Traditional Uses, Phytochemistry, and Pharmacological Activities of Fraxinus xanthoxyloides (G. Don) DC. (Oleaceae): A Review

The Oleaceae family comprises dicotyledonous flowering plants widely distributed in tropical and temperate regions. Globally, it includes approximately 25 genera and nearly 688 species (Huang et al., 2019). In Pakistan, eight genera and roughly 30 species are recorded, of which around 22 are cultivated (Grohmann, 1974). The distribution of this family extends to Afghanistan, India, Morocco, Algeria, and throughout the Hindu Kush and Himalayan ranges. Within Pakistan, Oleaceae representatives are commonly observed in areas such as Takht-i-Suleiman, the Toba Kakkar Range, Zhob (formerly Fort Sandeman), the western boundary of Waziristan, Safed Koh, Chitral, Malakand, Swat, Punjab, and Baluchistan, usually inhabiting temperate valleys between 1000 and 2500 meters in elevation (Ali and Nasir, 1989-1992; Ali et al., 2001; Ali and Qaiser, 1993-2007 (Ijaz et al., 2020), Outside South Asia, species of Oleaceae are also distributed in northeast Asia, China, North America, and parts of France (Sarfraz et al., 2017).

Fraxinus is a prominent genus within the subtribe Fraxininae and shares a close evolutionary relationship with the subtribe Oleinae of the tribe Oleeae (Ijaz et al., 2020). The genus was initially classified by Linnaeus in 1753, who described over 450 taxa, many of which are now considered synonyms. According to the most extensive monograph available, 64 species are currently recognized as valid, although additional species have been reported (Wallander, 2008). Within the Oleaceae family, Fraxinus is regarded as monophyletic and is characterized by relatively large imparipinnate leaves and one-seeded samaras.

Most Fraxinus species are medium-to large-sized trees, but some have adapted to arid environments as shrubs. Their leaves show remarkable variability, including differences in shape, texture, number of leaflets, margin type, petiolule length, hairiness, epidermal papillae, and the presence of rachis wings. Many synonyms in the genus arise from intraspecific variation. The flowers are generally small, with a single pistil and two stamens, consistent with other Oleaceae members. The corolla may be absent or composed of four (occasionally two) free, linear, white petals that are rarely fused. The calyx is usually small, synsepalous, dentate, and occasionally absent. The genus exhibits both insect-pollinated, petaliferous flowers that grow in large ornamental panicles from terminal buds alongside the leaves and apetalous, wind-pollinated flowers that develop in lateral or terminal inflorescences, usually preceding leaf expansion. The ovary is syncarpous with four ovules (two per locule), although typically only a single ovule develops into a one-seeded samara (Wallander and Albert, 2000), (Wallander, 2008).

F. xanthoxyloides (Figure 1), commonly known as Afghan ash, is a small tree native to dry hills. It is characterized by 5–11 ovate-lanceolate, toothed leaflets with a winged rachis, and flowers that occur in dense clusters. Although the genus Fraxinus has been studied globally, F. xanthoxyloides remains underexplored despite its ecological resilience in arid regions. This review differs from previous studies by providing the first integrated synthesis of its multi-organ ethnobotany, together with a comparative analysis of its secoiridoid and coumarin profiles.

Scientific literature indicates that F. xanthoxyloides has long been valued in traditional and folk medicine for addressing a wide spectrum of health conditions, such as malaria, pneumonia, jaundice, typhoid, bone fractures, traumatic injuries, internal wounds, and complications related to premature delivery. The use of plants as therapeutic agents dates back millennia, and according to the World Health Organization (WHO), nearly four-fifths of the world’s population continues to depend on traditional remedies for primary health care needs. Ethnobotanical investigations play a crucial role in recording indigenous medicinal practices (Idu et al., 2008). Research conducted in northern Pakistan shows that the root bark and leaves of F. xanthoxyloides are traditionally used to treat malaria, pneumonia, and jaundice, and its leaves also serve as animal fodder (Younis, 2017; (Khan et al., 2009). Additionally, an infusion prepared from 2-3 g of the stem bark is administered to ease labor pain (Shah and Hussain, 2012). Reports further highlight the use of the stem bark in managing typhoid fever and pneumonia (Khan and Khatoon, 2007), traumatic injuries, and premature infant expulsion (Younis, Rasul, et al., 2018). The wood of the plant is widely used for bone fracture healing, tool handles, and walking sticks (Sarangzai et al., 2013). It is also a major source of fuel (Khan and Khatoon, 2007). Moreover, a bark concoction is used to manage internal injuries (Singh, 2012) and to facilitate the expulsion of a deceased premature infant (Farrukh Hussain et al., 2007). Additionally, a stem decoction is administered for bone fractures and wounds in cattle (Mukerji, 2006), while the foliage serves as animal fodder (Sarangzai et al., 2013). Beyond ethnomedicinal uses, leaves and their fractions have been reported to exhibit significant anti-inflammatory and analgesic properties (Younis et al., 2016).

Table 1: Traditional uses of Fraxinus xanthoxyloides (G. Don) DC.

Traditional practitioners in the Hindu Kush region use various plant organs with high specificity. Nonetheless, the ethnobotanical record for this species is almost entirely descriptive: reported uses are rarely accompanied by a stated dose, preparation ratio, or duration of treatment, and no two sources employ a consistent unit of measurement. Although several reports describe its use for malaria and pneumonia, there is a lack of clinical validation for dose standardization in folk medicine (Table 1). In our view, this absence of standardized dosing is the single greatest obstacle to translating the ethnobotanical record into a testable pharmacological hypothesis.

One of the notable characteristics of plant physiology and phytochemistry is the determination of plant metabolites using sophisticated analytical methods. The initial step in establishing a comprehensive database that will be used to ensure quality evaluation and validation of herbal medicaments is phytochemical screening. It can also be used to predict potential biological effects and identify appropriate marker compounds (Jaina et al., 2013).

Over the last five years, the genus Fraxinus has received increased attention from researchers worldwide. One such species is F. xanthoxyloides, which is widely distributed and abundant in nature and has attracted significant phytochemical interest. According to the findings of these studies, the plant contains numerous bioactive compounds, including coumarins and secoiridoids (Huang et al., 2019), as well as rutin, quercetin, gallic acid, caffeic acid, kaempferol, catechins, and myricetin (Younis et al., 2020).

The presence of coumarins (such as cichoriin and fraxin) and secoiridoids (such as isofraxisecoside) is considered a characteristic feature of F. xanthoxyloides. These secoiridoids occur either as glucosides or as phenyl hydroxyphenolethylglycosesesters. In addition, lignans, flavonoids, and simple phenolic compounds are present, although their levels appear to be quite low (Kostova and Iossifova, 2007).A summary of all reported compounds is presented in Table 2.

Coumarins have been observed in F. xanthoxyloides in both glucosidic and free forms. Several species of this genus are known to be valuable natural sources of coumarins for commercial production. Several analytical techniques have been designed to study coumarin concentrations in plant extracts and fractions, both quantitatively in a quantitative and qualitative manner to facilitate this research (Hegnauer, 1969; Murray et al., 1982; Younis et al., 2016). Younis et al. (2016) stated that the plant itself contains simple coumarins, furanocoumarins, pyranocoumarins, chromones, and even dimeric coumarins. Moreover, isoferraxin was successfully extracted from the stem bark of F. xanthoxyloides (G. Don) Wall. ex (Hadroug et al., 2019). Previous phytochemical studies have also validated the existence of coumarins, secoiridoid glucosides (oleoside type), phenylethanoid glycosides, and other phenolic compounds in the bark of this plant (Iossifova et al., 2002).

Secoiridoid glucosides are among the most common phytochemical classes (Jin et al., 2020). These are monoterpene derivatives (cyclopentane) biosynthesized by the cleavage of cyclomethene oxime structures at the C-7 and C-8 positions. These metabolites are notorious for their highly diverse pharmacological activities, such as anti-diabetic, anti-inflammatory, immunosuppressive, neuroprotective, anti-cancer, and anti-obesity effects. A new secoiridoid, iso-fraxitelesecoside, was extracted from the stem bark of F. xanthoxyloides (Huang et al., 2019).

Rutin is a flavone derivative and a naturally occurring product that was first extracted from buckwheat in the 19th century. This low-molecular-weight polyphenolic compound is very common in fruits and vegetables, and

Table 2: Different compounds found in Fraxinus xanthoxyloides (G. Don) DC.

buckwheat is one of the major dietary sources of this compound. Rutin, a flavonoid glycoside also known as Vitamin P, has various biological effects, such as anti-platelet, antiviral, and antihypertensive effects and it can reinforce capillary walls. These effects are mainly due to its strong antioxidant and radical-scavenging characteristics (Guo et al., 2007). Besides, rutin has been linked with several pharmacological properties, including anti-inflammatory, anti-allergic, anti-bacterial, antiprotozoal, vasoprotective, antitumor, hypolipidemic, cytoprotective, antispasmodic and anticarcinogenic effects (Yang et al., 2008). It has been confirmed in extracts of F. xanthoxyloides by high-performance liquid chromatography (HPLC) analysis (Younis et al., 2020).

The most common plant flavonoid is quercetin, which can be found in tea and onions and is well known as a powerful antioxidant. The name quercetin name derives from the Latin quercetum (oak). It has a variety of pharmacological effects, such as anti-allergic, anti-inflammatory, anticancer, cardioprotective, antitumor, antiviral, anti-diabetic, immunomodulatory, antihypertensive, and gastroprotective activities. Besides its medicinal significance, quercetin is essential in plant physiology, including seed germination, pollen development, antioxidant defense mechanisms, and photosynthesis, which contribute to overall plant growth and development. It is also a powerful antioxidant that increases plant resistance to various biotic and abiotic stress factors (Derosa et al., 2021). Quercetin has been recognized and confirmed in F. xanthoxyloides by HPLC profiling (Younis et al., 2020).

Gallic acid, or 3,4,5-trihydroxybenzoic acid is a trihydroxybenzoic acid commonly found in plants worldwide. It has documented antioxidant properties and potential applications as an anticancer agent. Notably, gallic acid has been shown to induce cytotoxicity in specific types of cancerous cells without harming normal, healthy cells (Zhao and Hu, 2013). In the same context, Younis et al., (2020) used HPLC to determine the gallic acid content of F. xanthoxyloides.

Caffeic acid, chemically known as 3,4-dihydroxycinnamic acid, is a phenylpropanoid. It is one of the main derivatives of cinnamic acid and is found in plants (Riaz et al., 2019). Multiple studies have demonstrated its wide range of biological activities, such as antioxidant, antiviral, anticancer, and anti-inflammatory effects (Lin and Yan, 2012). Similarly, Younis et al. (2020) reported the presence of caffeic acid through F. xanthoxyloides HPLC analysis.

Kaempferol is a type of flavonoid belonging to the flavonol subclass, which is rarely found in a wide range of food plants. Due to its powerful antioxidant and anti-inflammatory actions, kaempferol has attracted considerable academic attention, especially regarding its molecular mechanisms and potential in the treatment of inflammatory diseases (Rajendran et al., 2014). Kaempferol has been determined in F.xanthoxyloides by HPLC profiling (Younis et al., 2020).

Catechins are defined as a category of polyphenolic metabolites belonging to the flavonoid family that often exist in large quantities in a variety of fruits, vegetables, as well as plant-derived beverages. They belong to the flavanol group (Gadkari and Balaraman, 2015). These compounds have been demonstrated to protect against a variety of degenerative diseases and to have antitumor and immune-modulating effects, especially in states of immune dysfunction (Boyer and Liu, 2004). Research also indicates that catechins have a higher ability to act as antibacterial agents against gram-positive bacteria than Gram-negative bacteria (Ikigai et al., 1993). Younis et al. (2020) have carried out widespread research on catechins in F. xanthoxyloides.

Myricetin is a naturally occurring bioflavonoid that is widely found in plant species. It has been known to possess good anticarcinogenic and antimutagenic effects, but ironically, it has also shown mutagenic effects in Ames test assays (Park et al., 2016)(Ong and Khoo, 1997). Younis et al., (2020) used HPLC to demonstrate the presence of myricetin in F. xanthoxyloides.

Nummularic acid (NA), chemically designated as (19S) 3β-hydroxy-urs-12-en-29β-oic acid, is a pentacyclic triterpenoid with the molecular formula C₃₀H₄₈O₃. It was first isolated in 2007 from Evolvulus nummularius (Dinda et al., 2007); (Younis, Khan, et al., 2018). Subsequent investigations by Younis and colleagues (2018) identified this compound as a key phytochemical constituent of F. xanthoxyloides, a species valued in traditional medicine for more than a hundred years. In addition, nummularic acid has been recognized for its notable anticancer potential.

Cisplatin is a highly effective chemotherapeutic drug, extensively employed in the treatment of testicular tumors. However, its clinical application is limited by a major drawback: the induction of oxidative stress, which leads to alterations in tissue physiology and biochemical composition (Yucel et al., 2019). In this context, Ijaz et al. (2020) investigated the therapeutic potential of F. xanthoxyloides extract in mitigating cisplatin-induced testicular damage in albino rats.

The molecules consist of a coumarin glucoside and a secoiridoid glucoside, connected through the glucose moiety of the coumarin and the carboxyl group at the C-7 position of the secoiridoid (Hadroug et al., 2019). Earlier phytochemical investigations on the bark of Fraxinus rhynchophylla Hance led to the identification of several coumarins, including fraxidin, fraxin, and fraxisecoside (Si et al., 2009). The occurrence of fraxisecoside in F. xanthoxyloides was likewise confirmed by Hadroug et al. (2019).

The compound ligstroside was first identified in Ligustrum obtusifolium Sieb. et Zucc. (Jensen et al., 2002). Subsequently, two new secoiridoid diglucosides (angustifolioside A and B) were obtained from the methanolic leaf extract of Fraxinus angustifolia, along with the previously recognized secoiridoid glucosides ligstroside and oleuropein (Calis et al., 1993). Consistent with these findings, Hadroug (2019) also confirmed the occurrence of ligstroside in F. xanthoxyloides.

The presence of oleuropein in F. xanthoxyloides was further verified by (Omar, 2010; Piroddi et al., 2017; Hadroug et al., 2019).

Esculetin is a naturally occurring compound widely distributed in medicinal plants such as Artemisia capillaries Miq., Artemisia scoparia Waldst. & Kitam., Citrus limonia Osbeck, and Ceratostigma willmottianum Stapf. It has been shown to possess multiple pharmacological properties, particularly anti-proliferative and antioxidant activities (Kok et al., 2009); (Kim et al., 2016). The presence of esculetin in F. xanthoxyloides was also confirmed by Hadroug et al. (2019).

Nicotiflorin is a naturally occurring flavonol glycoside that has been isolated from multiple plant species (Dehaghani et al., 2017). It is associated with diverse pharmacological properties, including reduction of arterial blood pressure and heart rate, hepatoprotection against CCl₄-induced liver injury, as well as pronounced antioxidant, anti-inflammatory, antinociceptive, antihypertensive, and antianaphylactic activities. In addition, nicotiflorin has shown neuroprotective potential by mitigating cerebral ischemic damage, memory impairment, and oxidative stress in multi-infarct dementia model rats (Gamal-Eldeen et al., 2004; Wang et al., 2015)(Dehaghani et al., 2017; Huang et al., 2007). Its occurrence in F. xanthoxyloides has also been validated by Hadroug et al. (2019).

Hadroug et al. (2019) also found the existence of 2-(3, 4-dihydroxy) phenylethyl-O-β-D-glucopyranoside in F. xanthoxyloides.

Salidroside is a principal bioactive compound in several medicinal plants and is recognized for a broad spectrum of pharmacological effects, including anti-aging, anti-fatigue, antioxidant, antiviral, and anti-inflammatory activities, along with notable neuroprotective and cardioprotective roles (Chen et al., 2009); Tan et al., 2009; (Zhu et al., 2011); (Xu et al., 2013); (Yuan et al., 2013). More recently, salidroside has been shown to inhibit cell proliferation and trigger apoptosis in various cancer cell lines, such as lung, bladder, neuroblastoma, and glioma, although its full anticancer mechanisms remain to be fully elucidated (Zhao et al., 2015). The detection of salidroside in F. xanthoxyloides was also confirmed by Hadrouget al. (2019).

Osmanthuside has previously been isolated from plants such as Tabebuia impetiginosa (Mart. ex DC.) Standl. (Warashina et al., 2006) and F. sieboldiana Blume (Lin et al., 2010), later Hadroug et al. (2019) identified this compound in F. xanthoxyloides.

Table 3: Proximate and Elemental composition of Fraxinus xanthoxyloides (G. Don) DC. (Zaman, 2021).

Calceolarioside B has demonstrated significant antiproliferative activity against a range of tumor cell lines, including prostate cancer, human bladder carcinoma (T-24), human gastric adenocarcinoma, uterine carcinoma, and murine melanoma (Khong and Judeh, 2017). Amorim et al. (2017) identified calceolarioside B from Sinningia hatschbachii Chautems through spectroscopic techniques, Verdan et al. (2015) identified it from Sinningia aggregate (Ker Gawl.) Wiehler. Similarly, Hadroug et al. (2018) identified calceolarioside B from F. xanthoxyloides.

Coniferin is a glucoside of coniferyl alcohol (Lemmich et al., 1983). Coniferin, a coniferyl alcohol glucoside, has been detected in the cambial sap of many tree species (Tsuyama et al., 2013). Coniferin has also been detected, though not quantified, in various foods including lemons, asparagus, soft-neck garlic, black salsify, and red beetroot. Consequently, it may serve as a potential biomarker for the dietary intake of these foods (Duke, 1994); (Shinbo et al., 2006). Similarly, Hadroug et al. (2019) studied coniferin in F. xanthoxyloides.

Syringin is a phenolic glycoside isolated from several medicinal plants, including Magnolia sieboldii K. Koch and Tinospora cordifolia (Willd.) Hook. f. & Thomson, although its biological activities have been described in only a limited number of studies (Cho et al., 2001; Liu et al., 2025). Syringin was isolated and characterized from Musa x paradisiaca L. tepal extract (MPTE) by Krishnan et al., (2014). Another plant from which syringin was extracted is Ilex rotunda Thumb (Wang et al., 2008). Similarly Hadroug et al. (2018) also analyzed syringin from F. xanthoxyloides.

8-Hydroxypinoresinol-4-O-β-D-glucoside is an active compound reported to possess antiarrhythmic properties in Valeriana officinalis L. (Liu et al., 2021) and antioxidant activity in Valeriana prionophylla Standl. (Li et al., 2022), Additionally, 8-hydroxypinoresinol-4′-O-β-D-glucoside has been isolated from F. xanthoxyloides (Hadroug et al. 2018).

8-Acetoxypinoresinol-4-O-β-D-glucoside is a lignan glycoside, a class of aromatic polycyclic compounds in which a lignan moiety is glycosidically linked to a carbohydrate unit (Yannai, 2012). Consistent with our findings, Hadroug et al. (2019) also reported the isolation of 8-Acetoxypinoresinol-4-O-β-D-glucoside from F. xanthoxyloides.

Plants not only provide essential nutrients such as fats, proteins, and carbohydrates, but also contain phytochemicals that play vital medicinal roles (Khan et al., 2020). Proximate analysis involves classifying plant components into categories such as moisture, ash, crude protein, lipids, fiber, and nitrogen-free extract (AOAC, 1990). Living organisms require approximately 40 macro- and microelements to survive, and an element is deemed essential when its deficiency leads to a decline in physiological processes (Zaman and Badshah, 2021). Zaman (2021) studied organic matter, moisture, ash, crude protein, fiber, fats, and nitrogen-free extract, along with eight elements (phosphorus, potassium, calcium, magnesium, iron, manganese, cadmium, and zinc) in F. xanthoxyloides from the Terich Valley, Hindukush range (Figure 1 and Table 3). The analysis revealed significant concentrations of calcium and magnesium, supporting the plant’s nutritional and medicinal potential. However, the presence of cadmium, even in trace amounts, suggests that the plant may act as a bio-accumulator. This poses a potential risk for long-term medicinal use, indicating that toxic elemental levels must be monitored in traditional remedies to ensure safety.

Leishmaniasis refers to a group of diseases caused by various species of Leishmania, each associated with distinct clinical manifestations. It is considered the third most prevalent vector-borne disease worldwide, after malaria and filariasis (Solano-Gallego et al., 2009). In 1960 leishmaniasis was initially reported in Pakistan in the north countries. The disease was once limited to these regions but has since spread across the nation. Both cutaneous and visceral leishmaniasis are health-threatening, and mucocutaneous leishmaniasis is infrequently reported (Bhutto et al., 2016); (Shah et al., 2014). The therapeutic efficacy of plant extracts against these infections can be attributed to their various bioactive substances in them, which include essential oils, phenolic compounds, and aldehydes(Sahreen et al., 2013). Methanolic leaf powder extract of F. xanthoxyloides has been investigated for its strong anti-leishmanial activity against Leishmania tropica in the promastigote stage (Younis et al., 2016)

The chloroform extract of F. xanthoxyloides (FXC) showed significant anti-leishmanial activity, with an LD50 of 15.23 ± 0.9 µg/mL compared with the reference drug glucantime, which had an LD50 of 5.6 ±0.4 µg/mL (Table 4). These findings suggest that the chloroform fraction of F. xanthoxyloides can be regarded as a viable source of anti-leishmanial compounds and further studies are needed to identify and characterize its bioactive components. The mechanism underlying this activity-whether via induction of oxidative stress in the parasite or disruption of the mitochondrial membrane-remains unresolved, and we note that an LD50 roughly three-fold higher than that of glucantime indicates that the extract is markedly less potent than the reference drug, not simply “promising” in absolute terms.

Plants are potential replacements for traditional insecticides and acaricides, serving as sources of bioactive substances that tend to act on a narrower range of target arthropods, biodegrade into non-toxic compounds, and are therefore considered greener; their compatibility with integrated pest management further supports sustainable use (Sarwar et al., 2025). The brine shrimp lethality assay was used to test the insecticidal activity of the methanolic leaf extract of F. xanthoxyloides. The chloroform fraction (FXC) was the most active, with an LD₅₀ of 28.15 ± 1.8 µg/mL, followed by the n-hexane fraction (FXH) at 67.59 ± 2.3 µg/mL; the remaining fractions showed relatively low activity. Correlation analysis indicated no significant relationship (p> 0.05) with insecticidal activity (Younis et al., 2016) (Table 4). These LD50 values were not benchmarked against a standard insecticide in the source study, so their practical significance for pest control cannot yet be assessed.

Table 4: Different biological activities of Fraxinus xanthoxyloides

Cytotoxicity is the capacity of a substance to cause toxicity in living cells (Riss and Moravec, 2004). The methanolic leaf extract of F. xanthoxyloides was evaluated for its anti-cytotoxic potential (Younis et al., 2016). Assessment of the extract/fractions was carried out using the sulforhodamine B (SRB) assay described by You et al. (1995). Results from Younis et al. (2016) demonstrated that F. xanthoxyloides exhibited no cytotoxic effects at a concentration of 20 µg/mL.

• Molecular target discovery: Investigate the specific pathways (e.g., mitochondrial disruption) through which F. xanthoxyloides secoiridoids exert anti-leishmanial effects.
• Standardization: Establish HPTLC fingerprints to reconcile the varying doses (2–3g) reported in ethnobotanical surveys into a pharmaceutical standard.
• Toxicity monitoring: Given the plant’s potential as a bio-accumulator, long-term studies must assess the safety of trace cadmium levels found in traditional preparations.

The current review concluded that F. xanthoxyloides has strong evidence of several pharmacological and biological activities. Different parts (root bark, stem bark, stem, wood, foliage, and leaves) of F. xanthoxyloides are used to treat different ailments (malaria, pneumonia, jaundice, labor pain, internal injuries, typhoid fever, trauma, premature birth expulsion of infants, bone fractures, and wounds in cattle). In total, 30 different compounds were studied in this plant for proximate and elemental composition, as well as different biological activities (anti-leishmanial, insecticidal, and cytotoxic). Considering its significant economic value and contribution to the germplasm resources of cultivated plants in Pakistan, it is imperative to acknowledge that northern Pakistan is a vital component of the Central Asian Center of Diversity.

The present study is a piece of research that synthesizes current work and establishes a new perspective.

Muhammad Adnan: Conceptualization, Data Curation, Formal analysis, Investigation.

Muhammad Nauman Khan: Conceptualization, administration and original draft writing, Investigation, Methodology, Resources, Project administration and original draft writing.

Akhtar Zaman: Conceptualization, Data Curation

Hansa Gul: Data Curation, Formal Analysis and Investigation, Software, validation, visualization.

Alevcan Kaplan: Formal Analysis and Investigation, Software, Validation, Visualization.

Sana Wahab: Investigation, Methodology, Resources, Writing review and editing.

Maryam Bibi: Writing review and editing.

Declaration Statements

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All data generated or analyzed during this study are included in this published article.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors declare no conflicts of interest.

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