Plants are the massive source of medicines because they produce a wide range of bioactive molecules, the majority of which act as chemical defence against predation or infection¹. Natural plant-based products are gaining popularity due to their low side effects and wide range of applications, which includes pharmaceuticals and cosmetics.

Medicinal and aromatic plants consist of a variety of phytochemicals, which includes alkaloids, phenolics, saponins, steroids, flavonoids, glucosides, terpenoids, tannins, aliphatic alcohols, acids and esters, and essential oils, that are used in a wide range of applications in pharmaceuticals². Limonia acidissima is a member of the Rutaceae family and also known as Feronia limonia, Elephant apple, Feronia elephantum, and Schinus limonia³. It is indigenous to Bangladesh, India, Pakistan, and Sri Lanka, and it is commonly known as wood apple⁴. Wood apple is widely used in Ayurvedic medicine to treat dysentery, peptic ulcers, chronic diarrhea, and other ailments. Limonia acidissima fruit is commonly used for preparing puddings, murabba, and fresh juice⁵. In Indian folk medicine, the decoction of L. acidissima leaf is used to treat constipation, vomiting, cardiotonics, and diuretics⁶.

In developed countries, cancer is a complex disease that is difficult to cure in most cases. During the last decade, there has been a growing demand for newer and safer chemotherapy agents. Cancer is the third leading cause of death worldwide, after cardiovascular, infectious, and parasitic diseases. Medicinal plant extracts contain a variety of compounds, including polyphenols, flavonoids, alkaloids, terpenoids, and saponins, which may have therapeutic benefits and prevent cancer formation. More than 60% of current cytotoxic agents are derived from natural sources such as plants, marine organisms, and microorganisms, either directly or through chemical synthesis using natural lead compounds. Natural products are widely used in cancer chemotherapy⁷.

Globally, Herbal plants contain a wide range of phytochemicals with anti-oxidative properties and have been used in cancer treatment to reduce side effects and toxicity⁸.

A thrombus, or blood clot, that forms in the circulatory system as a result of hemostasis failure blocks the blood vessels and can have fatal consequences in thrombolytic diseases like acute myocardial infarction or cerebral infarction⁹. Blood clot formation has been a severe blood circulation lately. A thrombus or embolus prevents blood flow by blocking a blood vessel, depriving tissues of oxygen and regular blood flow. These effects lead to tissue necrosis in that location. Thrombin is a protein that forms blood clots from fibrinogen and is lysed by plasmin. Tissue plasminogen activator (tPA) activates plasminogen. A fibrinolytic medication works by dissolving thrombin in acutely blocked coronary arteries, which improves prognosis, limits necrosis, and restores blood flow to the ischemic myocardium. Numerous thrombolytic drugs are used to treat myocardial infarction¹⁰. Plants consist of fibrinolytic protease enzymes that promote thrombolysis. Plasmin is produced by plasminogen activators like urokinase (UK)-type plasminogen and tissue-type plasminogen activator. Plasmin dissolves the fibrin network that causes blood clots and restores blood flow to affected tissues. Heart disease, caused by blood clots (thrombus), is a serious condition that has been on the rise in recent years. Thrombolysis is an emergency treatment used to dissolve blood clots in arteries feeding the heart and brain, which are the leading cause of heart attacks and ischemic strokes^11,12.

The aim of this study was to investigate the thrombolytic and anti-cancer properties of the Limonia acidissima extract.

2. MATERIALS AND METHODS:

2.1. Plant Material:

The fresh leaves of (Limonia acidissima L.) were collected from the local areas of Akluj, Solapur, Maharashtra (India). After collection of leaf of Limonia acidissima L. the taxonomical identification by Mr. Rajendra S. Suryavanshi Dept. of Botany, at Dr. Ganpatrao Deshmukh Mahavidyalaya Sangola. Then these leaf were dried under the shade dry for 5 days. This dried leaf was grinded to obtain fine powder, then this powder was stored in the polythene bag to protect it from the moisture at room temperature before extraction.

2.2. Preparation of Hydroalcoholic Leaf Extract:

The dried powder of leaf of Limonia acidissima L. was extracted hydroalcoholic cold maceration. The preparation of extracts were done by using two ratios. Hydroalcoholic solvent was prepared Methanol and distilled water with ratios 50:50(Methanol: Water) and 75:25 (Methanol: Water) respectively. 10gm of dried powder was taken in 100ml of hydroalcoholic solvent in a conical flask tied with filter paper by using tread. Then shake occasionally for 24hr., it was filtered through muslin cloth and the supernatant was collected and kept 24hr. air dry, and 15min at 70°C in a hot air oven. Then obtain dry residues. The residues were weighed to obtain the extractive yield of both the extracts and were stored in air tight bottles¹³.

2.3. Preliminary Phytochemical Testing:

The hydroalcoholic extracts of Limonia acidissima L. leaf were subjected to chemical identification tests in order to determine which major classes of phytochemicals were present¹⁴.

2.4. Anti-cancer Activity (Brine Shrimp Lethality Assay):

Brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of extracts of Limonia acidissima¹⁵.

2.4.1. Preparation of Sea Water:

38gm of NaCl (without iodine) and 0.006gm yeast were weighed, and transferred into 1000ml of volumetric flask. Then make up the volume by using distilled water and filtered off to get clear solution.

2.4.2. Hatching of Brine Shrimp:

Brine shrimp eggs collected from pet shops was used as the test organism. Seawater was taken in the small tank and shrimp eggs were added to the tank and then this tank placed under dark area, two days were allowed to hatch the shrimp and to be matured as nauplii. Constant oxygen supply was carried out through the hatching time. The hatched shrimps are attracted to the light (phototaxis) and so nauplii free from egg shell was collected from the illuminated part of the tank. The nauplii was taken from the fish tank by a pipette and diluted in fresh clear sea water to increase visibility and 10 nauplii were taken carefully by micropipette¹⁵.

2.4.3. Preparation of Test Solution:

0.1 mg plant extract were taken and dissolve in 0.2ml of pure dimethyl sulphoxide (DMSO) and finally the volume was made to 100ml to sea water. Thus, the concentration of the stock solution was 1000µg/ml. Then the solution was serial diluted to 10, 20, 30, 40, 50µg/ml with sea water. Then 2.5ml of plant extract solution was added to 2.5ml of sea water containing 10 nauplii. Percent mortality can be calculated by following

Formula:
No. of napuli taken - No.of live napuli)

% Mortality = ––––––––––––––––––––––––––––×100

No.of napuli taken

Figure no. 1: Live Nauplii with test sample

2.5. Thrombolytic Activity:

Aspirin is used as a standard to investigate thrombolytic activity.

2.5.1. Preparation of Reagents:

1000µg/ml stock solution of aspirin was prepared in hydroalcoholic medium. Three concentrations of 200, 500, and 1000µg/ml were prepared from the stock solution and 1000µg/ml hydroalcoholic test solution was made with leaf extracts. Three concentrations (200, 500, and 1000µg/ml) were prepared from the test solution.

2.5.2. Collection of Blood:

Whole blood was drawn from healthy human volunteers without a history of oral contraceptives or anti-coagulant therapy and 1ml of blood was transferred to the previously weighted sterile Eppendorf tubes and was allowed to form clots.

2.5.3. Determination of Thrombolytic Activity:

The transferred to different pre weighed sterilized micro-centrifuge tube (1 ml/tube). The Eppendorf tubes were placed into centrifuge machine at 37°C for 45 minutes to form clot. Serum was completely removed from the tubes without disturbing the clot formed and discarded. Each of the tube having clot was again weighed. Then prepared conc. of standard and test are added into labelled tube having clot accordingly. The tubes containing clot with conc. were incubated at 37°C for 90 minutes for observation of clot lysis. After supernatant fluid was completely removed and discarded, again weighed to observe the difference in weight after clot disruption. Finally, difference obtained in weight of the experimental tubes was calculated and the result was expressed as percentage of clot lysis by the following equation:^16,17

w₂-w₃

Percentage of clot lysis = –––––––––––––––––×100

w₂-w₁

Where,

w₁₌ Empty weight of Eppendorf tube.

w₂₌Weight after removal of serum (after centrifuge)

w₃₌Weight after clot lysis.

3. RESULTS AND DISCUSSION:

3.1. Phytochemical Screening of Leaf Extracts:

Phytochemical analysis of Limonia acidissima leaf extract showed the presence of major classes of phytochemicals such as flavonoid, glycosides, alkaloids, saponin, steroid, tannins. Hydroalcoholic ratio methanol: water (50: 50) contains active constituent flavonoid, glycosides, alkaloids, saponin, steroid, tannins and methanol: water (75: 25) contains flavonoids, alkaloids, steroids, tannins (Table 1).

Table no. 1: Results of phytochemical screening of hydroalcoholic extracts of Limonia acidissima L.

Phytochemical tests	Hydroalcoholic Extracts Methanol: Water
Phytochemical tests	50:50	75:50
Alkaloids	+++	++
Flavonoids	++ +	++
Glycosides	+++	++
Tannins and Phenols	+ +	++
Saponin	+ +	++ +
Steroids	+ +	+ +

++ indicates = Present , - indicates = Absent

3.2. Anticancer Activity:

Anticancer activity has been assessed by using brine shrimp lethality bioassay.

The hydroalcoholic extract of Limonia acidissima leaf showed significant result in comparison with the Standard (5-Fluorouracil) & that's why it can be assumed that extract is pharmacologically active.

Table no. 2: In- vitro anti-cancer activity of hydroalcoholic extract of Limonia acidissima L.

Sr. No.	Conc. µg/ml	Total Nauplii	No. of nauplii dead			No. of nauplii live	% Mortality Mean ±SEM	LC ₅₀ value µg/ml
Sr. No.	Conc. µg/ml	Total Nauplii	T1	T2	T3	No. of nauplii live	% Mortality Mean ±SEM	LC ₅₀ value µg/ml
1	10	10	2	2	3	23	20.333±0.881	40
2	20	10	3	4	3	20	30±1.154
3	30	10	4	4	4	18	40±0.577
4	40	10	5	5	6	14	50±1.154*
5	50	10	7	6	7	10	59.666±1.452**

Values are expressed as Mean ± SEM (n=3) Data was analyzed using one way ANOVA followed by Dunnett’s multiple comparison test by using graph pad prism 8.0.1. (*p<0.05, **p<0.01, ***p<0.001)

Table no. 3: In- vitro activity of Standard (5 fluorouracil) of Limonia acidissima L.

Sr. No.	Conc. µg/ml	Total Nauplii	No. of nauplii dead			No. of nauplii live	% Mortality Mean ±SEM	LC ₅₀value µg/ml
Sr. No.	Conc. µg/ml	Total Nauplii	T1	T2	T3	No. of nauplii live	% Mortality Mean ±SEM	LC ₅₀value µg/ml
1	10	10	3	2	3	22	25.333±0.666	32.21
2	20	10	4	3	4	19	35±0.577
3	30	10	5	4	5	16	47±0.577
4	40	10	5	7	6	12	60±1.154
5	50	10	8	6	7	9	70±1.154**

Values are expressed as Mean ± SEM (n=3) Data was analyzed using one way ANOVA followed by Dunnett’s multiple comparison test by using graph pad prism 8.0.1. (*p<0.05, **p<0.01, ***p<0.001)

Hydroalcoholic extract has significant activity with a % mortality of 20.333±0.881, 30±1.154, 40±0.577, 50± 1.154, 59.666±1.452 at concentration 10, 20, 30, 40, and 50µg/ml respectively and standard % mortality of 25.333 ±0.666, 35±0.577, 47±0.577, 60±1.154, 70±1.154 at concentration 10, 20, 30, 40, and 50µg/ml respectively. By plotting different concentration Vs % of mortality for test samples showed a linear correlation. From the graph, the median lethal concentration LC₅₀, the concentration at which 50% mortality of brine shrimp nauplii were determined. Hydroalcoholic extract and standard had LC₅₀ values of, 40µg/ml and 32.21µg/ml respectively. The LC₅₀ values of which extract is low that extract should be shows more significant activity. The crude hydroalcoholic extract of Limonia acidissima showed significant cytotoxic activity against brine shrimp nauplii (Table 2 and 3).

Figure no. 2: Anti-cancer activity of Limonia acidissima extracts presented as percent of mortality

3.3. Thrombolytic Activity:

Table no. 4: In-vitro % clot lysis of hydroalcoholic extract of Limonia acidissima L.

Sr. No.	Conc. µg/ml	%Clot lysis (Mean ± SEM)		IC₅₀
Sr. No.	Conc. µg/ml	Standard	Test	Standard	Test
1.	250	56±1.154	50±0.577	67.77	202.9
2.	500	70±1.154*	63±1.732*
3.	1000	83±1.154**	77.33±1.763^**

Values are expressed as Mean ± SEM (n=3) Data was analyzed using one way ANOVA followed by Dunnett’s multiple comparison test by using graph pad prism 8.0.1.(*p<0.05, **p<0.01, ***p<0.001)

Figure no. 3. Clot lysis

The thrombolytic agent works by activating the plasminogen enzyme, which can remove cross-linked fibrin mesh. This dissolves the clot, which is then subjected to more proteolysis by other enzymes, restoring blood flow over hindered blood arteries^11,12. Hydroalcoholic extract has significant activity with a % clot lysis of 50±0.577, 63±1.732, 77.33±1.763 at concentration 250, 500, 1000µg/ml respectively and standard % clot lysis of 56±1.154, 70±1.154, 83±1.154 at concentration 250, 500, 1000µg/ml respectively. Hydroalcoholic extract and standard had IC₅₀ values of, 202.9µg/ml and 67.77µg/ml respectively. The % clot lysis of the extract at various concentrations as well as a comparison to the standard medication, are indicated in (Table 4).

Figure no. 4: Thrombolytic activity of Limonia acidissima extracts presented as percent of clot lysis

4. CONCLUSION:

In this study, the leaf extracts of Limonia acidissima L. were investigated to preliminary phytochemical screening of hydroalcoholic extract with different ratio like Methanol: Water (50:50), Methanol: Water (75: 25) and Methanol: Water (50:50) among all the extract Methanol: Water (50:50) shown maximum number of active metabolite like Flavonoid, Glycosides, Alkaloids, Saponin, Steroid, Tannins so based on quantity of active metabolite present we use Methanol: Water (50:50) for further analysis. It was discovered that Methanol: Water (50:50) extracts had a higher concentration of Flavonoids than other extracts.

The hydroalcoholic extract of Limonia acidissima showed excellent anticancer activity against brine shrimp larvae. When compared to the standard extract (5-Fluorouracil), the results show pharmacological activity with LC₅₀ values of 32.21 and 40µg/ml, respectively. Additionally, the investigated extract revealed significant thrombolytic action (IC₅₀ 202.9µg/ml) in comparison with standard aspirin (IC₅₀ 66.77µg/mL). Further investigation is needed to isolate and assess the pure phytoconstituents identified in Limonia acidissima leaf, which could serve as natural anticancer and thrombolytic agents.

5. ACKNOWLEDGMENT:

The authors are grateful to the Chairman Dr. Dilipkumar Ingawale and Principal Dr. Manojkumar Patil for providing the facilities to conduct my research work in laboratory.

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Received on 05.06.2025 Revised on 12.08.2025

Accepted on 17.09.2025 Published on 20.01.2026

Available online from January 27, 2026

Asian J. Pharm. Tech. 2026; 16(1):25-29.

DOI: 10.52711/2231-5713.2026.00005

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