Introduction

Recently, great interest has been paid to plant-based phytochemicals including phenolic acids, flavonoids, steroids, terpenoids, and others, owing to their myriad health benefits1. Onagraceae also known as evening primrose or willowherb family, is a family of flowering plants widely distributed in every continent, from tropical to boreal regions. Onagraceae comprises about 17 genera and 650 species of trees, shrubs, and herbs distributed into two subfamilies and seven tribes2. Genus Ludwigia is a member of the Ludwigioideae subfamily distributed in South and North America, and comprises about 82 species of aquatics plants3.

Ludwigia species are reported for their diverse biological properties including antidiabetic, cytotoxicity, antioxidant, hepatoprotective antimicrobial, and anti-inflammatory activities. For instance, L. hyssopifolia (G.Don) Exell. aerial parts methanolic extract showed potent anti-inflammatory activity4 L. octovalvis (Jacq.) P.H.Raven aqueous ethanolic extract showed antidiabetic effect5and L. peploides (Kunth) P.H.Raven leaves methanolic extract revealed cytotoxic, analgesic, antimicrobial, antidiarrheal and hypolipidemic properties2. In the Egyptian flora, Ludwigia genus is represented by two species: L. erecta (L.) Hara. and L. stolonifera (Guill. & Perr.) P.H.Raven6.

Ludwigia adscendens subsp. diffusa Forssk. Also known as Ludwigia stolonifera, it emerged as one of the most important aquatic plants widely distributed in canals and drains crossing the cultivated lands in the Nile Delta. L. adscendens is well known for its economic importance, being used in water bioremediation to help in improving drinking water quality3. Owing to its ability to remove toxic contaminants including heavy metals such Pb, Cd, and Cr from aquatic ecosystems, L. stolonifera roots and leaves are used as water biofilters7. Moreover, L. stolonifera is rich in bioactive secondary metabolites which are imparted for their biological activities including antioxidant, antidiabetic, hepatoprotective, and cytotoxic activities3,8,9.

Nowadays, different metabolomics tools are widely applied to profile plant-based primary and secondary metabolites10. LC-MS is well-suited metabolomics approach suited for the identification of non-volatile secondary metabolites in herbal materials11. Several studies have demonstrated that variety of antioxidant phytoconstituents also display a potent anti-inflammatory effect12,13. L. adscendens ethyl acetate extract possesses antioxidant activity9 so this study focuses on investigation of its anti-inflammatory effect.

The main goal of the current study is to evaluate the phytochemical profile in L. adscendens subsp. diffusa aerial parts using UPLC-MS/MS in negative and positive modes. Further investigation of the anti-inflammatory activity L. adscendens extract was employed using nitric oxide inhibition assay.

Results and discussion

Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS/MS) analysis was employed for L. adscendens aerial parts methanol extract in both positive and negative modes (Fig. 1). Compounds were eluted within 25 min from the most polar to the least polarity ones according to the sequence of elusion. The identification was depended on comparison of the high-resolution mass spectra information with phytochemical dictionary of natural product databases and MS/MS and with that reported in the literature. A total of 168 metabolites were identified by UPLC-MS analysis in negative and positive modes (Table 1; Fig. 2).

Fig. 1
figure 1

UPLC-MS base peak chromatograms of L. adscendens aerial parts (A) BPC in negative mode (B) BPC in positive mode.

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Table 1 Chemical metabolites tentatively identified in L. adscendens aerial parts by UPLC-MS/MS at negative and positive modes.
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Fig. 2
figure 2

Representative bar-chart and pie chart of identified metabolites classes in L. adscendens aerial parts using UPLC-MS negative and positive modes.

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Chemical metabolites identified in Ludwigia stolonifera by UPLC-MS/MS negative and positive modes

UPLC-MS/MS analysis of L. adscendens aerial parts in negative and positive (Fig. 1A and B) revealed the annotation of 168 metabolites (Table 1; Fig. 2) belonging to several phytochemical classes including phenolics (57), flavonoids (26), terpenoids (25), sterols (22), fatty acids (11), coumarins (8) organic acids (5), sugar derivatives (5), lactones (4), acids (3), and glycoside (2). Flavonoids and phenolics were identified as the most abundant metabolite classes which enhance the biological properties of L. adscendens including antioxidant and anti-inflammatory effects.

Phenolic compounds

Phenolic compounds were identified as the most abundant class represented by 57 peaks. Phenolic compounds are ubiquitously distributed phytochemicals found in most plants and possess numerous bioactive properties including antioxidant, antimicrobial, and anti-inflammatory14. Peaks 60, 61, and 75 were assigned to gallic acid (m/z 169.0140, C7H6O5−)15 and its derivatives, gallic acid hexoside (C13H16O10−)16 and digallic acid (C14H10O9−), respectively. Gallic acid and its derivatives are potential biological importance including antioxidant, anti-inflammatory, and antimicrobial properties17. Methyl gallate (peak 67) and pyrogallol gallate (peak 66) appeared at m/z [M-H]− 183.0292 (C8H8O5−) and 293.0289 (C13H10O8−)18respectively. Gallate deratives were previously identified in L. adscendens aerial parts3 and has been reported to exhibit hepatoprotective and anticancer effects19. Peaks 73, 74, and 18 were annotated for ellagic acid (m/z [M-H]− 300.9983 C14H6O8)20 and its derivatives, such as gallagic acid and ellagic acid dihydrates, respectively. Ellagic acid and its derivatives are ellagitannins with potent antioxidant and antitumor properties21. Ferulic acid (peak 59) was identified (m/z [M-H]− 193.0498 C10H10O4−) is a hydroxycinnamic acid with antioxidant and anti-inflammatory properties22. Peak 70 were assigned to dihydroxycaffeic acid (m/z [M-H]− 211.0239 C9H8O6−) which possesses strong neuroprotective and anti-inflammatory properties23. Gingerol (Peak 59) was detected in L. adscendens aerial parts and is well known for its anti-inflammatory and antioxidant activities24. Simple phenolics were identified including pyrogallol (peak 86) [M + H]+ at m/z 127.0388 (C6H6O3+) and 2-hydroxyethyl gallate (peak 87) [M + H]+ at m/z 215.0525 (C9H10O6+) with antioxidant and anti-inflammatory properties25. Peak 107 was identified as feruloyl lactate and peak 98 was identified as Sinapyl aldehyde [M + H]+ at m/z 209.0782 (C11H12O4+)26. Sinapyl aldehyde is a precursor in lignin biosynthesis and contributes to plant cell wall integrity and well known for its antioxidant and anti-inflammatory properties26. Peak 102 was assigned to zingerone, which is a phenolic compound with anti-inflammatory and antioxidant properties27.

Flavonoids

Flavonoids, a group of natural substances with variable phenolic structures with potential health benefits owing to their anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties28. Flavonoids represented by 26 peaks were identified in L. adscendens aerial parts. Both O- & C-flavonoid glycosides were identified by different fragmentation pattern that distinguished between the two types of glycosidic linkages. Flavonols and flavone O-glycosides were identified according to neutral loss of sugar moieties; [M-H]− [179, 161, 149 & 131 amu] which assigned for ions loss of (hexose, deoxyhexose, and pentose units), respectively. Peaks 33 and 26 were annotated as quercetin (m/z [M-H]− 301.0342 C15H10O7−) and quercetin 3-O-hexoside (m/z 463.0862 C21H20O12−), respectively. Peaks 29, 31 and 46 were assigned to kaempferol-3-O-hexoside29 kaempferol-3-O-arabinoside30 and apigenin-8-C-hexoside31 respectively. Moreover, myricetin (peak 45) (m/z 317.0293 C15H10O8−) and myricetin-3-O-hexoside (peak 27) (m/z 479.0810 C21H20O13−) were identified in L. adscendens aerial parts. Peak 30 and peak 22 were annotated as quercetin 3-(2’’-galloyl-pentoside) (m/z [M-H]− 585.055 C27H22O15) and quercetin 3-O-(6’’-galloyl-hexoside) (m/z [M-H]− 615.0963 C28H42O16−) which is related to previously isolated compounds from in L. adscendens aerial parts3. Moreover, peaks 36, 38, and 40 were identified as quercetin derivatives26 and including quercetin 3-O-glucuronide [M + H]+ at m/z 479.0822 (C21H18O13+), quercetin 3-O-pentoside [M + H]+ at m/z 435.0923 (C20H18O11+), and quercetin 3-O-deoxyhexoside [M + H]+ at m/z 449.1078 (C21H16O13+)32 respectively. Quercetin derivatives are reported for their antioxidant and anti-inflammatory properties33. Quercetin has been reported to modulate signaling pathways involved in cancer progression32. Flavonoids can contribute to the biological importance of L. adscendens aerial parts owing to their myriad pharmacological properties.

Triterpenes and sterols

Triterpenoids represented by 25 peaks were identified in L. adscendens aerial parts. Triterpenoids play a pivotal role in human health owing to their pharmacological activities including antidiabetic properties, neuropharmacological, and anti-inflammatory effects34. Peaks 143, 148, and 160 were assigned to protobassic acid (m/z [M-H]− 503.3355 C30H48O6−), asiatic acid (m/z [M-H]− 487.3407 C30H48O5−), and betulinic acid (m/z [M-H]− 455.3513 C30H48O3−)35. Such triterpenoids were reported for their significant biological activities such as anti-inflammatory, anticancer, and hepatoprotective effects36. Peak 155 was annotated as maslinic acid (m/z [M-H]− 471.3461 C30H48O4−) which a pentacyclic triterpenoid with antioxidant and anti-inflammatory properties37. Hederagenin (peak 154) was previously identified as the aglycone of triterpenoid saponins isolated from L. adscendens aerial parts3. It has been reported for its potential pharmacological activities including antitumor, anti-inflammatory, antidepressant, antineurodegenerative, antihyperlipidemic, antidiabetic, and antiviral activities38. Moreover, triterpenoids were detected in positive mode among which peak 169, 170, and 171 were identified as ganoderic acid, asiatic acid triacetate and dihydrosarcostin, respectively. These triterpenoid compounds are found in medicinal plants and contribute to their antioxidant and anti-inflammatory properties36.

Likewise, sterols were identified by 23 peaks represented mainly by stoloniferone S (Peak 150) and viticosterone E (Peak 135) with potential bioactive properties39. Plant sterols play a pivotal role in human health through several biological properties including cardioprotective, neuroprotective, and anti-aging40. Moreover, peak 116 and 131 were assigned to momordicoside E (C37H60O12) and agosterol F (C31H50O7) which are steroidal saponin with anti-inflammatory properties41. Moreover, sterols were detected in positive mode represented by several peaks among which 117, 126, 128, and 129 were identified as sarcostin [M + H]+ at m/z 383.2404 (C21H34O6+), β-sitosterol-3-O-arabinobenzoate [M + H]+ at m/z 651.4591 (C41H62O6+), Stigmasterylferulate [M + H]+ at m/z 589.4283 (C39H56O4+), and stigmastadiene [M + H]+ at m/z 397.3827 (C29H48+).

Fatty acids

Fatty acids were detected mainly by 11 peaks, among which linolenic acid (peak 12)42 and ricinoleic acid (peak 13) were identified in negative mode. Linolenic acid [M-H]− at m/z 277.2163 (C18H30O2−) is an essential omega-3 fatty acid with anti-inflammatory and cardioprotective effects43. Moreover, ricinoleic acid [M-H]− at m/z 297.2422 (C18H34O3−) is a hydroxy fatty acid with antimicrobial and anti-inflammatory properties44. Peaks 15 and 20 were assigned to colneleic acid [M + H]+ at m/z 295.2268 (C18H30O3+)45 and stearidonic acid [M + H]+ at m/z 277.2164 (C18H28O2+)10,11.

Coumarins

Coumarins represented by 7 peaks were detected in L. adscendens aerial parts and detected only in positive mode. Coumarins are considered as biologically active metabolites with potential pharmacological properties including anticoagulant, anti-inflammatory, and anticancer46. Peaks 10, 4, 7, and 9 were identified as ethynyl coumarin [M + H]+ at m/z 171.0451 (C11H6O2+), coumarin-3-carboxylic acid, penicimarin F, and 7-methoxycoumarin [M + H]+ at m/z 419.0977 (C20H18O10+).

Organic acids

Aliphatic organic acids are the important bioactive compounds found in medicinal plants and play a key role in flavor, maintain nutritional value as well as their characteristic taste47,48. Among organic acids, malic and citric acids are mainly produced in the tricarboxylic acid cycle and accumulated in various plant species47. Peaks 55 and 56 were assigned to citric acid (m/z [M-H]− 191.0192 C6H8O7) and malic acid (m/z [M-H]− 133.0139 C4H6O5)42. These acids also contribute to the sour taste of plant tissues and play roles in pH regulation48. Peak 58 was assigned to dehydroascorbic acid m/z 173 C6H6O6−49 indicating the presence of ascorbic acid metabolism in L. adscendens aerial parts which imparts to its potent antioxidant potential.

Anti-inflammatory activity

In daily routine, the human body is largely exposed to inflammation by environmental pollutants, infections (bacteria, viruses, and fungi) and other physical and chemical agents50. Inflammation is a protective strategy that stimulate immune response to protect from tissue injury and other noxious conditions and promote the healing of damaged tissue50. Recently, several diseases were linked to the inflammatory response including atherosclerosis, Alzheimer’s disease, cancer, and cardiovascular diseases13. Elevation of NO level is used as a marker for inflammatory response as manifested by elevated exhaled nitric oxide (NO) in asthmatics which indicate airways inflammation13,51. NO is an important chemical mediator produced by endothelial cells, macrophages, and neurons and play a key role in the immune system’s host defense mechanism and regulate blood vessel tone in vascular systems52. NO is considered as a pro-inflammatory mediator that induces inflammation due to over production in abnormal situations13,53. Hence, inhibition of excess nitric oxide is one of the possible mechanisms of anti-inflammatory agents. Recently, plant-based phytochemicals identified in medicinal plants’ crude extracts and/or pure compounds, are widely used as potential sources of anti-inflammatory agents54. Several phytoconstituents widely distributed in plants and possess anti-inflammatory activity including phenolics, flavonoids, terpenoids, steroids, and saponins54,55. The ant-inflammatory activity of L. adscendens aerial parts methanol extract and ethyl acetate fraction was investigated via NO inhibitory assay (Table S1). Results revealed that methanol extract and ethyl acetate fraction showed potent anti-inflammatory with calculated IC50 of 26.4 and 23.9 µg/ml, respectively, compared to resveratrol as standard anti-inflammatory with IC50 value of 14.2 µg/ml (Fig. 3). Such anti-inflammatory activity of L. adscendens aerial parts is manifested by its richness with bioactive phytochemicals including phenolics, flavonoids, triterpenoids, and steroids. Several metabolites were identified and reported for their antioxidant and anti-inflammatory activities. Gallic acid and its derivatives are potential biological importance including antioxidant, anti-inflammatory, and antimicrobial properties17. Gallic acid can reduce inflammation via inhibition of proinflammatory cytokines56. Moreover, quercetin57 ellagic acid58 and betulinic acid59 contributes to the significant antioxidant, and anti-inflammatory activity33 of L. adscendens aerial parts. Such results are compatible with other previous studies on several species belonging to the family Onagraceae, which reported to exert a potent anti-inflammatory activity60. In another study, Jussiaea repens L. aerial parts ethylacetate extract revealed in vitro anti-inflammatory activity61. Epilobium angustifolium and Epilobium montanum aerial parts dichloromethane extracts were tested for the anti-inflammatory activity revealing a potent effect62.

Fig. 3
figure 3

Calculated IC50 (µg/ml) of L. adscendens aerial parts by NO inhibitory activity assay.

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Conclusion

Phytochemical profiling of L. adscendens aerial parts via UPLC-MS/MS analysis in negative and positive modes was introduced herein. A total of 168 metabolites were identified belonging to several phytochemical classes including phenolics, flavonoids, terpenoids, sterols, fatty acids, coumarins, organic acids, sugar derivatives, lactones, acids, and glycoside. Several metabolites were identified in L. adscendens aerial parts with significant biological importance including gallic acid and gallate derivatives, quercetin derivatives, ellagic acid, gingerol and betulinic acid which can contribute to the antioxidant and anti-inflammatory activities. Investigation of the anti-inflammatory activity of L. adscendens methanol and ethyl acetate extract via nitric acid inhibition assay revealed potent activity with IC50 of 26.4 and 23.9 µg/ml, respectively, compared to resveratrol with IC50 value of 14.2 µg/ml. These findings can highlight the importance of L. adscendens aerial parts as a potential source of bioactive metabolites. Further isolation and biological investigation of the bioactive metabolites using different chromatographic techniques are recommended in future work.

Materials and methods

Plant material

The aerial parts of L. adscendens subsp. diffusa (Forssk.) P.H. Raven were collected from the Nile River at El Qanatir Al-Khayriyah, El Qulyoubia governorate (30.193583°N 31.132064°E), Egypt in September 2024. The botanical identification of the plant was confirmed by Prof. Dr. Rim Hamdy, Professor of plant taxonomy, Botany Department, Faculty of Science, Cairo University, Egypt. A voucher specimen with the number Lus2/2024, has been deposited at the Pharmacy Department of the Faculty of Pharmacy, Egyptian Russian University.

Plant extraction

The air-dried aerial parts of L. adscendens (250 g) were macerated in methanol at room temperature, stirring occasionally, and the operation was repeated three times until being exhausted. The dried methanol extract was obtained by concentrating under reduced pressure using a Rotary evaporator at 50 oC to yield 30 g dry methanol extract. About 10 g was kept in closed contained and kept in the refrigerator for UPLC-MS analysis. About 20 g of the obtained methanol extract was suspended in distilled water and sequentially partitioned with different immiscible solvents (petroleum ether, chloroform and ethyl acetate solvents) starting with petroleum ether followed by fractionation with chloroform, and finally ethyl acetate. Ethyl acetate fraction (7 g) was obtained and used for further biological investigation.

Chemicals

HR-UPLC/MS/MS: Milli-Q water and solvents; formic acid and acetonitrile of LC-MS grade, J. T. Baker (The Netherlands). Nitric oxide and all chemicals used in biological investigation were supplied by Sigma Aldrich Chemie GmbH, St. Louis, MO.

HR-UPLC-MS/MS analysis

Dried methanol extract (10 mg) was extracted by adding 2 mL of 100% MeOH, containing 10 µgmL−1 umbelliferone as an internal standard and sonicated for 20 min with frequent shaking, then centrifuged at 12 000 × g for 10 min to remove debris. A sample of 3 µl of 100% methanol extract was subjected to chromatographic separation using an I-Class UPLC system (Waters Corporation, Milford, USA). The filtered extract through a 0.22-µm filter was subjected to solid-phase extraction using a C18 cartridge (Sep Pack, Waters, Milford, MA, USA) as previously described11. UPLC-ESI-qTOF-MS analysis was carried out using an ACQUITY UPLC system (Waters, Milford, MA, USA). Reversed-phase sorbent column: HSS T3 (C18, 100 × 1.0 mm), particle size: 1.8 μm: (Waters). The annotation of metabolites was based on full mass spectra, molecular formula with an (error < 5 ppm), and by comparing fragmentation pattern with available literature and phytochemical dictionary of natural products database63. Chromatographic separation was carried out at 40 °C, using a Waters HSS T3 column (1.0 mm × 100 mm, 1.8 μm) with mobile phases A (0.1% formic acid in water) and B (acetonitrile). The flow rate was set at 0.15 mL/min. The gradient profile was as follows: 0–1 min, 5–5% B; 1–11 min, 5–100% B; 11–19 min, 100% B; 19–20 min, 100%−5% B; 20–25 min, 5% B. Mass spectrometric detection was carried out on Waters Synapt XS mass spectrometer (Waters Corporation, Milford, USA) equipped with an ESI source. The full scan data were acquired from 50 to 1200 Da, using a capillary voltage of 4.0 kV for positive ion mode and 3.0 kV for negative ion mode, sampling cone voltage of 30 V for positive ion mode and 35 V for negative ion mode, extraction cone voltage of 4.0 V, source temperature of 140 °C, cone gas flow of 50 L/h, desolvation gas (N2) flow of 1000 L/h and desolvation gas temperature of 450 °C. The collision voltage was set as 5.0 eV for low-energy scan and 25–50 eV for high-energy scan. The collision energy settings (5.0 eV for low-energy scan and 25–50 eV for high-energy scan)11 were selected based on instrument manufacturer recommendations and prior optimization studies to ensure effective fragmentation of a wide range of metabolites with diverse structural properties. These values provide a balance between low-energy precursor ion detection and adequate high-energy fragmentation required for structural elucidation in data-independent acquisition (DIA) mode.

Nitric oxide (NO) Inhibition activity

NO inhibition activity of the tested sample was determined by using a sodium nitroprusside (SNP)64,65. NO radical generated from SNP in aqueous solution at physiological pH reacts with oxygen to produce nitrite ions that were measured by the Greiss reagent. The reaction mixture (2 mL) containing various concentrations of the tested samples and SNP (10 mM) in phosphate buffered saline (PBS; pH 7.4) was incubated at 25 ºC for 150 min. At the end of the incubation period, 1 mL of reaction mixture samples was diluted with 1 mL Greiss reagent (1% sulphanilamide (w/v) in 5% phosphoric acid (v/v) and 0.1% naphthyl ethylene diamine dihydrochloride). The mixture was incubated at 25 °C for a further 30 min. The absorbance of these solutions was measured at 546 nm against the corresponding blank solution (without sodium nitroprusside). Resveratrol was used as a reference standard. All the tests were performed in triplicate. The percentage inhibition activity was calculated using the formula:

$$\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\text{\%}\:=\left[\frac{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\right]\times 100$$

Where, A control is the absorbance of the control reaction at 546 nm and Atest represents the absorbance of a test reaction at the same wavelength. Tested material concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against concentration.

Statistical analysis

The results of biological investigation were analyzed in triplicate and displayed as average ± standard deviation of the mean (SD) (Table S1).