IngredientsMarsilea quadrifolia L. is an aquatic fern belonging to the family Marsileaceae. All parts of the plant possess a multitude of phytochemical secondary metabolites which impart an exceptional assortment of medicinal uses. The plant is classified under the Division Pteridophyta, Class Polypodiopsida, Order Salviniales (Marsileales), Family Marsileaceae, and Genus Marsilea. Marsilea quadrifolia L. is a leptosporangiate aquatic fern which has played a key role in the evolutionary history of plants, and is characterized by heterospory, the ancestral progressive trait that led to the evolution of seeds.
Its common names include "four leaf clover" and "European waterclover" (USA), even though it is not a species of clover. The plant is also commonly known as water clover, pepperwort, and water shamrock. In India it is called Sushni, and in Tamil Nadu it is known as Aarai keerai or Aala keerai.
Marsilea quadrifolia is a herbaceous plant found naturally in central and southern Europe, Caucasia, western Siberia, Afghanistan, south-west India, China, Japan, and Vietnam, though it is considered a weed in some parts of the United States, where it has been well established in the northeast for over 100 years. It is an aquatic fern that grows from creeping rhizomes anchored in the muddy bottoms of shallow ponds and lakes. The species has creeping, fleshy, adventitious roots containing multiple rhizomes. From the rhizomes, a four-leaf clover-like frond grows above the water level with a long petiole, at the base of which the sporocarps containing spores are located. Leaves may float in deep water or remain erect in shallow water or on land. Leaflets are obdeltoid, to 3/4 inch long and glaucous, with petioles to 8 inches long. The sporocarp is ellipsoid, dark brown, on stalks to 3/4 inch long, attached to the base of petioles.
Although its common names include "four leaf clover," Marsilea quadrifolia is not a species of clover (Trifolium). It is an aquatic fern bearing a 4-parted leaf that merely resembles a four-leaf clover (Trifolium). This distinction is important in supplement and ethnobotanical contexts, as the plant's chemistry and pharmacology are entirely distinct from those of Trifolium species.
Its documented uses include antibacterial, antioxidant, neurodegenerative disorder, anticonvulsive, and anticholinesterase applications. Several extract types — including aqueous, chloroform, ethanol, methanol, and petroleum ether extracts — have been used for different pharmacological activities in research. As a dietary supplement ingredient, the plant is most commonly encountered as a dried leaf powder, fresh whole plant, or as a standardized extract. In traditional food use, fresh leaves and young shoots are consumed directly.
In some places, Marsilea quadrifolia has been used as food for more than 3,000 years. The plant has diverse medicinal properties and has been in use for more than 3,000 years as part of food. It is used in the Ayurvedic System of Medicine for curing several ailments. In Asia it is used as a food source and in traditional Ayurvedic medicine.
Marsilea minuta L. (a closely related species within the same genus), commonly known as Sunishannaka in Ayurvedic medicine, is an aquatic fern traditionally used for the management of various neurological and inflammatory disorders. The leaves and young shoots of related Marsilea species are commonly consumed as vegetables and have long been used in traditional and ethnomedicine for managing various health conditions.
Marsilea quadrifolia is used in Ayurvedic practice to treat cough, bronchitis, diabetes, psychiatric diseases, eye diseases, diarrhoea, and skin diseases. In Indian traditional medicine it is referred to specifically as a sedative and anticonvulsant agent.
The plant is used in traditional medicine for its diuretic, anti-inflammatory, and depurative properties. The leaf extract of the plant is also applied externally to treat snake bites and skin injuries. Additionally, M. quadrifolia is traditionally used to treat fever, cold, cough, and for wound healing. A juice made from the leaves is employed as diuretic and febrifuge. The plant is also applied externally in the treatment of snakebites and skin injuries, including abscesses.
The seasonal variation in the nutrient composition of Marsilea quadrifolia, as an edible semi-aquatic plant, has been studied in order to promote its consumption as a green leafy vegetable. In parts of West Bengal, India, and other regions, the fresh fronds are consumed as cooked greens or incorporated into traditional cuisine.
The chemical composition of M. quadrifolia is diverse and includes phenolic compounds, tannins, saponins, flavonoids, steroids, terpenoids, alkaloids, carbohydrates, and several others that possess antioxidant properties. The plant contains tannins, flavonoids, betulinic acid, fatty acids, and sterols. It is also reported to contain quercetin, hentriacontane, and other important phytoconstituents with pharmacological properties.
Marsilea quadrifolia is an edible aquatic medicinal plant used as a traditional health food in Asia. Four new polyphenols — including kaempferol 3-O-(2″-O-E-caffeoyl)-β-d-glucopyranoside, kaempferol 3-O-(3″-O-E-caffeoyl)-α-l-arabinopyranoside, 4-methy-3'-hydroxypsilotinin, and (±)-(E)-4b-methoxy-3b,5b-dihydroxyscirpusin A — together with 14 known polyphenols, were isolated from the ethanol extract of M. quadrifolia. In DPPH and oxygen radical absorbance capacity antioxidant assays, some compounds showed stronger antioxidant activities, and quercetin was among the most active.
Qualitative analysis has identified tannins, saponins, flavonoids, steroids, terpenoids, triterpenoids, alkaloids, carbohydrates, proteins, and phenolic compounds in the plant. Tannins, saponins, flavonoids, steroids, alkaloids, carbohydrates, proteins, and phenolic compounds were all confirmed in both methanol and aqueous leaf and stem extracts. Quantitatively, phenolic compounds were measured at 8.34 ± 0.92 mg/g (leaf) and 7.31 ± 0.46 mg/g (stem), flavonoids at 7.46 ± 0.64 mg/g (leaf) and 6.45 ± 0.68 mg/g (stem), alkaloids at 6.12 ± 0.51 mg/g (leaf) and 5.89 ± 0.61 mg/g (stem), tannins at 6.58 ± 0.72 mg/g (leaf) and 6.07 ± 0.56 mg/g (stem), and saponins at 5.32 ± 0.48 mg/g (leaf) and 6.30 ± 0.58 mg/g (stem).
The plant material contains carbohydrate (19.5% g), protein (4.9%), fat (0.2%), amino acid (3%), flavonoids (0.3%), and saponins (0.3%). Seasonal variations in the compositions of sodium, potassium, calcium, phosphorus, and β-carotene contents in the whole plant have been reported. Total phenolics, flavonoids, alkaloids, tannins, and saponins in both leaves and stems have been separately evaluated. The plant also has higher amounts of thiamine, riboflavin, and vitamin C than other compared species. Marsilea quadrifolia exhibited wide fluctuations between seasons and was described as "not very promising in nutrient composition when compared to other commonly used green leafy vegetables" in one published analysis. Among assessed freshwater macrophytes, carbohydrate levels were highest in M. quadrifolia.
M. quadrifolia has been characterized as a potent source of polyphenols with antioxidant properties that may be utilized for alleviating oxidative stress. Its significant antioxidant potential is attributed to the presence of bioactive compounds, particularly quercetin. Antioxidant activity using DPPH was found to increase in a concentration-dependent manner. All three tested extracts — ethyl acetate, aqueous, and methanol — exhibited antioxidant activity with IC50 values of 10, 125, and 25 µg/mL respectively, compared to the standard BHT with an IC50 value of 7.5 µg/mL.
It is plausible that the neuroprotective changes seen with chloroform extract of M. quadrifolia (CEMQ) may be attributed to N-methyl-D-aspartate (NMDA) receptor antagonistic properties. The neurotrophic activity of the plant is attributed to quercetin, a potent antioxidant that is also neuroprotective. Quercetin derivatives may be present in the extract, which may bind with the NMDA receptor and antagonize it, causing improvement against excitotoxicity. Findings from in silico studies indicated that both quercetin and the standard drug memantine exhibited excellent binding affinities with the NMDA glutamate receptor (5EWJ).
A published study (Bhadra S, Mukherjee PK, Bandyopadhyay A., Natural Product Research, 2012) documented cholinesterase inhibition activity of Marsilea quadrifolia Linn., an edible leafy vegetable from West Bengal, India. This mechanism is of interest in the context of age-related cognitive disorders, though no human clinical work on this specific endpoint has been published.
The anti-inflammatory effect of ethanolic extract of Marsilea quadrifolia has been evaluated in vitro against the human red blood cell (HRBC) membrane stabilization method, inhibition of protein denaturation method, and neutralization of anti-coagulation activity. Results exhibited a concentration-related inhibition of HRBC membrane stabilization, protein (albumin) denaturation, and neutralization of anticoagulant. The effect of the standard drug was found to be slightly greater than that of the Marsilea quadrifolia test extract, leading to the conclusion that aerial parts of M. quadrifolia possessed marked in vitro anti-inflammatory and anti-venom effect.
Traditional basis: Marsilea quadrifolia is a common Indian hydrophytic fern referred to in Indian traditional medicine as sedative and anticonvulsant, and leaf extracts were reported to protect epileptic rats against electroconvulsions in early studies (Satyavati et al., 1987).
Preclinical (animal) evidence: Water and ethanol extracts of M. quadrifolia at doses of 200 and 400 mg/kg (water extract) and 100, 200, and 400 mg/kg (ethanol extract) significantly increased the latency of seizure compared to respective vehicle controls. Seizure severity scores were decreased significantly in a dose-dependent manner. Duration of epileptic seizure was also decreased significantly for both extract types compared to their respective vehicle control groups. This work, published in the Journal of Ethnopharmacology (Sahu et al., 2012), employed a pentylenetetrazole (PTZ)-induced seizure rat model and assessed behavioral performance and electroencephalographic (EEG) outcomes.
Further investigation of phytochemicals in the methanolic leaf extract of M. quadrifolia (MQLE) showed diverse medicinal properties with antiepileptic efficacy in PTZ-induced rat models of epilepsy. The protective role of MQLE was investigated against PTZ-induced seizure, with behavioral performance tested using the open field, elevated plus maze, learning, and memory by Morris Water Maze tests.
Evidence strength: All anticonvulsant evidence is limited to animal models. No human clinical trials have been conducted on this endpoint, and translation to humans remains undemonstrated.
Preclinical evidence: In a rat model of monosodium glutamate (MSG)-induced excitotoxicity, animals treated with MSG showed greater reduction in locomotor score, impairment in memory and learning, increased blood levels of calcium and sodium, and neuronal disorganization with cerebral edema and neuronal degeneration. These changes were significantly improved in animals that received either the standard drug memantine (20 mg/kg) or chloroform extract of M. quadrifolia (CEMQ) at 200 and 400 mg/kg. Overall, this study indicated that M. quadrifolia ameliorated MSG-induced neurotoxicity. Future investigations are required to explore the neuroprotective mechanism of M. quadrifolia and its active constituents. This study was published in Frontiers in Pharmacology (2023) and indexed in PubMed (PMID: 37781695).
Evidence strength: Preclinical animal data only; the mechanism appears plausible via NMDA antagonism and antioxidant activity of quercetin derivatives, but no human studies exist.
Marsilea quadrifolia is traditionally used for treatment of diabetes by the natives of Jharkhand in India.
Preclinical evidence: The antidiabetic and antioxidant principles of the methanolic extract of Marsilea quadrifolia (MEMQ) were evaluated in an alloxan-induced diabetic rat model. The hypoglycemic effect was assessed by oral administration of plant extract at a dose of 300 mg/kg body weight in fasting glucose-loaded rats and in alloxan-induced (110 mg/kg body weight, intraperitoneal) diabetic rats, in comparison with the reference drug metformin hydrochloride (100 mg/kg) during a 3-day study period. The extract at doses of 100 and 200 mg/kg body weight showed glucose level reduction of 31.87% and 46.97%, respectively, in alloxan-induced diabetic mice, while metformin achieved 50.74% reduction after 2 weeks.
Marsilea quadrifolia has a folk reputation in central and southern India as a hypoglycemic agent. One study evaluated the effect of methanolic leaf and stem extracts on carbohydrate metabolic enzymes in alloxan-induced diabetic rats. Blood glucose levels were elevated in alloxan-induced diabetic rats compared to normal control rats, and a decreased level of blood glucose was observed in diabetic rats treated with leaf and stem extracts of M. quadrifolia. A dose of 300 mg/kg body weight in rats was reported to revert the levels of activities of hexokinase, pyruvate kinase, and glucose-6-phosphate dehydrogenase.
Additional work has focused on optimization of the extraction process and validation of the traditional antidiabetic claim in streptozotocin-nicotinamide-induced type 2 diabetic rats. Box-Behnken Design (BBD) software was used for optimization of extraction process and total phenolic content.
Evidence strength: All evidence is from animal (rodent) models. No human clinical trials exist. The mechanism appears to involve modulation of carbohydrate metabolic enzymes and direct antioxidant protection of pancreatic beta cells; however, direct extrapolation to humans is not warranted without clinical data.
In vitro evidence: Antioxidant potential of ethanolic extract of the plant was assessed by DPPH free radical scavenging assay at 517 nm and total antioxidant capacity by phosphomolybdenum method at 695 nm. The extract showed a moderate free radical scavenging capacity (IC50 value of 243.82 µg/mL for the plant extract, compared to 58.92 µg/mL for ascorbic acid) and moderate total antioxidant capacity compared to ascorbic acid.
In summary of that study, the extract showed significant antidiabetic and antioxidative potency, and the investigation suggests that MEMQ may be a potential source of natural antioxidant with good hypoglycemic activity.
Evidence strength: Primarily in vitro. In vivo antioxidant effects in rodents have been documented, but human trial data are absent.
In vitro evidence: The antibacterial and antifungal activity of hexane, ethyl acetate, ethanol, and methanol extracts of Marsilea quadrifolia was explored against 31 species of bacteria and 7 species of fungi. Antibacterial activity was assessed by the Kirby-Bauer disc diffusion method, with extract concentrations of 1.25, 2.5, and 5 mg/disc. Antifungal activity testing showed that hexane extract (0.002 g/mL) was effective against Aspergillus flavus and Candida albicans. Ethyl acetate extract (0.002 g/mL) was effective against Aspergillus flavus and Trichophyton rubrum.
Antibacterial and antifungal activities of methanol, ethanol, diethyl ether, and aqueous extracts of leaf and stem of M. quadrifolia against selected human bacterial and fungal pathogens have been reported in peer-reviewed literature.
Evidence strength: In vitro only. No clinical or in vivo antimicrobial trials have been published. The relevance of these MIC values to infectious disease treatment in humans cannot be established from current evidence.
The cytotoxic activity of extracts of Marsilea quadrifolia on MCF-7 cells from human breast cancer was investigated in vitro using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This laboratory study explored whether plant extracts could suppress the viability of cancer cell lines in cell culture. No animal studies or human trials on this endpoint have been identified in the published literature. Evidence is therefore preliminary and exploratory only.
No established human clinical dosage exists for Marsilea quadrifolia. The following dosages reflect those reported in preclinical studies only and cannot be extrapolated as human recommendations.
An acute oral toxicity study determined that the LD50 of the methanol extract of aerial parts of M. quadrifolia was greater than 2,000 mg/kg body weight. There was no significant variation found in body weight or organ-to-body mass index. No mortality or behavioral changes were observed in treated animals. All animals belonging to the treated group survived throughout the 14-day observation period after dosing. The methanol extract of aerial parts of Marsilea quadrifolia Linn had a nontoxic effect on the biochemical and haematological parameters studied up to a dose of 2,000 mg/kg. This study was conducted according to OECD guideline No. 425 and published in the Journal of Drug Delivery and Therapeutics (2019).
Based on an acute toxicity study following OECD guidelines (2022), the LD50 value of the chloroform extract of M. quadrifolia was also found to be 2,000 mg/kg body weight in rodents.
In comparison with the control group, there was a significant increase in levels of ALT, AST, bilirubin, total proteins, globulin levels, urea, cholesterol, triglycerides, LDL, platelet count, MCV, MCH, WBC count, and lymphocytes, whereas ALP and MCHC levels were reduced significantly. No significant changes were observed in hemoglobin, total RBC, total bilirubin, or albumin. These biochemical changes at the maximum tested dose warrant attention if considering higher or chronic dosing, and have not been evaluated in humans.
No published human clinical trials, controlled observational studies, or formal case series evaluating the safety of Marsilea quadrifolia preparations as a supplement have been identified in peer-reviewed literature. All toxicological data are from rodent models and cannot be directly applied to human exposure.
As Marsilea quadrifolia is listed in Appendix I of the Bern Convention and in Annexes II and IV of Directive 92/43/EEC as a strictly protected species, in situ and ex situ conservation activities have been conducted in most European countries. Marsilea quadrifolia is defined as an aquatic pteridophyte that has historically been considered a paddy weed but is currently threatened in Europe due to habitat loss and unsustainable agricultural practices. This protected status in Europe may affect the legality of harvesting or commercialization of wild-sourced material in those jurisdictions.
The body of scientific literature on Marsilea quadrifolia as a dietary supplement or medicinal ingredient is entirely preclinical. Evidence for antioxidant activity derives from in vitro assays; evidence for anticonvulsant, neuroprotective, antidiabetic, and antimicrobial effects derives exclusively from animal models, primarily rats and mice.
Marsilea quadrifolia holds great potential for applications in modern medicine and nutrition, though further clinical trials are necessary to support its potential therapeutic uses. Future investigations are required to explore the neuroprotective mechanism of M. quadrifolia and its active constituents, which will provide exciting insights in the therapeutic management of neurological disorders.
The following specific gaps exist: (1) no published randomized controlled trials in humans for any endpoint; (2) no established human-equivalent dosing regimens; (3) no formal assessment of drug-herb interactions; (4) no long-term toxicological studies beyond single-dose acute rodent experiments; (5) no standardized extract formulation approved by any regulatory body.
Health conditions that Four-leaved grass may help support.
Body systems that Four-leaved grass may help support.
Go back