Since ancient times, medicinal plants have been used as the main source of remedies. Their application in traditional medicine is still essential to healthcare, especially in underdeveloped nations with limited access to contemporary drugs. The World Health Organization (WHO) estimates that around eighty percent of the world’s population trust on plant-derived medicines for their primary health needs. There is need for ongoing research into the medicinal properties of plants, to both validate traditional uses and discover new potential therapeutics [1].

The resurgence of interest in medicinal plant research is largely driven by the limitations of synthetic drugs, such as side effects, resistance, and high costs. Medicinal plants often contain a wide range of bioactive compounds that work synergistically, providing a holistic approach to disease prevention and treatment. Furthermore, with the emergence of antibiotic-resistant pathogens and chronic diseases like cancer, diabetes, and cardiovascular conditions, there is an urgent need for alternative treatment options that are both safe and effective [2-8].

Phytochemicals, the bioactive component in medicinal plants, have been shown to exhibit various pharmacological activities, including anti-inflammatory, antimicrobial, anticancer, and antioxidant effects. These compounds include alkaloids, flavonons, terpenoids, and polyphenols, many of which have already been isolated and are being studied for their therapeutic potential [9-11].

Recent advances in technology, including high-throughput screening and bioinformatics, have accelerated the process of discovering new bioactive compounds from plants. These technologies allow researchers to isolate, characterize, and test hundreds of compounds in a short period, thus speeding up the drug discovery process. However, the biodiversity of plants is vast, with only a small fraction of the estimated 250,000 to 500,000 plant species on Earth having been scientifically investigated. This leaves a wealth of untapped potential for discovering new treatments. In addition to direct therapeutic applications, medicinal plants are also important for their role in sustainable development. Many plant species are grown and harvested in a way that supports local economies, particularly in rural and indigenous communities. By promoting the cultivation and research of medicinal plants, it is possible to create a system of medicine that is both effective and sustainable [12-13].

However, it is important to approach medicinal plant research with a scientific rigor that ensures the safety and efficacy of plant-based treatments. Standardization of herbal medicines, clinical trials, and toxicity studies are essential to establish their reliability as alternatives to synthetic drugs [14-15].

Necessity of Membrane Stabilizing Activity Studies

Membrane stabilizing activity (MSA) studies are essential in the evaluation of anti-inflammatory and anti-hemolytic properties of medicinal plants. A complicated biological reaction to damaging stimuli which is known as inflammation and it is often associated with various chronic diseases, such as arthritis, cardiovascular diseases, and autoimmune disorders. The stabilization of cell membranes, particularly red blood cells (RBCs), can inhibit the release of inflammatory mediators, thereby reducing inflammation [16].

Many medicinal plants exhibit membrane stabilizing properties, which contribute to their anti-inflammatory effects. For instance, plant extracts containing flavonoids, alkaloids, and saponins have been shown to prevent haemolysis and stabilize cellular membranes under stress conditions. This protective effect is crucial in maintaining the integrity of cells during inflammatory responses and helps prevent tissue damage [17].

Membrane stabilizing studies provide insight into the mechanism of action of plant-based anti-inflammatory agents. By inhibiting the lysis of cells, particularly erythrocytes, these studies demonstrate the potential of medicinal plants to serve as natural alternatives to non-steroidal anti-inflammatory drugs (NSAIDs), which are often associated with side effects such as gastrointestinal irritation and cardiovascular risks [18-19].

Furthermore, the MSA of plants is also relevant in the context of immune modulation, where maintaining membrane integrity is vital for proper immune function. Continued research into the membrane stabilizing effects of medicinal plants might result in safer and more efficient inflammatory disease treatments.

Necessity of Thrombolytic Activity Studies

Since cardiovascular diseases (CVDs) are one of the leading causes of death globally, research on the thrombolytic action of plants has garnered attention. Heart attacks and strokes are commonly caused by thrombus development in blood arteries. Despite its effectiveness, traditional thrombolytic medicines like urokinase and streptokinase have disadvantages such as high cost, restricted accessibility, and adverse effects, including bleeding issues. Therefore, a promising strategy for creating thrombolytic medicines that are affordable, easily accessible and safer is to seek natural substitutes from plants.

The bioactive substances flavonoids, alkaloids, and saponins found in plant-derived thrombolytic medicines have been demonstrated to prevent or dissolve blood clots by enhancing fibrinolysis and inhibiting platelet aggregation. Research on medicinal plants with thrombolytic properties can help develop novel treatments that have fewer adverse effects and may even reduce cost. xploring thrombolytic properties in plants is essential for expanding therapeutic options and addressing the global burden of CVDs [20-22].

General Information about the Plant

Boeica filiformis, belonging to the genus Boeica, is scientifically classified as Boeica filiformis C.B. Clarke. Boeica filiformis produces small, pink-purple flowers arranged in loose, thread-like panicles that emerge from the leaf axils. The flower cluster stalks are slender and typically exceed 7 cm in length. Both the stalks and branches of the inflorescence are hairless, contributing to the plant's fine texture. The flowers are tubular with a 5-parted calyx, and they contain four stamens, each with short filaments and two-celled anthers. The ovary is stalkless. Its leaves are narrow-elliptic, tapering at the ends, measuring about 10-15 cm in length and 2.5-4 cm in width, with approximately nine lateral veins on each side. The fruit is a linear capsule, 1-1.5 cm long, containing small, ellipsoid, smooth seeds [23-26].

Preparation of Plant Extract

The following procedures were employed in the preparation of plant extracts, unless otherwise specified.

Selection of Plant

Based on extensive literature review, Boeica filiformis from the family Gesneriaceae was selected for biological investigation due to its reported biological and chemical properties. Whole plant of Boeica filiformis were chosen for chemical analysis.

Collection and Identification of the Plant

When the plant was collected from Moulvibazar in November 2023, a taxonomist at the National Herbarium of Bangladesh in Mirpur, Dhaka, identified it. The voucher specimen was deposited in the herbarium for future reference, with the assigned accession number DACB 87894.

Drying, Grinding, and Storage

The freshly collected plant materials were thoroughly washed with water to remove dirt, followed by sorting to eliminate dead leaves. The clean materials were cut into small pieces and air-dried under sunlight for 10-15 days. After the plant material had completely dried, it was ground into a coarse powder with a grinding machine and kept in sealed containers for additional testing.

Extraction of Plant Materials

Approximately 80 grams of the powdered plant material was soaked in 500 mL of methanol in an amber-colored bottle. The mixture was sealed and left to macerate for 14 days, with periodic shaking and stirring. The extract was then filtered through filter paper.

Evaporation of Solvent

The methanolic extract was concentrated using a rotary evaporator under reduced pressure at 50°C. The remaining solvent was evaporated under a ceiling fan, yielding a gummy concentrate. Approximately 5-6 grams of methanolic extract were obtained for further analysis.

Qualitative Phytochemical Analysis

Phytochemical screenings of the plant extracts were performed to identify the presence of various bioactive compounds such as triterpenes, steroids, alkaloids, coumarins, anthraquinones, flavonoids, tannins, saponins and phenolic acids. The tests relied on the development of specific color changes or precipitates, which served as analytical indicators.

Detection of Alkaloids: After dissolving the extracts in diluted hydrochloric acid, they were filtered. The filtrates were subjected to Mayer's, Wagner's, and Hager's tests. In Mayer’s test, a yellow cream precipitate indicated alkaloids, while Wagner’s test showed a brown or reddish precipitate. Hager’s test resulted in a yellow precipitate, confirming the presence of alkaloids.

Detection of Carbohydrates: For this study, extracts were dissolved well in distilled water and filtered. Molisch’s test indicated carbohydrates by a violet ring formation. Benedict’s test produced an orange-red precipitate for reducing sugars, while Fehling’s test resulted in a red precipitate, verifying that reducing sugars are present.

Detection of Glycosides: Plant extracts were treated with ferric chloride and sodium hydroxide. Dark green and yellow colors indicated the presence of tannins and glycosides, respectively.

Detection of Saponins: Froth and foam tests confirmed saponins by the persistence of foam after shaking the extracts with water.

Detection of Flavonoids

Alkaline reagent and lead acetate tests produced intense yellow color and yellow precipitate, respectively, indicating the presence of flavonoids.

By assessing the reduction capability of phenolic compounds, the Folin-Ciocalteu technique was used to determine the total phenolic content of the B. filiformis methanol extract. By dissolving 7.5 g of sodium carbonate in 100 mL of distilled water, a 7.5% sodium carbonate solution was created. Gallic acid (0.025 g in 5 mL of 100% ethanol) was converted into a stock solution to reach a concentration of 5 µg/µL. From there, several concentrations (200, 100, 50, 25, 12.5, and 6.25 µg/mL) were created. Additionally, methanol was used to dissolve each root extract, producing a stock solution with the same concentration that was subsequently diluted for the test.

 For the assay, 1.0 mL of the root extract or standard solution was placed in a test tube, followed by the addition of 5 mL of diluted Folin-Ciocalteu reagent and 4 mL of the 7.5% sodium carbonate solution. The mixtures were incubated for 30 minutes at 20°C for the standard solutions and for one hour for the extract solutions to ensure complete reaction. The absorbance was measured at 765 nm using a UV-spectrophotometer against a blank containing the solvent. Using the formula C = (c × V) / m, where C is the total phenolic content in milligrams per gram of plant extract, c is the concentration of gallic acid from the calibration curve, V is the volume of extract in milliliters, and m is the weight of the crude plant extract in grams, the total phenolic content was expressed in gallic acid equivalents (GAE).

Preliminary phytochemical screening

Table 1: Results of phytochemical screening of methanol extract of Boeica filiformis

• Absent -- , Low concentration +-, High concentration ++

Several bioactive components were found via preliminary phytochemical screening like  alkaloids,  carbohydrates, alkaloids,  glycosides, Tannins, proteins and amino acids.

Determination of total phenolics content

The Folin-Ciocalteu reagent was used to measure the extract's phenolic content. The extract's phenolic content was determined using the gallic acid standard curve, which is displayed in Table 2 and Figure 1. The findings were reported as gallic acid equivalent (GAE) in milligrams per gram of dried extractives.

Table 2: Gallic acid absorbance at various concentrations following the Folin-Ciocalteu reagent reaction

Figure 1: The Calibration curve of Gallic acid for the determination of total phenolic

Table 3: Determination of total phenolic content of the methanol extract of B.filiformis

The extracts' phenolic content was measured in milligrams of galic acid equivalent per gram, and against the absorbance of 0.330, which signifies the presence of phenolic content, it is 31.8 GAE/g.

Membrane Stabilizing Activity

Different partitionates of B. filiformis crude methanol extract effectively prevented the haemolysis of HRBC membrane produced by hypnotic solution at 2 mg/ml, according to in vitro membrane stabilizing activity, as compared to the standard.

Table 4: Percentage of inhibition of hypotonic solution induced haemolysis of erythrocyte membrane by standard and crude methanol extract of B.filiformis.

Figure 2: Percentage of inhibition of hypotonic solution induced haemolysis of erythrocyte membrane by standard and methanol extract of B. filiformis.

According to the findings of this investigation, the methanol extract of B. filiformis protected the haemolysis of the HRBC membrane by demonstrating a 67.70% suppression of the haemolysis caused by the hypnotic solution. The methanol extract of B. filiformis may therefore have anti-inflammatory properties through inhibiting erythrocyte lysis.

Thrombolytic activity

Table 5: Data analysis of thrombolytic activity

Figure 3: Percentage of clot lysis Plant extract, standard and blank

The methanol extract of B.filiformis exerted 81.64% lysis of the blood clot in thrombolytic activity test while 72.89% and 20.45% lysis were obtained for positive control (streptokinase) and negative control respectively.

The findings from this study provide substantial insights into the phytochemical composition and biological activities of the methanol extract of Boeica filiformis, highlighting its potential therapeutic applications. The preliminary phytochemical screening revealed the presence of various bioactive components, including alkaloids, carbohydrates, glycosides, tannins, and proteins, which have been linked to numerous health benefits. The high concentration of alkaloids detected using multiple tests (Mayer’s, Wagner’s, Hager’s, and Dragendorff’s) is particularly noteworthy, as these compounds are known for their diverse pharmacological properties, including analgesic and anti-inflammatory effects. The presence of carbohydrates and glycosides, albeit at varying concentrations, suggests that B. filiformis may possess additional nutritional and medicinal benefits, supporting traditional uses of this plant in various cultures.

The total phenolic content of 31.8 mg GAE/g of extract signifies that B. filiformis is a valuable source of phenolic compounds, which are known for their health-promoting properties, including anti-inflammatory and anticancer effects. The correlation between phenolic content and antioxidant activity suggests that these compounds could be integral to the therapeutic efficacy of B. filiformis.

The membrane stabilizing activity exhibited by the methanol extract, with a 67.70% inhibition of haemolysis at a concentration of 2 mg/ml, supports the potential anti-inflammatory effects of B. filiformis. This activity, comparable to the standard aspirin (80.23% inhibition), indicates that the extract may offer protective effects against cellular damage caused by inflammatory agents, possibly due to its phytochemical constituents that enhance membrane integrity [27].

Furthermore, the thrombolytic activity observed, with 72.89% lysis of the blood clot in comparison to the positive control, suggests that B. filiformis may have significant implications in cardiovascular health, potentially providing an alternative to conventional thrombolytic therapies. The results support the traditional use of this plant in managing conditions related to blood clotting and cardiovascular diseases, warranting further exploration into its mechanisms of action and active components responsible for such effects [28].

In summary, the comprehensive analysis of Boeica filiformis reveals a promising profile of phytochemicals that confer anti-inflammatory, and thrombolytic activities. These findings not only validate the traditional medicinal uses of this plant but also open avenues for further research into its application in modern therapeutic contexts. Future studies should aim to isolate and identify specific bioactive compounds within the extract, elucidating their individual contributions to the observed biological activities, and exploring their potential for use in nutraceutical or pharmaceutical formulations. Such investigations could significantly enhance our understanding of the therapeutic roles of B. filiformis and contribute to the development of novel health-promoting products.

The findings from this study highlight the substantial pharmacological potential of the methanol extract of Boeica filiformis, particularly its antioxidant, anti-inflammatory, and thrombolytic activities, underpinned by a rich profile of bioactive compounds. Preliminary phytochemical screening revealed the presence of alkaloids, carbohydrates, glycosides, proteins, and amino acids, suggesting that B. filiformis is a promising source of various therapeutic agents. The quantification of total phenolic content, calculated as 31.8 mg gallic acid equivalent per gram of dried extract, underscores the extract’s significant phenolic richness, known for its association with antioxidant properties. The extract exhibited considerable membrane-stabilizing activity, with 67.70% inhibition of hypotonic solution-induced haemolysis, indicating its potential as an anti-inflammatory agent. This aligns with the idea that stabilizing erythrocyte membranes could play a pivotal role in mediating anti-inflammatory responses, thereby offering insights into its mechanism of action. The thrombolytic activity of the extract was also promising, with a lysis percentage of 72.89%, which, while lower than the standard (81.64% lysis by streptokinase), demonstrates a significant capacity for clot disruption. This suggests that B. filiformis may have therapeutic applications in managing conditions related to thrombus formation, further broadening its medicinal potential. Collectively, these findings support the traditional use of B. filiformis in herbal medicine and lay the groundwork for further investigations into its active constituents and their specific biological mechanisms. The evidence of various bioactive compounds and their associated pharmacological activities may not only validate its traditional applications but also contribute to the development of new therapeutic agents derived from natural sources. Future studies should focus on isolating these active components, elucidating their structures, and exploring their individual and synergistic effects, which could facilitate the advancement of B. filiformis into modern medicinal formulations. The promising outcomes of this study highlight the need for more extensive research, including in vivo studies, to fully understand the therapeutic potential and safety profile of B. filiformis, aiming ultimately to integrate such natural products into conventional treatment protocols.

AUTHOR CONTRIBUTIONS

Sabiha Ferdowsy Koly created concept and supervised study, Sanjana Hoque Jui conducted the lab experiment, Israt Jahan Bulbul and Nusratun Nahar drafted the manuscript, Sonia Zaman reviewed the manuscript.

FUNDING

This research received no external funding.

ACKNOWLEDGMENTS

Authors offer sincere gratitude to Department of Pharmacy for all kind of assistance.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The ethical review committee of the pharmacy department at Southeast University in Dhaka, Bangladesh, gave its approval for this study's use of human blood in the assays. Additionally, donors' informed agreement was sought before using their blood in this study.

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