phytolacca americana, leaf and soft stem, South Korea	In vitro	[87]
	MIC = 0.20	Hexane fraction
	MIC = 0.20	CHCl3 fraction
Pseudomonas aeruginosa	BI: 29.60	Isoamoritin isolated from the hexane–acetone subfraction.	Amorpha fruticosa, root, South Korea	In vivo	[76]
	BI: 21.00	Dalbinol isolated from the hexane–acetone subfraction.
Pseudomonas aeruginosa ATCC 27853	MIC = 31.25	Crude 80% methanolic extract, macerated.	Ailanthus altissima, leaf, Blida, Algeria	In vitro	[70]
	MIC = 16.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	MIC = 125.00		Ailanthus altissima, leaf, Bizerte, Tunisia
	MIC = 72.50		Ailanthus altissima, leaf, Sousse, Tunisia
	MBC = 250.00		Ailanthus altissima, leaf, Tlemcen, Algeria
Staphylococcus aureus ATCC 25923	MIC = 4.00	Crude 80% methanolic	Ailanthus altissima, leaf, Blida, Algeria	In vitro	[70]
	MIC = 16.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	MIC = 4.00		Ailanthus altissima, leaf, Bizerte, Tunisia
	MIC = 4.00		Ailanthus altissima, leaf, Sousse, Tunisia
	MBC = 16.00		Ailanthus altissima, leaf, Blida, Algeria
	MBC = 72.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	MBC = 16.00		Ailanthus altissima, leaf, Bizerte, Tunisia
	IZ = 8.00	Hydroethanolic crude extract of dried summer leaves.	Ailanthus altissima, leaf, Romania	In vitro	[73]
	IZ = 8.00	Hydroethanolic crude extract of dried autumn leaves.
Staphylococcus aureus Clinical isolate	IZ = 8.00	Hydroethanolic crude extract of frozen summer leaves.	Ailanthus altissima, leaf, Romania	In vitro	[73]
	IZ = 8.00	Hydroethanolic crude extract of frozen autumn leaves.
	IZ = 10.00	Hydroethanolic crude extract of dried summer leaves.
	IZ = 10.00	Hydroethanolic crude extract of dried autumn leaves.
Staphylococcus epidermidis ATCC 2059	MIC = 72.25	Crude 80% methanolic extract, macerated.	Ailanthus altissima, leaf, Blida, Algeria	In vitro	[70]
	MIC = 8.00		Ailanthus altissima, leaf, Tlemcen, Algeria
	MIC = 16.00		Ailanthus altissima, leaf, Bizerte, Tunisia
	MIC = 16.00		Ailanthus altissima, leaf, Sousse, Tunisia
	MBC = 125.00		Ailanthus altissima, leaf, Blida, Algeria
	MBC = 125.00		Ailanthus altissima, leaf, Tlemcen, Algeria
Streptococcus mutans UA159 ATCC 700610	MIC = 1.80 (44%)	Crude 80% methanolic	Phytolacca americana, leaf and soft stem, South Korea	In vitro	[87]
	MIC = 0.20	Hexane fraction
	MIC = 0.20	CHCl3 fraction
Streptococcus pyogenes ATCC 19615	IZ = 8.00	Hydroethanolic crude extract of dried autumn leaves.	Ailanthus altissima, leaf, Romania	In vitro	[73]

MIC = minimum inhibitory concentration expressed in mg/mL. MBC = minimum bactericidal concentration expressed in mg/mL. IZ = inhibition zone expressed in mm. CHCl₃ = chloroform/trichloromethane; BI = biofilm inhibition in %; IC₅₀ = half maximal inhibitory concentration expressed in μM. 13-HODE = (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid. 9-HODE = (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid.

.

The most predominant plant material screened among the selected studies is the aerial part of Ailanthus altissima (bark and leaves). It was the only plant with studies assessing how the phytochemical composition and the activity can differ in material collected from different locations [70] or collected during different seasons and processed differently before the extraction [73]. Other INPS could also benefit from similar studies of the variation of the phytochemical content and the biological activity depending on the location, time of harvest, and pre-extraction processing techniques. Such studies would lead to a better understanding of the best time for harvesting to maximise their value and applicability, depending on each specific geographic location.

In terms of the bacteria and methods selected in the presented studies, the range varied greatly. A study conducted by Patra JK et al. assessed the bacterial growth inhibition effect of Phytolacca americana crude extracts and some of their fractions on two bacteria involved in periodontal diseases and the formation of oral caries, with the main findings being that they were highly active on P. gingivalis and moderately active on S. mutans. Therefore, it was concluded that the extract could be formulated in the form of oral hygiene products or drugs for oral diseases [87]. However, the study used no antibiotic control as a reference except for the solvent and inoculum controls. Although the bactericidal effect was evaluated, only the minimum inhibitory concentrations and growth inhibition percentages were determined. Future studies could benefit from including the determination of the minimum bactericidal concentrations, the use of antibiotic controls, and in vivo determinations.

Another interesting approach for determining antibacterial activity, aside from the conventional micro-dilution method that is most often employed, was used by Kim YS et al., where rotenoids and flavanones isolated from the root of Amorpha fruticosa were assessed for their bacterial neuraminidase inhibition capabilities. Among the isolated compounds, amorisin was 30 times more potent in inhibiting bacterial neuraminidase originating from Clostridium perfringens than the quercetin control, an already established neuraminidase inhibitor. The promising results led to determinations of the inhibition of biofilm production on live bacteria (Pseudomonas aeruginosa), in which two of the compounds isolated, isoamoritin and dalbinol, showed the best efficiency [76].

Generally, the selected studies focused on testing some common bacterial strains, both Gram-positive and Gram-negative, used in antibacterial testing and utilised the micro-dilution assay method on microplates to help determine the MIC of the tested plant extracts or isolated compounds. The limitations of these studies include the fact that they took place in the laboratory under controlled conditions. In vivo studies should also be employed to determine their activity with biological variability in mind, and studies should be performed to evaluate the mechanism of actions, pharmacokinetics, and toxicology for the most promising agents discovered. The real applicability of the results should also be assessed by comparing the results to standard antibiotic controls, where relevant.

Interestingly, some studies showcase a synergistic effect of plant extracts, particularly those rich in phenolic compounds, with conventional antibiotics, which have proved to help combat various bacterial infections, as they can potentiate the effect of antibiotics and even combat antibacterial resistance [98,99,100]. This presents another valuable research opportunity for the selected plants, given the lack of published data on this topic.

3.3.2. The Antiviral Activity of INPS

Viral infections are a significant cause of concern globally, with many causing annual epidemics that result in a loss of lives. Managing epidemics and pandemics is a challenging task; therefore, there is a constant need to find new antiviral agents, especially since viruses may adapt to classic antiviral drugs [101,102,103,104]. As with antibacterial agents, screening plants and isolating phytochemicals from them is a good strategy for discovering and developing new antiviral agents [105].

From the identified studies assessing the antiviral activity of the selected plants, the majority of the them were focused on testing the effect of pokeweed antiviral protein (PAP) on inhibiting the growth of a few viruses, such as Japanese encephalitis virus (JEV), human immunodeficiency virus 1 (HIV-1), human T-cell leukaemia virus I (HTLV-I), and Epstein–Barr virus (EBV). PAP is a plant-derived N-glycosidase ribosomal-inactivating protein, and it is isolated from the plant Phytolacca americana. Once purified from the plant, PAP can also be obtained through cloning, which is useful in laboratory settings to produce larger quantities for research purposes. Ailanthus altissima stem bark crude methanolic extract was also screened for its potential effect against HIV-1, and some isolated compounds from the aerial parts of the same plant were screened against the Epstein–Barr virus. The main results for each study are summarised in

Table 3. Summary of antiviral activity reported in selected studies, including the targeted viruses, type of extract or isolated compound, experimental model (in vitro or in vivo), and references. Only results demonstrating effective antiviral potential are included.

Virus	Result	Extract Type/Isolated Compound	Tested Cell Line/Organism	In Vivo/In Vitro	Reference
EBV	IC50 = 221.00	Ailantinol E from the methanolic extract of aerial part of Ailanthus altissima.	EBV-EA-positive Raji cells.	In vitro	[50]
	IC50 = 180.00	Ailantinol F from the methanolic extract of aerial part of Ailanthus altissima.
	IC50 = 285.00	Ailantinol G from the methanolic extract of aerial part of Ailanthus altissima.
HIV-1	450-fold reduction in virus production due to altering the splicing of RNAs	PAP (0.50, 1.00, or 2.50 μg)	Human embryonic kidney 293T cells transfected with a proviral clone of HIV-1.	In vitro	[84]
	HIV-1 fusion inhibition of 74.90%	Ailanthus altissima stem bark methanolic extract (D = 100.00 μg/mL).	HeLa-CD4+ cell lines, recombinant vaccinia virus vPE 16 with the expressed HIV-1 envelope protein gp120/41.	In vitro	[52]
HTLV-I	Suppression of HTLV-I gene expression at translational and transcriptional levels, diminishing virus production. Absence of cytotoxicity.	PAP (10.00, 25.00 and 50.00 ng)	Human embryonic kidney 293Tcells, Jur- KAT cells, and HTLV-I-infected human T-cell line.	In vitro	[83]
JEV	IC50 = 23.10 (inhibitory value of JEV titre)	PAP (0.10–50.00 μg/mL)	JEV-infected BHK-21 cells.	In vitro	[82]
	SP = 87.50	PAP (pre-infection, intraperitoneally, D = 1.00 mg/kg)	2-week-old BALB/c mice challenged with a lethal dose of JEV.	In vivo
	SP = 85.70	PAP (post-infection, intraperitoneally, D = 1.00 mg/kg)

IC₅₀ = half maximal inhibitory concentration expressed in nM (unless otherwise specified). PAP = pokeweed antiviral protein, found in Phytolacca americana. BHK-21 cells = baby hamster kidney cells. D = dose. SP = survival percentage in %; EBV-EA = Epstein–Barr virus early antigen.

.

Aside from the growth inhibition effects, Ishag HZ also noted a prophylactic effect of PAP when it was tested in vivo against JEV infection. When PAP was administered in mice before the injection of a lethal dose of JEV, a survival rate of 87.50% was obtained, which was higher than the survival rate when PAP was administered post-infection (85.70%). PAP also dose-dependently inhibited JEV replication in vitro with an IC₅₀ of 23.10 nM. PAP also showed inhibitory activity on both HIV-1 and HTLV-1 [82]. Zhabokritsky A et al. performed a mechanistic study on the effect of PAP on HIV-1, and the authors determined through Western blotting that the protein can reduce HIV-1 production by altering the splicing of viral mRNA through targeting Rev, a regulatory protein of HIV-1. Therefore, the authors concluded that PAP should be further studied for its antiviral activity against HIV-1 [84].

Another selected study by Chang YS et al. determined that the Ailanthus altissima stem bark methanolic extract was able to inhibit HIV-1 fusion by 74.90% at a dose of 100.00 μg/mL, concluding that there is a need to isolate the active components from the tested extract to find potential new anti-HIV candidates [52]. Isolated compounds from the aerial parts of Ailanthus altissima, namely the quassinoids ailantinol E, F, and G, were studied by Tamura S et al. The authors determined that they exhibited an inhibitory effect on Epstein–Barr virus early antigen activation in Raji cells (in vitro), which may prevent EBV-associated cancers [50]. Similarly, antiviral activity combined with possible cancer prevention effects was also determined by Mansouri et al., who found that PAP diminished virus production of HTLV-1 by affecting it at both the translational and transcriptional levels [83].

The antiviral potential can be further explored by isolating additional compounds from promising extracts and testing their antiviral activity. Furthermore, employing in vivo, mechanism of action, pharmacokinetic, and clinical studies can help develop compounds with potential as novel antiviral agents.

3.3.3. The Antimalarial Activity of INPS

In the screening process carried out, we found one study that assessed the in vitro antimalarial activity of both the ethanolic seeds and seedlings extracts, as well as the fractions and isolated compounds, of two quassinoids (ailanthone and 6α-tigloyloxychaparrinone) from Ailanthus altissima. The authors chose a chloroquine-sensitive strain (HB-3) and a chloroquine-resistant strain (Dd-2) of Plasmodium falciparum. In addition to the antimalarial assay, the authors also performed a cytotoxic in vitro assay. Both ailanthone and 6α-tigloyloxychaparrinone exhibited antiplasmodial activity against sensitive and resistant Plasmodium falciparum strains, with ailanthone yielding superior results (0.003 μg/mL against HB-3 and 0.037 μg/mL against Dd-2) compared to those of 6α-tigloyloxychaparrinone (0.061 μg/mL against HB-3 and 0.062 μg/mL against Dd-2). However, in the cytotoxic assay on the Vero cell line, ailanthone showed a low cytotoxicity at 200.00 μg/mL, whereas 6α-tigloyloxychaparrinone showed no toxicity at the same concentration [51]. Additional studies, both in vitro and in vivo, should be performed to assess the potential of ailanthone and 6α-tigloyloxychaparrinone as antimalarial/antiplasmodial agents. Unfortunately, the evidence of antimalarial activity is limited when it comes to Ailanthus altissima, considering that the one selected study did not describe a mechanism of action for the two quassinoids, nor did it compare the results to standard antiplasmodial drugs. Moreover, there were no in vivo, bioavailability, and toxicity studies performed, which would otherwise provide more clues into the safety profile and actual applicability of the in vitro results in clinical settings.

3.4. INPS and Their Antioxidant Potential

Oxidative stress is caused by an imbalance of pro-oxidant and antioxidant molecules, which results in disruptions of the redox balance in the organism and cell damage. The human organism is equipped with endogenous antioxidant enzymes that can deal with oxidative stress, such as glutathione peroxidases (GSH-Px), superoxide dismutases (SOD1 and SOD2), and catalases (CAT). Even though pro-oxidant molecules are needed, to some degree, for normal body function, specifically in redox processes, in some cases, excess reactive oxygen species (ROS) production can overwhelm the organism and its endogenous enzymes, leading to cell damage [106]. In such cases, supplementing the organism with antioxidant compounds that can assist in capturing free radicals generated by ROS can help prevent cell damage. An excess amount of ROS and oxidative damage to cells has been linked to various pathologies such as cancer, cardiovascular diseases, and diabetes, to name a few [107]. Plants are naturally equipped with many phytocomponents that can deal with oxidative stress, as this is their way of adapting to the environmental stress factors (UV rays, predators, heat, cold, drought, flooding, soil composition, etc.), thus making them good candidates for the extraction of antioxidant compounds [108,109].

In terms of the assays used for determining the antioxidant capacity of various extracts and compounds isolated from the selected plants, most of which are found in the selected studies, in vitro methods that use generated free radicals that are then partially captured by the antioxidant molecules were commonly employed. However, these assays do not generally correlate well with in vivo results because they cannot predict the antioxidant effect in the context of the complex biological processes in the human body and the capacities of the endogenous enzymes to deal with pro-oxidant molecules; this may lead to potential overestimation of the results [110,111]. Consequently, in vitro assays remain the initial step in determining the antioxidant potential of compounds/extracts to identify the most promising types. Thus, to more accurately predict the significance of the results and their antioxidant potential in therapy, in vivo tests are required. Unfortunately, most of the selected studies fall short in this regard; thus, they can be considered only preliminary results. The main findings regarding antioxidant potential can be found in

Table 4. Overview of antioxidant activity recorded in selected studies, including assay methods, observed results, extract types or isolated compounds, plant material and origin, experimental model (in vitro or in vivo), and reference. Only results demonstrating effective antioxidant potential are presented.

Assay Method	Result	Extract Type/Isolated Compound	Plant Material/Origin	In Vivo/In Vitro	Reference
ABTS	IC50 = 643.21 μg/mL	Methanolic extract	Ailanthus altissima, bark, Pakistan	In vitro	[69]
	c = 299.54 mmol TE/g DW	Ethanolic extract	Ailanthus altissima, leaf, Bulgaria	In vitro	[71]
	c = 893.14 mmol TE/g DW		Ailanthus altissima, flower, Bulgaria
	c = 31.24 mmol TE/g DW		Ailanthus altissima, stem bark, Bulgaria
	IC50 = 18.43 μg/mL	Methanolic extract	Phytolacca americana, leaf, Bulgaria	In vitro	[86]
	IC50 = 112.49 μg/mL		Phytolacca americana, fruit, Bulgaria
	IC50 = 2.93 μg/mL	Methanolic extract	Amorpha fruticosa, leaf, Bulgaria	In vitro	[86]
	IC50 = 2.90 μg/mL		Amorpha fruticosa, fruit, Bulgaria
CUPRAC	c = 548.07 mmol TE/g DW	Ethanolic extract	Ailanthus altissima, leaf, Bulgaria	In vitro	[71]
	c = 789.54 mmol TE/g DW		Ailanthus altissima, flower, Bulgaria
	c = 10.22 mmol TE/g DW		Ailanthus altissima, stem bark, Bulgaria
DPPH	IC50 = 88.79 μg/mL	Methanolic extract	Phytolacca americana, leaf, Bulgaria	In vitro	[86]
	IC50 = 412.06 μg/mL		Phytolacca americana, fruit, Bulgaria
	IC50 = 16.00	Americanoic Acid A Methyl Ester	Phytolacca americana, seed, Japan	In vitro	[85]
	IC50 = 38.00	Isoamericanoic Acid A Methyl Ester
	IC50 = 9.00	9′-O-Methylamericanol A
	IC50 = 11.00	Americanin-type 4
	IC50 = 39.00	Isoamericanin-type 5
	IC50 = 5.00	Americanol A
	IC50 = 16.00	Isoamericanol A
	IC50 = 10.00	Americanin A
	IC50 = 16.00	Isoamericanin A
	IC50 = 741.74 μg/mL	Methanolic extract	Ailanthus altissima, bark, Pakistan	In vitro	[69]
	c = 225.62 mmol TE/g DW	Ethanolic extract	Ailanthus altissima, leaf, Bulgaria	In vitro	[71]
	c = 729.72 mmol TE/g DW		Ailanthus altissima, flower, Bulgaria
	c = 24.96 mmol TE/g DW		Ailanthus altissima, stem bark, Bulgaria
	SR = 20.95% (c = 15.63 µg/mL)	Ethanolic extract	Ailanthus altissima, fruit, Bozhou Baohua Pharmaceutical Co., Ltd.	In vitro	[66]
	SR = 91.97% (c = 0.50 mg/mL)
	SR = 97.90% (c = 1.00 mg/mL)
	IC50 = 11.23 μg/mL	Methanolic extract	Amorpha fruticosa, leaf, Bulgaria	In vitro	[86]
	IC50 = 9.83 μg/mL		Amorpha fruticosa, fruit, Bulgaria
FIC	IP = 54.94% (c = 1 mg/mL)	Ethanolic extract	Ailanthus altissima, fruit, Bozhou Baohua Pharmaceutical Co., Ltd.	In vitro	[66]
FRAP	c = 906.01 mmol TE/g DW	Ethanolic extract	Ailanthus altissima, leaf, Bulgaria	In vitro	[71]
	c = 661.48 mmol TE/g DW		Ailanthus altissima, flower, Bulgaria
	c = 16.65 mmol TE/g DW		Ailanthus altissima, stem bark, Bulgaria
	IC50 = 508.81 μg/mL	Methanolic extract	Amorpha fruticosa, leaf, Bulgaria	In vitro	[86]
	IC50 = 642.95 μg/mL		Amorpha fruticosa, fruit, Bulgaria
O2−	IC50 = 412.06 μg/mL	Americanoic Acid A Methyl Ester	Phytolacca americana, fruit, Bulgaria	In vitro	[85]
	IC50 = 64.00	Isoamericanoic Acid A Methyl Ester
	IC50 = 8.00	9′-O-Methylamericanol A
	IC50 = 9.00	Americanin-type 4
	IC50 = 29.00	Isoamericanin-type 5
	IC50 = 24.00	Americanol A
	IC50 = 23.00	Isoamericanol A
	IC50 = 9.00	Americanin A
	IC50 = 58.00	Isoamericanin A
OH	IP = 42.34% (c = 1.00 mg/mL)	Ethanolic extract	Ailanthus altissima, fruit, Bozhou Baohua Pharmaceutical Co., Ltd.	In vitro	[66]

IC₅₀ = half maximal inhibitory concentration expressed in μM (unless specified otherwise), DPPH = 2,2-Diphenyl-1-picrylhydrazyl, O₂⁻ = superoxide anion, c = concentration, TE = Trolox equivalents, DW = dry weight, SR = scavenging rates, IP = inhibition percentage, ABTS = 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, CUPRAC = cupric ion-reducing antioxidant capacity, FRAP = ferric-reducing antioxidant power, FIC = ferrous ion chelating, and OH = hydroxyl.

.

Among the selected studies, few assessed the in vivo antioxidant activity. Popovici LF et al. performed a comprehensive in vivo study on the anxiolytic, neuroprotective, and antioxidant effects of Phytolacca americana hydroethanolic fruit extracts on scopolamine-treated zebrafish. The authors found that the extracts showed the ability to enhance the enzymatic activity of the endogenous SOD, CAT, and GSH-Px, while at the same time reducing lipid and protein oxidation markers. The results were linked to a neuroprotective effect, considering the design of the study and the fact that oxidative stress is associated with neurodegenerative diseases [89]. A similar approach was employed by Rahman HMA et al., in which scopolamine-induced male Sprague Dawley rats were treated with Ailanthus altissima methanolic bark extracts in doses of 50.00, 100.00, and 200.00 mg/kg for 28 days. The antioxidant activity of the extracts was then determined on isolated brains obtained from the studied specimens. The results showed a significant dose-dependent increase in the activity of both SOD and GSH-Px enzymes in the groups treated with Ailanthus altissima bark extract compared to the results for the controls. Moreover, malondialdehyde (MDA), a marker of lipid peroxidation and oxidative stress, was significantly reduced in brains treated with bark extracts compared to the levels in the scopolamine controls [63]. Once again, these results showcased an in vivo antioxidant capacity of the extract that can be correlated to a subsequent neuroprotective effect.

Most of the studies focus on the in vitro antioxidant activity of the selected plants, which constitutes a limitation. More studies aiming to assess in vivo antioxidant activities should be performed to demonstrate the potential therapeutic application in the biological context, starting with mechanism of action studies and animal studies and moving on to clinical trials, if the initial results are promising. However, it is necessary to continue preliminary in vitro screening of the antioxidant potential of plants.

3.5. INPS and Their Cytotoxic Potential

Plant extracts contain various components that exhibit cytotoxic activity, generally depending on their concentration [112,113,114]. Cytotoxicity comprises two critical aspects; one is a toxicological aspect, in which extracts and contained compounds can be assessed for their capacity to damage normal human cells at specific concentrations, and this is used to determine their safety profile. The other aspect is assessing their cytotoxicity on tumoral cells at various concentrations, with potential applications and benefits in cancer therapy, adjuvants, and alternative treatments. It is essential to determine both aspect to ensure a good safety profile for administration, but also for ensuring effectiveness against tumoral cells. Plant extracts and compounds may exhibit specificity for different tumour cells and pathological markers, and lower specificity and toxicity on normal human cells, which we will discuss in the following paragraphs. As with all the other assessed activities, it is just as essential to correlate in vitro assays with in vivo ones, and if deemed safe and effective, clinical trials may be performed [115,116,117]. Among the selected studies, the distribution of in vitro and in vivo cytotoxicity studies is visualised in Figure 3, highlighting the need for more in vivo studies. Furthermore, the studies that reported cytotoxic activity among the selected plants is summarised in

Table 5. Overview of cytotoxic activity reported in selected studies, including tested cell lines or organisms, observed results, extract types or isolated compounds, assay methods, plant material and origin, experimental model (in vitro or in vivo), and reference. Only results demonstrating effective cytotoxic potential are presented, ordered after the tested cell line or organism.

Tested Cell Line/Organism	Result	Extract Type/Isolated Compound	Assay	Plant Material/Origin	In Vivo/In Vitro	Reference
2008/MRP1	IC50 = 7.08	Ailanthone	MTT	Puruifa Science & Technology Development Co., Chengdu, China	In vitro	[39]
A2780	IC50 = 10.60 μg/mL	Crude ethanolic	MTT	Ailanthus altissima, bark, South Korea	In vitro	[62]
	IC50 = 89.50 μg/mL	n-Hexane fraction
	IC50 = 8.60 μg/mL	EtOAc fraction
	IC50 = 7.10 μg/mL	n-BuOH fraction
	IC50 = 25.70 μg/mL	Water fraction
Cal-27	ACP = 39.00 (24 h)	Ailanthone (D = 4.00 µM)	Apoptosis	Jiangxi Herb Tiangong Technology, Jiangxi, China	In vitro	[43]
	IC50 = 0.84	Ailanthone	MTT
HCT-116	IC50 = 1.79	Dalbin	MTT	Amorpha fruticosa, seed, China	In vitro	[77]
	IC50 = 1.98	8′-O-β-D-glucopyranosyl-amorphigenin
	IC50 = 0.6	Ailanthone	CCK-8	Shanghai Yiyan Biotechnology Co., Ltd., Shanghai, China	In vitro	[42]
HEK293/R2	IC50 = 4.77	Ailanthone	MTT	Puruifa Science & Technology Development Co., Chengdu, China	In vitro	[39]
HeLa	CV = 6.00	Chloroform crude extract	Trypan blue staining	Ailanthus altissima, root, Italy	In vitro	[53]
	CV = 6.00	Chloroform fraction
	CV = 9.00	1-Methoxy-canthin-6-one isolated from the chloroform fraction
	ACP = 41.00 (48 h)	Chloroform crude extract	Cell apoptosis
	ACP = 28.00 (48 h)	Chloroform fraction
	ACP = 27.00 (48 h)	1-methoxy-canthin-6-one isolated from the chloroform fraction
Hep3B	IC50 = 0.37	Altissinol A	MTT	Ailanthus altissima, bark, China	In vitro	[56]
	IC50 = 0.48	Ailanthone
	IC50 = 0.98	13,18-Dehydroglaucarubinone
	IC50 = 2.17	(-)-Cha-parrinone
	IC50 = 0.05	6a-Tigloyloxychaparrinone
	IC50 = 8.01	Shinjulactone A
	IC50 = 2.36	Altissinol B
	IC50 = 2.43	6a-Tigloyloxychaparrin
	IC50 = 23.46	Glaucarubin
	IC50 = 47.08	Chouchunsteride B	MTT	Ailanthus altissima, leaf, China	In vitro	[68]
	IC50 = 31.52	Chouchunsteride D
	IC50 = 31.49	6-dehydropregnenolone
	IC50 = 44.52	20S- Hydroxyergosta-4,6,24(28)-trien-3-one
	IC50 = 39.88	3-O-β-D-glucopyranosyl-16-dehydropregnenolone
	IC50 = 45.21	Altissimacoumarin C	MTT	Ailanthus altissima, root bark, China	In vitro	[65]
	IC50 = 0.54	Ailanthone	MTT	Jinan University, Guangzhou, China	In vitro	[37]
HepG2	IC50 = 0.28	Altissinol A	MTT	Ailanthus altissima, bark, China	In vitro	[56]
	IC50 = 0.24	Ailanthone
	IC50 = 1.15	13,18-Dehydroglaucarubinone
	IC50 = 1.20	(-)-Cha-parrinone
	IC50 = 0.55	6a-Tigloyloxychaparrinone
	IC50 = 4.67	Shinjulactone A
	IC50 = 1.22	Altissinol B
	IC50 = 10.54	6a-Tigloyloxychaparrin
	IC50 = 35.57	Glaucarubin
	IC50 = 4.03	Chouchunsteride A	MTT	Ailanthus altissima, leaf, China	In vitro	[68]
	IC50 = 7.62	6-dehydropregnenolone
	IC50 = 13.43	20S- Hydroxyergosta-4,6,24(28)-trien-3-one
	IC50 = 66.47	(+)-7S,8R-ailanthussin A	MTT	Ailanthus altissima, bark, China	In vitro	[61]
	IC50 = 29.53	(−)-7R,8S-ailanthussin A
	IC50 = 0.63	Ailanthone	MTT	Jinan University, Guangzhou, China	In vitro	[37]
HepG2/ADM	IC50 = 4.03	Chouchunsteride A	MTT	Ailanthus altissima, leaf, China	In vitro	[56]
	IC50 = 7.62	6-Dehydropregnenolone
	IC50 = 13.43	20S- Hydroxyergosta-4,6,24(28)-trien-3-one
HL-60	IC50 = 5.99	Ailanthone	MTT	Jinan University, Guangzhou, China	In vitro	[38]
	ACP = 42.02	Ailanthone (5.00 μM)	Cell apoptosis
	ACP = 52.05	Ailanthone (10.00 μM)
	ACP = 56.69	Ailanthone (20.00 μM)
Huh7	IC50 = 0.35	Ailanthone	MTT	Jinan University, Guangzhou, China	In vitro	[37]
Ishikawa cell line	IC50 = 3.00 nM	GnRH-PAP	Translation	N/A	In vitro	[81]
K562/A02	IC50 = 2.21	Ailanthone	MTT	Puruifa Science & Technology Development Co., Chengdu, China	In vitro	[39]
MCF-7	IC50 = 3.90	Amorphasidase	MTT	Amorpha fruticosa, seed, China	In vitro	[86]
	IC50 = 1.50	Dalbin
	IC50 = 0.45	8′-O-β-D-glucopyranosyl-amorphigenin
	IC50 = 0.95	Amorphin
	IC50 = 34.08	6′-O-β-D-glucopyranosyl-12a-hydroxydalpanol
	CV = 64.36	Ailanthone (0.50 µg/mL)	MTT	Ailanthus altissima, bark, China	In vitro	[60]
	CV = 62.48	Ailanthone (1.00 µg/mL)
	CV = 57.64	Ailanthone (2.00 µg/mL)
	CV = 50.24	Ailanthone (4.00 µg/mL)
	CV = 43.24	Ailanthone (8.00 µg/mL)
	ACP = 22.68	Ailanthone (0.50 µg/mL)	Cell apoptosis
	ACP = 27.99	Ailanthone (1.00 µg/mL)
	ACP = 35.88	Ailanthone (2.00 µg/mL)
	ACP = 49.77	Ailanthone (4.00 µg/mL)
	ACP = 75.51	Ailanthone (8.00 µg/mL)
	BBR = 0.53	Ailanthone (0.50 µg/mL)	Bax/Bcl-2 protein expression levels
	BBR = 0.56	Ailanthone (1.00 µg/mL)
	BBR = 0.80	Ailanthone (2.00 µg/mL)
	BBR = 0.93	Ailanthone (4.00 µg/mL)
	BBR = 1.25	Ailanthone (8.00 µg/mL)
NCM460	IC50 = 1.76	Ailanthone	CCK-8	Shanghai Yiyan Biotechnology Co., Ltd., Shanghai, China	In vitro	[42]
NSCLC Lewis cells	IC50 = 7.70 (24 h)	Ailanthone	MTT	Chengdu Alfa Biological Technology Co., Ltd., Chengdu, China	In vitro	[47]
OVCAR3	IC50 = 34.70 μg/mL	Crude ethanolic	MTT	Ailanthus altissima, bark, South Korea	In vitro	[62]
	IC50 = 28.00 μg/mL	EtOAc fraction
	IC50 = 22.50 μg/mL	n-BuOH fraction
	IC50 = 44.20 μg/mL	Water fraction
SKOV3	IC50 = 34.70 μg/mL	Crude ethanolic	MTT	Ailanthus altissima, bark, South Korea	In vitro	[62]
	IC50 = 28.00 μg/mL	EtOAc fraction
	IC50 = 22.50 μg/mL	n-BuOH fraction
	IC50 = 44.20 μg/mL	Water fraction
SW620	IC50 = 1.01	Ailanthone	CCK-8	Shanghai Yiyan Biotechnology Co., Ltd., Shanghai, China	In vitro	[42]
Tca8113	ACP = 17.00 (24 h)	Ailanthone (D = 4.00 µM)	Apoptosis	Jiangxi Herb Tiangong Technology, Jiangxi, China	In vitro	[43]
	IC50 = 0.79	Ailanthone	MTT
U87MG	ACP = 20.00 (48 h)	Chloroform crude extract	Cell apoptosis	Ailanthus altissima, root, Italy	In vitro	[53]
	ACP = 8.00 (48 h)	Chloroform fraction
	ACP = 9.00 (48 h)	1-methoxy-canthin-6-one isolated from the chloroform fraction
U937	ACP = 19.00 (48 h)	Chloroform crude extract	Cell apoptosis
	ACP = 11.00 (48 h)	Chloroform fraction
	ACP = 11.00 (48 h)	1-methoxy-canthin-6-one isolated from the chloroform fraction

GnRH-PAP = gonadotropin-releasing hormone fused with pokeweed antiviral protein, MTT = 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, MCF-7 = breast cancer cell line, IC₅₀ = half maximal inhibitory concentration expressed in μM (unless specified otherwise), HCT-116 = human colorectal carcinoma cell line, CCK-8 = cell counting kit-8, Hep3B = human hepatoma cell line, HepG2 = human hepatoma cell line, HepG2/ADM = multidrug resistant human hepatoma cell line, CV = cell viability expressed in percentage after 72 h of treatment (unless specified otherwise), ACP = apoptotic cells percentage after 48 h of treatment (unless specified otherwise), BBR = Bax/Bcl2 ratio, HCT-116 = colon cancer cell line, A2780, SKOV3, OVCAR3 = ovarian cancer cell lines, HeLa = immortalized regular human cell line, U87MG = glioblastoma cell line, U937 = histiocytic lymphoma cell line, Tca8113, Cal-27 = squamous cell carcinoma of the human tongue cell lines, SW620 = colon cancer cell line, NCM460 = normal human colon mucosal epithelial cells, NSCLC Lewis cells = non-small cell lung cancer cell line, HL-60 = human promyelocytic leukaemia cell line, Huh7 = immortalized human hepatoma cell line, K562/A02 = doxorubicin-resistant human leukaemia cell line, 2008/MRP1 = MRP1 (multidrug resistance-associated protein 1)-overexpressing derivative of human ovarian carcinoma cell line, HEK293/R2 = human embryonic kidney 293 cell lines transfected with BCRP (breast cancer resistance protein), EtOAc = ethyl acetate, n-BuOH = n-butanol, and N/A = not applicable.

.

Ailanthone isolated from Ailanthus altissima (Table 5) is most often studied in the context of cytotoxic antiproliferative activities among the selected plants. We also identified a few more studies that further observe its activities and elucidate its mechanisms of action. Cucci MA et al. determined that ailanthone is capable of reducing the proliferation and invasion rates of A2780/CP70 ovarian cancer cell lines that are resistant to cisplatin (CDDP) treatment in vitro through a varied mechanism of action, specifically by inhibiting Nrf2 (nuclear factor erythroid 2-related factor 2) and YAP/TEAD (yes-associated protein 1 and the transcription factor) transcriptional activity and by reducing UCHL1 (ubiquitin carboxy-terminal hydrolase L1) expression, which both lead to increased oxidative stress in the cancerous cells [41]. Similarly to Cucci MA et al., Daga M et al. determined the in vitro activity of ailanthone on bladder cancer cell lines that are resistant to CDDP treatment. They observed that it was able to inhibit the proliferation and invasion of the cancerous cell lines by reducing the expression levels of the Nrf2, YAP, and c/Myc (an oncogene). Moreover, the authors determined that ailanthone showed a low cytotoxic profile on normal kidney cells, highlighting a specific selectivity of the compound for cancerous cells [40]. Both these studies could serve as a basis for future in vivo studies in this context, since they highlight ailanthone’s potential in treating tumours that are resistant to conventional treatment.

Another compound of interest that was isolated from Ailanthus altissima was 1-methoxy-canthin-6-one, with some of its activities presented in Table 5. In addition, a study conducted by Ammirante M et al. also highlighted its apoptotic properties on a handful of cancerous cell lines, namely on the Jurkat cell line (leukaemia), the NPA and ARO cell lines (thyroid carcinoma), and the Huh7 cell line (hepatocellular carcinoma). For all tested cell lines, apoptosis was evident at a concentration of under 10 µmol/L, with the IC₅₀ being approximately 40 µmol/L. The authors also noted that apoptosis occurred through the MAPK (mitogen-activated protein kinase) pathway, since 1-methoxy-canthin-6-one modulated JNK (c-Jun NH₂-terminal kinase), while at the same time synergising with the hrTRAIL (human recombinant TNF-related apoptosis-inducing ligand) [55]. Thus, 1-methoxy-canthin-6-one might also be potentially helpful in anticancer therapy.

Furthermore, we identified six studies assessing cytotoxic activity in vivo, all of them being focused on ailanthone isolated from Ailanthus altissima. Fang C et al. determined that ailanthone was able to effectively inhibit NSLCL subcutaneous tumour growth and metastasis in tumour-bearing mice at doses of 0.10 mg/kg, 1.00 mg/kg, or 2.00 mg/kg, with the best result at a dose of 2.00 mg/kg. Treatment with ailanthone also enhanced the survival rate of the tested specimens. Moreover, the authors found that the antitumour mechanism of ailanthone was based on modulating the UPF1/GAS5/ULK1 (up-frameshift protein 1/growth arrest specific 5/unc51-like autophagy activating kinase 1) signalling pathway [46]. Liang J et al. studied the effects of ailanthone on lung metastasis associated with osteosarcoma. Here, the authors found that the compound was able to suppress metastasis by targeting the KMT2A-MEN1 gene complex and inhibiting the SSP (serine synthesis pathway) in vivo in mouse models, without exhibiting significant toxicity on normal cells. Also, the authors noted that ailanthone exhibited a different mechanism pathway compared to that of conventional osteosarcoma therapeutic agents [48]. Similarly, Zhang Y et al. studied the effect of ailanthone on osteosarcoma, finding that when administered, it significantly inhibited tumour growth in Saos-2 mice xenografts, without causing significant weight difference between the ailanthone-treated lot and the DMSO (dimethyl sulfoxide) control lot, thus displaying good tolerability. The authors also showed that the SSP pathway was most significantly modulated by ailanthone, which was also one of the findings in the study conducted by Liang J et al. [44,48]. Li J et al. studied the in vivo cytotoxic activity of ailanthone on subcutaneous bladder cancer mouse models. They determined that treatment with the compound resulted in reduced tumour growth by modulating the JAK/STAT3 (Janus kinases/signal transducer and activator of transcription 3) pathway, suppressing its activity [49]. Wang Y et al. sought to determine the cytotoxic activity of ailanthone in the context of breast cancer-related bone metastasis, finding that ailanthone significantly suppressed the growth of bone metastasis cells in the tested mouse models when compared to the results for the control group. Together with data from in vitro studies, the authors concluded that ailanthone was capable of blocking osteoclast differentiation in bone metastasis by inhibiting cytokines that are secreted by the breast cancer cells, mainly through the RANKL (receptor activator of nuclear factor kappa B ligand)-dependent pathway [45]. Finally, the last in vivo cytotoxic activity assessment that we identified among the selected studies was conducted by Zhuo Z et al., in which ailanthone was tested for its effects against hepatocellular carcinoma. The study performed on tumour xenografts in nude mice showed that ailanthone was capable of significantly inhibiting tumour growth compared to the results for the control group, while also maintaining a good safety profile. The authors also observed that ailanthone increased the apoptosis marker TUNEL (terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling) in vivo and that it induced G₀/G₁-phase cell cycle arrest, observed through the significantly lower expression levels of the key regulatory protein cyclin-dependent kinase 4 (CDK4) in the treated group compared to those in the control group [37].

Crude extracts were not often studied in this antiproliferative context. However, a few data points about their cytotoxicity are tabulated. In addition, we identified a study by Cui X et al., which focused on determining the antiproliferative activity of the 90% crude ethanolic extract obtained from Amorpha fruticosa leaves on HCT116 and HepG2 cell lines. The authors reported a cell viability percentage of under 60% for the extract concentrations of 100.00 µg/mL, 25.00 µg/mL, and 5.00 µg/mL on the HCT116 cells; however, no activity was observed on HepG2 cells [78].

Concerning the Phytolacca americana plant, one study conducted by Saleri FD et al. showed a possible anticancer activity of the crude ethanolic extract on two cell lines, namely SCG-7901 and Hep G2; however, the study was designed as a comparative assessment with other Phytolacca species. As such, it was concluded that in this specific context, Phytolacca acinosa exhibited greater antiproliferative activity (IC₅₀ = 27.20 ± 1.60 µg/mL for the SCG-7901 cell line and IC₅₀ = 25.59 ± 1.63 µg/mL for the Hep G2 cell line) when compared to that of Phytolacca americana, for which the authors provided no conclusive values [88].

Notably, plant-derived compounds display certain limitations in cancer therapy, mainly, as discussed previously, the issue of promising in vitro results that do not translate well into real-life settings and in actual clinical practice. There are a few factors, such as limited bioavailability, poor solubility, high toxicity, incompletely deciphered mechanisms of action, and the possibility of drug resistance development, that limit this translation. Therefore, more research is needed in this area, including the preliminary screening of promising compounds, complete elucidation of the mechanisms of action, identifying target molecules/pathways, determining safety profiles, and establishing the actual applicability of potential novel therapeutic agents in clinical settings.

3.6. INPS and Their Other Therapeutic Potentials

Among the selected studies, we have identified even more therapeutic applications of broad applicability; however, there is a need for more studies in these areas to properly assess the therapeutic potential in clinical practice. The possible therapeutic applications identified ranged from enzyme inhibition, which was focused on acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and tyrosinase inhibition, as well as anti-inflammatory, antidiabetic, neuroprotective, neurotrophic, and DNA protective potentials and potential application in the cardiovascular system. Data concerning enzyme inhibition, anti-inflammatory, and neuroprotective activities are summarised in

Table 6. Summary of enzyme inhibition, anti-inflammatory, and neuroprotective activities identified in selected studies, detailing assay types, biological effects, tested extracts or isolated compounds, plant material and origin, experimental model (in vitro or in vivo), and reference. Only results demonstrating significant biological activity are included.

Activity	Assay	Result	Extract Type/Isolated Compound	Plant Material/Origin	In Vivo/In Vitro	Reference
Neuroprotective	MTT assay on H2O2-induced SH-SY5Y cells	CV = 70.50%	7S,8R-Guaiacylglycerol-8-acetovanillone ether (50.00 μM)	Ailanthus altissima root bark, China	In vitro	[64]
Enzyme inhibition	AChE inhibition	IP = 76.00	Methanolic crude extract	Amorpha fruticosa, flower, Czech Republic	In vitro	[80]
		IP = 9.10	(E)-N6-(Z)-di-p-Coumaroylputrescine (100.00 μM)
		IP = 1.10	N1,N6-(E)-di-p-Coumaroylputrescine (100.00 μM)
		IP = 14.10	N1-(E)-N5,N10-(Z)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 26.60	N1,N5-(Z)-N10-(E)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 47.90	N1,N5,N10-(E)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 17.90	cis-12a-Hydroxymunduserone (100.00 μM)
		IP = 37.90	6-Deoxyclitoriacetal (100.00 μM)
		IP = 22.40	12α-Hydroxy-α-toxicarol (100.00 μM)
		IP = 25.43	Methanolic crude extract (0.17 mg/mL)	Amorpha fruticosa, leaf, Bulgaria	In vitro	[86]
		IP = 48.86		Amorpha fruticosa, fruit, Bulgaria
		IC50 = 3.28	Chouchunionone A	Ailanthus altissima, leaf, China	In vitro	[72]
		IC50 = 16.31 μg/mL	Methanolic crude extract	Ailanthus altissima, bark, Pakistan	In vitro	[69]
	BuChE inhibition	IP = 90.00	Methanolic crude extract	Amorpha fruticosa, flower, Czech Republic	In vitro	[80]
		IP = 8.10	(E)-N6-(Z)-di-p-Coumaroylputrescine (100.00 μM)
		IP = 4.80	N1,N6-(E)-di-p-Coumaroylputrescine (100.00 μM)
		IP = 4.50	N1-(E)-N5,N10-(Z)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 19.10	N1,N5-(Z)-N10-(E)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 43.80	N1,N5,N10-(E)-tri-p-Coumaroylspermidine (100.00 μM)
		IP = 23.80	cis-12a-Hydroxymunduserone (100.00 μM)
		IP = 25.60	6-Deoxyclitoriacetal (100.00 μM)
		IP = 37.60	Amorphispironone (100.00 μM)
		IP = 23.60	Tephrosin (100.00 μM)
		IP = 22.80	12α-Hydroxy-α-toxicarol (100.00 μM)
	Tyrosinase inhibition	IC50 = 20.28	Chouchunionone C	Ailanthus altissima, leaf, China	In vitro	[72]
Anti-inflammatory	NO production inhibition in LPS-induced RAW 264.7 cells	IC50 = 15.09	(R)-5-(1-Hydroxyethyl)-canthin-6-on	Ailanthus altissima, stem bark, South Korea	In vitro	[58]
		IC50 = 9.09	Canthin-6-one
		IC50 = 7.73	9-Hydroxycanthin-6-one
		IC50 = 12.01	10-Hydroxycanthin-6-one
		IC50 = 5.92	Sinapaldehyde
		IC50 = 10.69	Erythro-guaiacylglycerol-β-O-41-coniferyl ether
		IC50 = 63.50	Canthin-6-one-1-O-b-D-apiofuranosyl-(1->2)-b-D-glucopyranoside	Ailanthus altissima, stem bark, South Korea	In vitro	[57]
		IC50 = 85.00	Canthin-6-one-1-O-[6-O-(3-hydroxy-3-methylglutaryl)]-b- D-glucopyranoside
		IC50 = 63.10	Shinjudilactone
		IC50 = 5.18	Ailanthone
		IC50 = 56.40	Shinjulactone A
		IC50 = 72.80	4-Hydroxybenzoic acid
		IC50 = 23.20	Vanillic acid
		IC50 = 43.80	3-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one
		IC50 = 73.40	p-coumaric acid
		IC50 = 71.60	trans-4-O-b-D-Glucopyranosyl ferulic acid
		IC50 = 21.60	Syringaresinol
	COX-2 inhibition in BMMC cells	IC50 = 47.40 µg/mL	Ethanol crude extract	Ailanthus altissima, leaf and branch, South Korea	In vitro	[54]
	COX-1 inhibition in BMMC cells	IC50 = 131.66 µg/mL
	LTC4 suppression in BMMC cells	IC50 = 25.70 µg/mL
	β-HEX release inhibition in BMMC cells	IC50 = 27.30 µg/mL

AChE = acetylcholinesterase, IP = inhibition percent expressed in %, BuChE = butyrylcholinesterase, IC₅₀ = half maximal inhibitory concentration expressed in μM (unless specified otherwise), NO = nitric oxide, LPS = lipopolysaccharide, RAW 264.7 = murine macrophage cell line, COX-2 = cyclooxygenase-2, COX-1 = cyclooxygenase-1, LTC4 = leukotriene C4, BMMC = primary bone marrow mononuclear spherical cells, β-HEX = β-hexosaminidase, SH-SY5Y = human neuroblastoma cell line, and CV = cell viability.

.

Weidner C et al. and Lee W et al. both tested isolated compounds from Amorpha fruticosa for their antidiabetic activity [75,79]. Weidner C et al. focused on amorfrutins and determined that they were capable of selectively binding and activating PPARγ (peroxisome proliferator-activated) receptors, and when amorfrutin 1 was administered to C57BL/6 mice with high-fat diet-induced obesity, it was able to reduce insulin resistance, with an effect equal to that of rosiglitazone (used as a control). Amorfrutin 1 also enhanced glucose tolerance, increased insulin sensitivity, and lowered plasma levels of triglycerides, free fatty acids, insulin, and glucose. All the results were comparable to those for rosiglitazone, with the absence of a hepatotoxic effect. Moreover, treatment with amorfrutin 1 (3 weeks) also significantly reduced body weight gain in the tested mice when compared to the results for the rosiglitazone control. When amorfrutin 1 was tested against db/db mice with a leptin-receptor deficiency, a genetic model for type II diabetes, the results showed a more substantial reduction in insulin concentrations in the plasma compared to those for rosiglitazone. Moreover, its administration had no significant effect on body weight gain compared to that for the rosiglitazone-treated mice, who displayed an increase of 30% in body weight after administration. Similar to the levels for the diet-induced obesity mouse models, amorfrutin 1 also lowered plasma triglycerides, fatty acids, and glucose in db/db mice and enhanced insulin sensitivity. The authors noted a preservation of the pancreatic function in db/db mice treated with amorfrutin 1 as well, which was connected to improvements in insulin levels in the treated group compared to those in the control [75]. Lee W et al. focused on a different compound isolated from the fruits of Amorpha fruticosa, namely 5,7-dihydroxy-6-geranylflavanone (DGF). Through in vitro determinations, the authors found that DGF binds and activates PPARα and PPARγ receptors, promoting fatty acid oxidation (through activation of PPARγ receptors), improving insulin sensitivity, and enhancing glucose uptake mediated by the IR-Akt (insulin receptor- protein kinase B) signalling axis. No in vivo experiments were performed in this study [79].

Next, we have identified some studies focusing on neurological applications, ranging from neuroprotective to neurotrophic effects, as well as anxiolytic effects of certain extracts or compounds obtained from the selected plants. Some of these can be found in Table 6 for the AChE and BuChE in vitro inhibition assays. The inhibition of both enzymes has been linked to a neuroprotective effect and therefore, to the prevention of neurodegenerative diseases, although in vivo tests should also be employed to assess the actual applicability of these results [118,119,120]. Some other studies related to neurological applications are discussed in the following paragraphs.

A study conducted by Kim SR et al. focused on determining both the in vitro and in vivo anti-inflammatory effects of the ethanolic Ailanthus altissima leaf extract on LPS (lipopolysaccharide)-induced primary astrocytes and on LPS-injected mice brains in a neurological context. During the in vitro studies, the authors found that the extract was able to suppress inducible nitric oxide synthase (iNOS) production in LPS-induced astrocytes, thus inhibiting nitric oxide (NO) release; inhibit the upregulation of TNF-α, IL-1β, and IL-6 expression; inhibit ROS production; inhibit NF-κB (nuclear factor kappa B) translocation, which in turn suppressed proinflammatory cytokines; and inhibit the activation of the MAPK pathway through suppressing the activation of ERK (extracellular signal-regulated kinase) and JNK (c-Jun NH2-terminal kinase). In vivo neuroinflammation studies were performed on LPS-injected mice who were subjected to the Y-maze and three-chamber tests after being treated with the extract. The authors concluded that the extract was able to lower LPS-induced memory and social impairments in mice, and further Western blot studies confirmed some of the effects of the extract that were observed in the in vitro studies, namely that in the LPS-injected brain tissue of extract-treated mice, the iNOS and COX-2 expression was suppressed, and the MAPK pathway activation was inhibited. Thus, the authors concluded that Ailanthus altissima may have potential applications in the treatment of diseases associated with neuroinflammation [67].

Another interesting approach in the neurological area was published by Takahasi et al., who performed an in vitro neurotrophic study on cortical neurons from rats using 1,4-benzodioxane derivatives isolated from the seeds of Phytolacca americana. Among the tested compounds, americanoic acid A methyl ester exhibited the most potent neurite outgrowth promotion activity, and americanin-type four also induced neuritogenesis. Thus, the authors concluded that with more evidence, these compounds could potentially be considered as neurotrophic agents [85].

Next, we have identified two in vivo neuroprotective studies among those selected. First off, Popovici LF et al. studied the effects of Phytolacca americana fruit extract on a scopolamine-induced memory-impaired zebrafish (Danio rerio) model by performing various in vivo behavioural assays and an AChE activity determination on brain samples collected from the tested specimens. After the assays were performed, the authors concluded that the fruit extracts exhibited an anxiolytic effect, as shown by the lowering of the anxiogenic impact of scopolamine and improved scopolamine-induced cognitive impairment during behavioural tasks. Coupled with the data obtained from determinations on the zebrafish brain that showed a reduction of brain oxidative stress and the inhibition of AChE activity, the authors concluded that Phytolacca americana fruit extracts could potentially be used in treating anxiety and cognitive impairment associated with amnesia [89]. Muhammad Abdur Rahman H et al. performed behavioural studies similar to those of Popovici LF et al., using bark extracts from Ailanthus altissima on Sprague Dawley rats. They used scopolamine to induce amnesia and cognitive impairment, as needed. The results of the behavioural experiments showed an anxiolytic effect and improvement of memory in the treated group compared to the results for the control. Moreover, the analysis of brain isolates from the tested specimens showed an inhibition of the activity of both MDA and AChE, which may explain the effects noticed in the behavioural tests [69].

We also identified one study that focused on some potential therapeutic applications for the cardiovascular system. Rahman HMA et al. aimed to assess the hypotensive, smooth muscle relaxant, and anticoagulant effects of Ailanthus altissima bark extracts by employing specific assays in vitro on isolated organs from rabbits, Sprague Dawley rats, and BALB/c mice. The hypotensive effect was also tested in vivo on rats. Based on the results, the authors concluded that the extracts might possess calcium channel blocking compounds. However, further studies should be conducted to isolate the specific compounds responsible for this effect, to determine their exact mechanisms, and to perform more in vivo pharmacological and toxicological studies to assess their true potential in this context [63].

Finally, Andonova T et al. determined the in vitro DNA protective effect of several extracts from Ailanthus altissima, finding that flower extracts displayed the most promising results in the DNA nicking in vitro assay [71]. Similar to other identified potential applications, more studies should be undertaken to properly assess the potential of this plant in DNA protection. More specifically, the compounds that are responsible for this effect could be isolated, and mechanistic studies could be conducted on them. Further studies could consist of safety profiles, in vivo testing, and clinical studies to understand the true potential of the DNA protective effect.

4. Study Limitations

The present review has several limitations that are highlighted further:

• Most of the included research is based on in vitro assays, which may not accurately reflect therapeutic efficacy in vivo;
• The exclusion of non-English publications and reliance on a single database (PubMed) may have led to the omission of relevant studies.
• The lack of standardised methodologies across the reviewed articles limits direct comparability of results;
• The absence of toxicological and pharmacokinetic data for many compounds restricts the assessment of their clinical applicability.

Thus, the study provides a valuable foundation for future research and highlights the biomedical potential of INPS.

5. Conclusions

The world of plants has proved to be a valuable source of bioactive compounds with various applications in therapy, as they offer alternatives to conventional treatments. The spread of INPS can have adverse effects on the environment and society; therefore, valorising their biomass through therapeutic applications presents a dual benefit: ecological management and biomedical innovation. To achieve this valorisation, we must understand what is currently known about INPS in this context and determine the gaps and future directions. Our review has focused on four selected INPS, but a clear prioritisation of species and compounds based on therapeutic promise is still needed. For example, ailanthone from Ailanthus altissima and amorfrutins from Amorpha fruticosa emerged as up-and-coming candidates, yet their clinical relevance remains underexplored.

As this present study’s findings suggest, Ailanthus altissima and its isolated compound ailanthone have been the topic of most of our selected studies, which focused on the antiproliferative activity, anti-inflammatory properties, and antimicrobial potentials. Amorpha fruticosa has potential uses in managing metabolic diseases such as type II diabetes, while also exhibiting antiproliferative and neuroprotective effects. Phytolacca americana may find potential uses in pathologies related to increased oxidative stress, neurological diseases, and in formulating novel antiviral agents through its pokeweed antiviral protein. Finally, due to the set eligibility criteria, none of the retrieved papers on the topic of Asclepias syriaca were suitable for inclusion.

In summary, we acknowledge that all the selected INPS contain potentially valuable bioactive compounds that can exert various therapeutic effects that may be useful in the medical and pharmaceutical fields. However, more comprehensive studies are needed to properly assess their biological potential, from complete screenings of the entire plant material harvested from various geographical regions to preliminary in vitro assessments that should be completed by in vivo determinations to further broaden the knowledge and understanding of how the plant material can be best utilized in the context of therapeutical applications in the medical and pharmaceutical fields. For those compounds and/or extracts that show the most potential, pre-clinical and clinical studies should be employed to determine their actual applicability.