Evaluation of in vitro cytotoxicity of Asphodelus tenuifolius and Leptadenia pyrotechnica extracts in equine wound healing

Introduction

Wound healing is a complex and dynamic biological process involving inflammation, cell proliferation, extracellular matrix deposition, angiogenesis, and tissue remodeling. In equines, wound management poses particular challenges due to the species' tendency for exuberant granulation tissue formation, infection susceptibility, and delayed epithelialization, especially in distal limb injuries (Fatima et al., 2024) [1] . As such, there is a growing interest in identifying novel, effective, and natural therapeutic agents to enhance the wound healing process and minimize complications in horses. Horses are frequently found to be more prone to secondary complication of wound healing such as excessive granulation tissue (proud flash) formation (Dedar et al., 2020a; Harman et al., 2021) [2,3] . Horses are also sensitive for the development of allergic dermatitis, photodermatitis (Williams et al., 2011) [4] and skin tumors (MacGillivray et al., 2002; Smith, 2008) [5,6] . Medicinal plants have historically played a crucial role in traditional medicine systems, offering bioactive phytocompounds with anti-inflammatory, antimicrobial, and regenerative properties (Rates, 2001; Bhatt et al., 2019) [8,7] . Two such plants A. tenuifolius and L. pyrotechnica are of particular ethnomedicinal significance in South Asia and the Middle East. A. tenuifolius, commonly known as wild onion, is traditionally used for its wound healing, antibacterial, and anti-inflammatory effects (potential). It contains a range of phytochemicals including flavonoids, saponins, and phenolic compounds, which are known to augments cell signaling and tissue regeneration (Bibi et al., 2011) [9] . Recent in vitro studies have demonstrated its antioxidant and anti-proliferative activities, suggesting potential therapeutic applications. L. pyrotechnica found throughout the state of Rajasthan and seen in dry habitats particularly in desert zones. It is currently grown in forests, farm and road sides (Singh et al., 2007; El-Fitiany et al., 2023) [11,10] . It has been documented for its wound healing, anti-inflammatory, and immunomodulatory properties (El-Fitiany et al., 2023) [10] . Preliminary studies in animal models have shown improved wound contraction and collagen deposition following topical application of L. pyrotechnica extracts (Shaw et al., 2014) [12] . Despite these promising attributes, the potential of these medicinal plants for wound management, particularly in equine cells, remains insufficiently explored. Plant-derived extracts may contain a diverse range of bioactive compounds with antioxidant, anti-inflammatory, antimicrobial, and tissue-protective properties that could contribute to wound healing. However, before their therapeutic application, it is essential to estab lish their cellular safety and determine the concentrations that do not adversely affect cell viability. In vitro cytotoxicity assessment therefore provides an important preliminary step in evaluating the suitability of plant extracts for potential wound-healing applications. Accordingly, the objective of the present study was to evaluate and compare the in vitro cytotoxicity of polar and non-polar extracts of Asphodelus tenuifolius and Leptadenia pyrotechnica using the MTT assay. The findings may provide a scientific basis for assessing the safety and therapeutic potential of these plant extracts for the development of plant-derived approaches to equine wound management.

Materials and Methods

The present study was undertaken to comparatively evaluate the in vitro cytotoxicity of leaf extracts of Asphodelus tenuifolius and Leptadenia pyrotechnica using cultured equine dermal fibroblasts. fresh leaves of plant were collected manually from Bikaner region. Asphodelus tenuifolius identified by Botany Department, Government Dungar College, Bikaner. Leptadenia pyrotechnica identified by Central Arid Zone Research Institute, jodhpur. The collected leaves were thoroughly cleaned to remove adhering dust and foreign matter and were dried in a hot-air oven at 50 °C. The dried leaves were subsequently ground into a fine powder using a mixer grinder and stored appropriately until extraction. Analytical-grade chemicals and reagents were used throughout the study. The experimental work involved the preparation of plant extracts, culture and maintenance of equine dermal fibroblast cells, treatment of cells with different concentrations of the plant extracts, and assessment of cell viability using the MTT assay.

Revival and cultivation of equine fibroblast cells

The current study involves the utilization of cryopreserved equine dermal fibroblast cells (third generation), which were previously stored at EPC-NRCE in Bikaner. In order to revive and facilitate the cultivation of these fibroblast cells, the cryopreserved cells were extracted from the liquid nitrogen cryocanister and immediately subjected to a 37 °C water bath for thawing purposes. The cryogenic containers were gently agitated until all ice crystals had dissipated. The contents of each cryovial were then transferred to 15 ml centrifuge tubes containing cell culture medium, and subsequently underwent two rounds of centrifugation for 3 min at 1000 rpm to generate a cell pellet. The supernatant was subsequently discarded and replaced with fresh medium. Following another round of centrifugation for 3 min at 1000 rpm and subsequent removal of the supernatant, the resulting cell pellet was suspended in 6 ml of culture medium. This suspension was then gently centrifuged, and 1 ml was extracted for cell counting purposes. The remaining cell suspensions were then transferred to culture flasks and placed in an incubator containing 5% CO 2 and maintained at a temperature of 37 °C.

Preparation of petroleum ether extract

Exactly 50 gm of powder of each plant was taken in a glass bottle of 1000 ml capacity. Added petroleum ether (PE), to fill the bottle. It was kept at room temperature for 5 days. PE extract of A. tenuifolius and L. pyrotechnica having concentration of 1 mg/ml was diluted with ethanol 1000 times to make concentration 100 µg/ml in first well. Further 2-fold serial dilutions in ethanol were 100 µg/ml to 0.045 µg/ml made till 8th well.

Preparation of methanol ether extract

The remaining unfiltered sediment of petroleum ether extract is dried by a rotary evaporater to make powder. Exactly 50 gm of powder was taken in a glass bottle of 1000 ml capacity. Added methanol, to fill the bottle. It was kept at room temperature for 5 days. methanolic extract of A. tenuifolius and L. pyrotechnica 1 mg/ml in ethanol was diluted in ethanol 1000 times to make 100 µg/ml to 0.045 µg/ml concentration of 100 µg/ml in first well. further 2-fold serial dilutions in ethanol were made till 8th well.

Preparation of aqueous ether extract

The remaining unfiltered sediment of methanolic extract is dried with a rotary evaporater to make powder. Exactly 50 gm of powder was taken in a glass bottle of 1000 ml capacity. Added water, to fill the bottle. It was kept at room temperature for 5 days. Aqueous extract of A. tenuifolius and L. pyrotechnica having concentration of 1 mg/ml of ethanol was diluted with ethanol 1000 times to make concentration 100 µg/ml in first well. Further 2-fold serial dilutions in ethanol were 100 µg/ml to 0.045 µg/ml made till 8 th well.

Cytotoxicity analysis

Cytotoxicity MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) was used (Nirwana et al., 2021). Equine dermal fibroblasts maintained at EPC-NRCE, Bikaner, were trypsinised, seeded into culture plates, and allowed to attach. Cells were then treated with different concentrations of petroleum ether, methanolic, and aqueous extracts of A. tenuifolius and L. pyrotechnica. Following incubation, cell viability was determined by the MTT assay and expressed as a percentage of the untreated control. Extract concentrations maintaining > 80% cell viability were considered non-cytotoxic. The percentage of viable cells was calculated using the following formula :

% Living cells = OD treatment-OD media OD cell control-OD media × 100

Results

The cytotoxicity of various extracts from A. tenuifolius and L. pyrotechnica was evaluated using the MTT assay. Briefly, dermal fibroblast cells were treated with 2-fold serial dilutions (ranging from 100 µg/ml to 0.457 µg/ml) of the extracts for 72 hours in a CO 2 incubator (at 5% CO 2 and 37 °C temperature), alongside a vehicle control or culture medium only. The results of the cytotoxicity study for all extracts are provided below.

Cytotoxicity assay for Petroleum ether extract of A. tenuifolius

-50 grams of A. tenuifolius leaf powder were used to prepare the Petroleum ether extract. The MTT assay indicated significant cytotoxicity at concentrations of 100 µg/ml (~91%), 33.3 µg/ml (~78%), Therefore, a concentration of 1.23 µg/ml was considered non-cytotoxic for dermal fibroblast cells. 11.11 µg/ml (~28%), and 3.70 µg/ml (~5%). Below 1.23 µg/ml concentration the cell viability were comparable with control and vehicle control (Table 1).

Cytotoxicity assay for Methanolic extract of A. tenuifolius -

Similarly, fifty grams of A. tenuifolius leaf powder were used to prepare the methanolic extract. Significant cytotoxicity was observed at concentrations of 100 µg/ml (~90%), 33.3 µg/ml (~85%), 11.11 µg/ml (~57%), 3.70 µg/ml (~29%), 1.23 µg/ml (~27%), and 0.41 µg/ml (~8%). Below 0.14 µg/ml concentration the cell viability were comparable with control and vehicle control (Table 2). Hence, a concentration of 0.14 µg/ml was deemed non-cytotoxic for dermal fibroblast cells. 1.23 µg/ml (~26%), 0.41 µg/ml (~26%), 0.14 µg/ml (~29%), and 0.05 µg/ml (~28%). Surprisingly, the aqueous extract showed higher toxicity compared to the petroleum or methanolic extracts (Table 3). A concentration 0.05 µg/ml was considered non-cytotoxic for dermal fibroblast cells. 4). Hence, a concentration of 3.70 µg/ml was considered non-cytotoxic for dermal fibroblast cells. 5). However, below 0.05 µg/ml concentration the cell viability were comparable with control and vehicle control. Therefore, a concentration of 0.05 µg/ml was deemed non-cytotoxic for dermal fibroblast cells.

Discussion

Wound healing is a complex and highly coordinated biological process involving inflammation, proliferation, tissue formation and remodelling. Dermal fibroblasts are important cells in this process because they contribute to extracellular matrix formation, collagen synthesis and restoration of connective tissue during wound repair. Therefore, in vitro fibroblast-based models are useful for preliminary evaluation of the cellular compatibility and therapeutic potential of plant-derived extracts intended for wound management (Eming et al., 2014; You and Han, 2014) [13,14] . In the present study, the cytotoxicity of petroleum ether, methanolic and aqueous leaf extracts of A. tenuifolius and L. pyrotechnica was evaluated in equine dermal fibroblasts using the MTT assay. Cytotoxicity assessment was performed before subsequent biological investigations to identify concentrations that were well tolerated by the cells. The extracts were tested over a wide concentration range from 0.05 to 100 µg/ml. The results demonstrated clear differences in cellular viability depending on the plant species, extraction solvent and extract concentration. The petroleum ether extract of A. tenuifolius showed pronounced cytotoxicity at the higher concentrations tested. Cell viability was 9.19% at 100 µg/ml and 21.48% at 33.33 µg/ml, whereas a progressive increase in viability was observed with decreasing extract concentration. At 3.70 µg/ml, 1.23 µg/ml, 0.41 µg/ml, 0.14 µg/ml and 0.05 µg/ml, cell viability increased to 95.34%, 105.04%, 103.04%, 122.03% and 110.48%, respectively. These findings indicate that the cytotoxic effect of the petroleum ether extract was strongly concentration-dependent, with substantially better cellular compatibility at lower concentrations. The methanolic extract of A. tenuifolius exhibited a similar concentration-dependent response. Cell viability was markedly reduced at 100 µg/ml (10.32%) and 33.33 µg/ml (14.33%), followed by progressive recovery at lower concentrations. At 11.11 µg/ml, viability increased to 42.38%, while concentrations of 3.70 µg/ml and below resulted in considerably higher viability. At the lowest concentration tested (0.05 µg/ml), cell viability reached 99.27%. Thus, although the methanolic extract exhibited substantial cytotoxicity at higher concentrations, it was considerably better tolerated by equine dermal fibroblasts at lower concentrations. The aqueous extract of A. tenuifolius also showed a concentration-related reduction in cell viability at higher concentrations. Cell viability was 10.20% at 100 µg/ml and 10.90% at 33.33 µg/ml. However, viability increased at lower concentrations, reaching 73.20%, 73.63%, 70.52% and 72.15% at 1.23, 0.41, 0.14 and 0.05 µg/ml, respectively. Compared with the petroleum ether and methanolic extracts, the aqueous extract showed a somewhat different response pattern, suggesting that the biological effects of A. tenuifolius are influenced by the polarity and chemical composition of the extraction fraction. A comparatively different cytotoxicity profile was observed for L. pyrotechnica. The petroleum ether extract showed marked cytotoxicity at 100 µg/ml and 33.33 µg/ml, with cell viabilities of 9.68% and 10.16%, respectively. However, viability increased sharply at lower concentrations, reaching 99.26% at 11.11 µg/ml and remaining above 99% at several subsequent concentrations. At 0.05 µg/ml, cell viability was 103.75%. This indicates that the petroleum ether extract of L. pyrotechnica was highly cytotoxic at the highest concentrations but showed good cellular compatibility at lower concentrations. The methanolic extract of L. pyrotechnica demonstrated relatively favourable cytocompatibility at concentrations below 11.11 µg/ml. Cell viability increased from 8.70% at 100 µg/ml to 86.56% at 11.11 µg/ml and 90.91% at 3.70 µg/ml. At 1.23, 0.41, 0.14 and 0.05 µg/ml, viability was 90.59%, 92.86%, 85.53% and 105.89%, respectively. These findings suggest that the methanolic extract was comparatively well tolerated by equine dermal fibroblasts within the lower concentration range. Similarly, the aqueous extract of L. pyrotechnica exhibited a concentration-dependent improvement in cell viability. At 100 µg/ml, cell viability was only 9.07%, whereas it increased to 72.01% at 11.11 µg/ml and 85.78% at 3.70 µg/ml. At lower concentrations, viability remained relatively high, reaching 92.27%, 91.35%, 86.92% and 101.89% at 1.23, 0.41, 0.14 and 0.05 µg/ml, respectively. Overall, the aqueous extract demonstrated good cellular compatibility at the lower concentrations evaluated. The concentration-dependent cytotoxicity observed in the present study may be attributed to differences in the type and relative abundance of biologically active phytochemicals present in the extracts. Different extraction solvents selectively recover phytoconstituents according to their polarity and physicochemical characteristics, thereby producing extracts with distinct chemical profiles and biological activities. Previous investigations have demonstrated that solvent selec-tion can substantially influence the phenolic and flavonoid composition and, consequently, the biological properties of plant extracts (Khasawneh et al., 2011; Benariba et al., 2021) [16,15] . Therefore, cytotoxicity observed with a crude plant extract should not necessarily be regarded as a fixed characteristic of the plant species itself, but rather as a response influenced by extraction solvent, phytochemical composition and concentration. Asphodelus tenuifolius Cav., belonging to the family Asphodelaceae, is an annual medicinal herb traditionally used for several therapeutic purposes, including inflammatory, rheumatic, digestive and cardiometabolic conditions. Various pharmacological investigations have reported antibacterial, antifungal, anti-inflammatory, antioxidant, diuretic and other biological activities associated with this plant (Mahmood et al., 2011; Laouini et al., 2015; Aslam et al., 2016; Ouhaddou et al., 2020) [18,19,17,20] . More recent phytochemical investigations using chromatographic and mass-spectrometric approaches have further demonstrated the presence of diverse phenolic and flavonoid constituents in A. tenuifolius extracts, supporting the considerable chemical diversity of this species (Benariba et al., 2021) [15] . Such phytochemical diversity may contribute to the biological responses observed in the present cytotoxicity study. Previous phytochemical studies of A. tenuifolius have reported phenolic compounds, flavonoids, tannins, alkaloids, steroids and other secondary metabolites in different extracts. Compounds such as caffeic acid, chlorogenic acid, rutin, apigenin, luteolin and related polyphenolic constituents have been reported from the plant (Laouini et al., 2015; Benariba et al., 2021) [19,15] . The biological effects of these constituents may vary according to their concentration and combination within a particular extract. Phenolic compounds and flavonoids are well known for their antioxidant, anti-inflammatory and other pharmacological activities; however, at sufficiently high concentrations, some phytochemicals may interfere with cellular metabolic processes and adversely affect cell viability. Thus, the concentration-dependent reduction in cell viability observed in the present study may reflect the cumulative effects of multiple constituents rather than the action of a single phytochemical. The cytotoxic response of A. tenuifolius extracts may therefore be explained by differences in phytochemical composition among the petroleum ether, methanolic and aqueous fractions. Non-polar solvents are more likely to extract lipophilic constituents, whereas methanol and water preferentially recover relatively polar compounds, including phenolic acids, flavonoids and other hydrophilic metabolites. Consequently, differences in cellular response among the three extracts may reflect differences in the quantity and composition of the compounds extracted. Similar solvent-dependent variation in the phytochemical profile and biological activity of A. tenuifolius has been reported previously (Benariba et al., 2021) [15] . Leptadenia pyrotechnica (Forssk.) Decne is a medicinal plant traditionally associated with several therapeutic applications. Older literature places the species within Asclepiadaceae, whereas current taxonomic treatments generally include it within Apocynaceae. The plant has been investigated for antioxidant, anti-inflammatory, antimicrobial and other pharmacological activities. Its biological properties have been associated with a chemically diverse range of secondary metabolites, including phenolic acids, flavonoids, alkaloids, terpenoids, sterols and glycosidic constituents (Khasawneh et al., 2011; Javid et al., 2019; Alqahtani et al., 2022) [16,22,21] . Importantly, Khasawneh et al. (2011) [16] demonstrated considerable variation in the polyphenolic composition of different fractions of L. pyrotechnica. Gallic acid, vanillic acid, caffeic acid, epicatechin and quercetin-3-β-D-glucoside were identified in the extracts, with substantial differences in their concentrations among ethanol, ethyl acetate, n-butanol and aqueous fractions. The ethyl acetate fraction contained particularly high levels of total phenolics and showed pronounced biological activity, demonstrating that the pharmacological properties of L. pyrotechnica can vary according to the fractionation procedure and chemical composition (Khasawneh et al., 2011) [16] . More recent phytochemical studies have also confirmed the presence of caffeic, gallic, vanillic, ferulic, p-coumaric and cinnamic acids, together with flavonoid and terpenoid constituents in different extracts of the plant (Alqahtani et al., 2022; Al-Salman et al., 2024) [21,23] . The presence of these phytochemicals may partly explain the concentration-dependent cellular response observed in the present study. Phenolic acids and flavonoids can exert beneficial biological effects at suitable concentrations, whereas increasing concentrations may alter cellular redox status, membrane integrity or metabolic activity. The observed reduction in cell viability at higher extract concentrations is therefore consistent with the possibility that increasing concentrations result in greater cellular exposure to biologically active constituents. However, because the present study evaluated crude extracts rather than individual purified compounds, the contribution of specific phytochemicals to cytotoxicity cannot be established directly. When the two plant species were compared, L. pyrotechnica generally demonstrated better cellular compatibility at several of the lower concentrations tested, particularly in its methanolic and aqueous extracts. In contrast, A. tenuifolius, particularly its petroleum ether and methanolic extracts, showed comparatively greater cytotoxicity at the highest concentrations. Nevertheless, both plant species exhibited substantial recovery of cell viability following reduction in extract concentration. These findings indicate that cytocompatibility was influenced not only by plant species but also by extraction solvent and concentration. Such solvent-dependent differences are consistent with previous studies demonstrating that different fractions of medicinal plants can possess markedly different phytochemical compositions and biological activities (Khasawneh et al., 2011; Benariba et al., 2021) [16,15] . The MTT assay provides an indirect measure of cellular viability and metabolic activity through the reduction of the tetrazolium salt MTT to a coloured formazan product by metabolically active cells (Mosmann, 1983) [24] . Therefore, the marked decrease in MTT-derived viability at higher extract concentrations indicates reduced cellular metabolic activity under those conditions. However, because the MTT assay reflects cellular metabolic activity rather than viability alone, the results should be interpreted as an indicator of metabolic cytotoxicity or reduced cellular activity. Additional assays assessing membrane integrity, apoptosis or oxidative stress would be useful to confirm the mechanism underlying the observed reduction in viability.

From the perspective of equine wound management, cytocompatibility is an important preliminary criterion for evaluating plant-derived candidates for topical wound-healing applications. Fibroblasts play a central role in extracellular matrix deposition, collagen synthesis, tissue remodelling and wound contraction. Therefore, an extract intended for further wound-healing evaluation should ideally maintain adequate fibroblast viability at concentrations that are capable of producing beneficial biological effects. The present findings indicate that both A. tenuifolius and L. pyrotechnica contain extract fractions that can be tolerated by equine dermal fibroblasts when applied at appropriately selected concentrations. This provides a preliminary basis for subsequent investigation of their effects on wound-healing-associated cellular responses. The comparatively favourable cytocompatibility of L. pyrotechnica at lower concentrations is noteworthy because previous investigations have demonstrated considerable phenolic diversity in this plant and have identified compounds such as epicatechin, vanillic acid, caffeic acid and quercetin derivatives (Khasawneh et al., 2011) [16] . Similarly, the phytochemical diversity of A. tenuifolius may account for the biological activity observed in its different solvent extracts (Laouini et al., 2015; Benariba et al., 2021) [19,15] . Nevertheless, the relationship between individual phytochemicals and fibroblast viability requires further investigation using chemical profiling and purified compounds. Overall, the present cytotoxicity study demonstrated a clear concentration-dependent response to petroleum ether, methanolic and aqueous extracts of both A. tenuifolius and L. pyrotechnica. Higher concentrations generally produced greater reductions in cell viability, whereas lower concentrations were comparatively better tolerated. Among the extracts evaluated, L. pyrotechnica generally showed comparatively favourable cytocompatibility at lower concentrations. The differences observed between extracts may be associated with variation in phytochemical composition resulting from both plant species and extraction solvent. These findings support the selection of appropriately tolerated, non-cytotoxic concentrations for subsequent evaluation of the wound-healing potential of these medicinal plants in equine dermal fibroblasts. Further studies integrating phytochemical profiling with mechanistic cellular assays would be valuable for identifying the constituents responsible for the observed biological effects.

Conclusion

The extracts of Asphodelus tenuifolius and Leptadenia pyrotechnica showed concentration-dependent cytotoxicity in equine dermal fibroblasts. Lower concentrations were generally well tolerated, with L. pyrotechnica extracts demonstrating comparatively favourable cytocompatibility. These findings provide a basis for selecting safe concentrations for further evaluation of their potential in equine wound healing.

Acknowledgement

AcknowledgmentsWe extend our sincere gratitude to Prof. (Dr.) Satish K. Garg, Hon'ble Vice-Chancellor, RAJUVAS, Bikaner, and Dr. Tarun Kumar Bhattacharya, Director, ICAR-NRCE, Hisar, for providing institutional support and facilities. We are thankful to Dr. S. C. Mehta, Officer In-Charge, ICAR-NRCE, EPC, Bikaner, for laboratory and equipment access, and Dr. Ramesh Kumar Dedar, Senior Scientist, for his valuable research guidance. We also acknowledge Dr. Assim Verma and Dr. Garvit (SRF, NRCE) for their assistance during the experimental work

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24. How to cite this article: Gahlot C, Sankhala LN, Dedar RK, Kant L, Gahlot A. Evaluation of in vitro cytotoxicity of Asphodelus tenuifolius and Leptadenia pyrotechnica extracts in equine wound healing. International Journal of Advanced Biochemistry Research. 2026;10(9):798-805. doi: https://www.do i.org/10.33545/26174693.2026.v10.i9j.10181