Recently, the herbal remedies have been recognized as alternative medical treatments for managing primary healthcare among 80% of the world’s populations, especially in low- and medium-income countries (El-Nashar et al., 2021; El-Shawi et al., 2023). It is due to the fact that most often synthetic drugs result in many unavoidable adverse events, grow resistance (e.g., antibiotics) and tolerance (e.g., narcotic drugs) due to repeated uses, and thereby reduce safety and efficacy (Ishaq et al., 2022; El-Nashar et al., 2023b; Younis et al., 2023).

The use of pteridophytes (e.g., aka, ferns and fern allies) is ancient (>2000 years) due to their many beneficial properties. Pteridaceae is recognized as an important family among the pteridophytes. Many species in this family are rich with alkaloids, glycosides, and flavonoids and have promising medicinal features, such as antioxidant, anti-cancer, anti-inflammatory, antimicrobial, antidiabetic, and neurobiological effects (Bhuia et al., 2023a). Cheilanthes tenuifolia (Burm.f.) Swartz, which belongs to the family Pteridaceae, is a petite, evergreen fern that has the ability to reach a height of 70 cm. The delicate lip fern is a species of fern loacal to North America that is considered an ornamental plant (Mahfuz et al., 2019). It grows properly in open, warm, moist, shady, rocky regions and is often found in small crevices high up on cliffs (Sen and Mukhopadhyay, 2014; Patel and Reddy, 2018). It is found in many regions of the world, including Australia, China, Malaysia, Bangladesh, Nepal, Cambodia, Laos, New Zealand, the Philippines, Polynesia, Sri Lanka, Taiwan, Uruguay, Thailand, Vietnam, Tasmania, and India. September to November is considered the growing season for the plants. In India, there are over 30 species of this plant. The fronds are 63 cm long and 17 cm wide, with a dark red-brown stipe and rachis that are smooth or have sparse hairs consisting of 2–13 cells and very few slender scales. The lamina is pentagonal, triangular, or ovate, with 3-4 pinnates at the base and 3 pinnates for most of its length. The larger pinnae are triangular-ovate, the pinnules are lanceolate or ovate, and the ultimate pinnules may have a slightly caudate shape. The margins are either lobed or entire. The upper and lower surfaces of the lamina have very few, short (less than 0.5 mm), pointed hairs consisting of 2 or 3 cells, and are occasionally almost hairless. The spores are tetrahedral or rounded-tetrahedral, granulose, and trilete, with a varying degree of reticulate-echinate ornamentation, and have a diameter of 38–53 µm with 32 per sporangium.

Ancient people (prehistoric times) used the rhizome juice of ferns for gastrointestinal disorders (including peptic ulcer), cuts, and wounds. Traditionally, C. tenuifolia leaf juice is mixed with hot water and honey to cure throat pain (Augustin and Thomas, 2015). Its leaf and stem decoctions are used for healthy hair (Hanum and Hamzah, 1999). The tribes of Northeast India use its rhizomes and root extracts as general tonics (Benniamin, 2011; Singh and Upadhyay, 2014). One study reports that the plant contains important phytochemical groups, like steroids, alkaloids, flavonoids, triterpenoids, phenolic compounds, and tannins (Ghorpade et al., 2015). To date, two important flavonoids such as quercetin and rutin have been extracted from an ethyl acetate-soluble extract of C. tenuifolia (Jarial et al., 2018). The plant-derived compound quercetin is a natural aglycone of rutin. It is frequently used in dietary supplements due to its many important and promising bioactivities, including antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, anti-cancer, antidiabetic, antiallergy, antihypertensive, and organ-protective (e.g., brain, heart, liver, kidney, and GIT tract) effects (Ahmed et al., 2022). A recent investigation proposes that methanolic whole plant extract possesses a significant amount of phenolics and flavonoids and has demonstrated strong antioxidant, cytotoxic, membrane-stabilizing, and thrombolytic activity (Mahfuz et al., 2019). This study also confirmed that the plant contains stigmasterol. Therefore, there is a lack of adequate scientific studies on this hopeful medicinal plant and there is no detailed information on isolated phytochemicals, anti-inflammatory activity and its underlying mechanisms, antifungal properties, antibacterial effects against huge number of pathogenic bacteria and the molecular mechanisms of each activity.

Knowing the overall facts, this study aimed to do a preliminary phytochemical analysis along with the assessment of the radical scavenging, anti-inflammatory, antimicrobial, and clot lysis capacities of Cheilanthes tenuifolia leaf extracts and this study also evaluated the rectitude of the previous findings of the therapeutic activity of the plant.

Among the natural products, plants are common sources of traditional healing agents worldwide, especially in the poor and less developed countries (Ashmawy et al., 2023; Mia et al., 2023). On the other hand, people in developed countries are now conscious of the adverse events of modern drugs, thus there is a developing enthusiasm for research into and utilization of plant-mediated products or preparations (Abdelazim et al., 2024; Shill et al., 2024). Plant extracts contain numerous groups and components, including alkaloids, glycosides, phenolics and flavonoids, vitamins, and minerals (Gunathilake et al., 2018). Certain groups of compounds have diverse bioactivities, for example, phenolics and flavonoids have promising antioxidant and anti-inflammatory activities (Tlili et al., 2013) and play many important roles in biological systems such as anti-inflammatory (Ginwala et al., 2019), antibacterial, anti-cancer (Khalil et al., 2020), and anti-atherosclerotic effect (Salvamani et al., 2014). A certain study reported that certain organic polar solvents, such as ethanol and methanol, are the best solvents for the extraction of phenolics from natural sources (Ballard et al., 2009). Alkaloids, on the contrary, play vital roles in plants and humans. It is because these are considered defensive compounds. Alkaloids also regulate the growth of plants (Chik et al., 2013). Furthermore, glycosides have many significant therapeutic potentials, including antifungal (Khan et al., 2017) and anticancer (Khan et al., 2019) activities. Current drug development methodologies and modern medicine do not use complete plant extracts and instead rely on specific compounds. Taking the whole plant or extracts without isolating components as practiced in traditional medicine has a stronger therapeutic impact than specific substances (Wen et al., 2005; Thomford et al., 2018). Therefore, the isolated chemical constituents are important for developing novel drugs.

The plant is evidently composed of steroids, alkaloids, flavonoids, triterpenoids, phenolic compounds, and tannins (Ghorpade et al., 2015). Another study reports that the plant is rich in phenolics and flavonoids. It contains quercetin, rutin, and stigmasterol (Mahfuz et al., 2019). Our study also confirmed that the plant contains stigmasterol. In this study, we have seen that ethanolic leaf fractions of C. tenuifolia contain flavonoids. Additionally, we have also found that the plant leaf contains alkaloids, steroids, saponins, glycosides, and reducing sugars. Thus, the compounds of these phytochemical groups may be linked to the observed bioactivities. However, safety pharmacology is critical throughout the drug discovery and development process. Prior to first-in-human investigations, safety pharmacology assays, tests, and models anticipate the clinical risk profile of a possible new medicine (Morimoto et al., 2015). According to the findings of our study the crude ECT up to a 3,000 mg/kg oral dose did not show any behavioral changes or toxicological symptoms, or even death, in Swiss mice. Previous study on the plant extract showed that the LC₅₀ values of the chloroform, n-hexane, methanol soluble and ethyl acetate extracts were 34.493 μg/mL, 205.984 μg/mL, 751.169 μg/mL and 66.235 μg/mL respectively on shrimp nauplii (Mahfuz et al., 2019).

Oxidative stress from various sources due to an overabundance of reactive species production (e.g., ROS, RNS) is considered a precursor of diseases and disorders in humans. This can lead to neurological and cardiovascular diseases, cancer, diabetes, etc. (Rekatsina et al., 2020; Forman and Zhang, 2021). Antioxidants are the substances that act against oxidative stress. External antioxidants are required once our body’s defensive mechanisms, including physiological antioxidants, fail to manage an overabundance of free radicals. Plant-based natural antioxidants play significant roles in managing this situation (Jamshidi-Kia et al., 2020). It is due to their being readily available, economic, and biocompatible. Moreover, we can readily access these compounds through our daily diets (Landete, 2013). In a recent experiment, Mahfuz et al. (2019) demonstrated that methanolic whole plant extract exhibited strong (IC₅₀ = 9.926 μg/mL) DPPH free radical scavenging capacity. Another investigation of the plant’s methanolic extract found that the two flavonoids (rutin and quercetin) present in the extract showed considerable in vitro anti-oxidant activity; in this regard, the DPPH scavenging capability of quercetin (86.1%) was higher than that of rutin (73.2%) (Jarial et al., 2018; Daryono and Rhomawati, 2020; Daryono and Rhomawati, 2020). Another bioactive triterpenoid isolated from the n-hexane extract of the plant also has potent antioxidant capacity such as stigmasterol (Mahfuz et al., 2019), which significantly reduced the ROS generation in different in vitro investigation (Agatonovic-Kustrin et al., 2018; Bakrim et al., 2022).

In this investigation, we have seen that all the fractions of C. tenuifolia revealed significant DPPH free radical scavenging capacity in a concentration-dependent manner, with the IC₅₀ values estimated for the ECT, HCT, and CCT within the range of 22.17 ± 1.90 to 78.13 ± 2.08 μg/mL, respectively. ECT exhibited a better DPPH radical scavenging effect than the other two fractions. It is because of the large amount of phenolics and flavonoids in this fraction such as rutin and quercetin as well as terpenoids including stigmasterol (Jarial et al., 2018; Mahfuz et al., 2019). The promising DPPH radical scavenging effect of other species of Cheilanthes such as C. anceps also reported by (Chowdhary et al., 2010).

Oxidative stress can provoke inflammatory cascades. Thus, antioxidants have protective effects in a biological system. Certain antioxidants, such as polyphenols, have an anti-ageing effect (Moliner et al., 2020), and these can prevent or delay many disease conditions, including neurological diseases and disorders, cardiovascular diseases, diabetes, and so on (Rodríguez-Yoldi, 2021; Bhuia et al., 2023b). A study performed by Mahfuz and his coworkers in 2019 suggests that the methanol, n-hexane, ethyl acetate, and chloroform fractions of C. tenuifolia showed significant membrane stabilizing capacity in the HRBC assay, where the percent inhibition of hemolysis was determined within the range of 2.97 and 73.97 (Mahfuz et al., 2019). The n-hexane fraction showed the highest percent inhibition against hypnotic solution-induced hemolysis (73.97%), while the ethyl acetate fraction showed the highest percent inhibition of heat-induced hemolysis (67.27%). Our study demonstrated that the organic fractions of C. tenuifolia, especially its ECT, exhibited significant egg protein and HRBC protection capabilities. Another study by Akbor et al. (2023) also stated that methanolic extract of the plant has a notable membrane lysing inhibitory activity (1.42% ± 0.02%) on HRBCs (Akbor et al., 2023). One report suggested that the species C. farinose is traditionally used to manage inflammation (Yonathan et al., 2006). Thus, our present findings are in agreement with the traditional values and scientific reports observed in the databases. According to the findings of different studies the bioactive substances (rutin, and quercetin) of the plant has potent anti-inflammatory activity and liable for diminishing different inflammatory markers such as cytokines (Abd Nikfarjam et al., 2017; Bhuia et al., 2023c).

Infectious diseases due to microbial attacks (e.g., bacteria, fungi, viruses, and parasites) in humans are a common consequence in the world. Among the wide variety of natural products, plants are considered major sources of antimicrobial agents (Touati et al., 2018). The species of the Pteridaceae family are known for their diverse bioactivities, including antioxidant, anti-inflammatory, antimicrobial, anti-cancer, antidiabetic, and neurobiological properties (Baskaran et al., 2018). To date, a number of bioactive compounds have been introduced from the Cheilanthes genus, for example, glycosides of apigenin, chrysoeriol, luteolin, kaempferol, and quercetin from C. concolor, C. flexuosa and C. goyazensis (Salatino and Prado, 1998). In this study, we have seen that all the organic fractions of C. tenuifolia acted against both gram (+) and gram (−) species as well as many pathogenic fungi, suggesting its broad-spectrum anti-microbial effects. Conventional antimicrobial agents have some potential adverse effects, for example, allergic reactions and enhanced resistance (Mohsen et al., 2020). For example, chloramphenicol is widely used in meningitis, but it increases the risk of aplastic anemia. Another example is sulfonamides, which were considered “wonder drugs,” but these are evidently contributing to severe skin reactions (King et al., 2018). Therefore, it is crucial to inhibit microbial pathogenesis using appropriate and safe antimicrobials (French, 2005). Previous study by Jarial et al. (2018) also manifested the antimicrobial properties of the plant and the study also revealed that isolated flavonoids such as quercetin and rutin from C. tenuifolia showed inhibitory effects against Staphylococcus aureus and Enterobacter sp. with MIC values of (2.25 and 0.45 μg/mL), respectively (Jarial et al., 2018).

Atherothrombosis is one of the major causes of morbidity and mortality in the world. Chronic pathology is responsible for vascular remodeling through various pathways, including oxidative stress (Martin-Ventura et al., 2017). ROS and RNS play important pathophysiological roles in vascular diseases, such as atherothrombosis (Chen et al., 2018). It is also evident that microorganism-mediated infections result in the production of potentially reactive molecules in our body through oxygen metabolism (Brown et al., 2014), which is responsible for many pathological conditions, including inflammation, atherosclerosis, carcinogenesis, and so on (Aruoma, 1998). The methanolic whole plant extract is evidently able to exert significant clot lysis capacity at 100 µg/tube (Mahfuz et al., 2019). The authors demonstrated that the ethyl acetate, n-hexane, methanol, and chloroform fractions exhibited 31.59, 12.10, 17.01, and 41.26% clot lysis capacities, respectively. Another study by Akbor et al. (2023) also reported that the methanolic extract of the plant hindered hemolysis in a concentration-dependent manner and inhibited 78.93% ± 0.01% hemolysis (IC₅₀ = 46 ± 2.11 μg/mL) at the higher concentration (160 μg/mL) (Akbor et al., 2023). Findings of our study suggest that ECT, CCT, and HCT showed clot lysis capacities of 61.71 ± 0.03, 37.80 ± 0.02, and 15.19% ± 0.04% at 500 µg/tube. ECT showed better clot lysis capacity than the other two fractions. Due to the presence of different phytochemicals such as alkaloids, flavonoids, and steroids (Uddin et al., 2019; Tabassum et al., 2022), the bioactive phytoconstituents of the plant such as rutin, quercetin, and stigmasterol also have remarkable hemolytic activity (Zaragoza et al., 2021; Bari et al., 2022; Chen et al., 2022). We suppose that the observed clot lysis capacity, especially by the ECT, might be linked to the existence of phytochemical groups such as alkaloids, glycosides, and flavonoids and bioactivity such as radical scavenging, anti-inflammatory, and anti-microbial activities. Natural products and their structural counterparts have historically made significant contributions to pharmacology, particularly in cancer and infectious disorders (El-Nashar et al., 2024). Nonetheless, natural products pose hurdles for drug development, such as technical barriers to screening, isolation, characterization, and optimization, which have contributed to a drop in their pursuit by the pharmaceutical industry since the 1990s (Atanasov et al., 2021; El-Nashar et al., 2023a).

As a conclusion, the organic fractions of C. tenuifolia leaf extract remain rich in many valuable phytochemical groups, including alkaloids, tannins, glycosides, flavonoids, and saponins. All the fractions exhibited concentration-dependent and significant radical scavenging, anti-inflammatory, and anti-microbial effects against different gram (+) and gram (−) bacteria, as well as a number of pathogenic fungi, and clot lysis capacities. ECT exhibited better anti-radical (IC₅₀ = 22.17 ± 1.90), anti-inflammatory, anti-bacterial, and clot-lysis capacity than the CCT and HCT. We suppose that all the activities, because of the presence of alkaloids, glycosides, and flavonoids within the organic fractions of C. tenuifolia leaf extract and the bioactive compound rutin, quercetin and stigmasterol play an important role in exerting therapeutic activities against oxidative stress and inflammation related diseases and disorders, pathogenic infections and a good source of thrombolytic agents. However, additional research is deemed necessary to conduct the isolation, identification, and clarification of the underlying molecular mechanisms responsible for each individual bioactivity as well as clinical investigation of the plant’s extract and exploited phytochemicals which would be useful in producing plant-based pharmaceuticals to treat numerous complicated human diseases.