Abstract Nymphaea ‘Blue Sakura’ (NB) and Nymphaea ‘Chularath’ (NC) are newly developed hybrid cultivars of Nymphaea nouchali Burm. F.; belonging to Nymphaeaceae family. The aims of this study were to investigate the phytochemicals of stamen, petal, and leaf extracts from three water lily strains including NB, NC, and Nymphaea nouchali Burm. (NN), to elucidate the structure of isolated Compound 1, and to determine the biological activities including anti-oxidative, anti-α-glucosidase, anti-α-amylase, and anti-tyrosinase activities. Tannins, terpenoids, and cardiac glycosides were found in all parts of water lily extracts. Phenolic compounds, especially gallic acid, were detected and correlated with the TLC fingerprint, HPLC and NMR spectra. Compound 1 structure was revealed to be a gallic acid that can be used as a chemical marker for quality control of the extracts. NN- and NC-stamen extracts showed higher anti-oxidative activity than that of their petal and leaf extracts. While NB-petal extract showed higher anti-oxidative activity than that of its stamen and leaf extracts. NN-stamen extract (EC₅₀ of 50.48 ± 3.30 µg/ml) showed the higher anti-α-glucosidase activity than the NB- and NC-stamen extracts. Whereas the NC-stamen extract (EC₅₀ of 408.21 ± 16.00 µg/ml) showed the higher anti-α-amylase activity. For the NB, its stamen extract showed stronger anti-α-glucosidase activity and anti-α-amylase activity than the petal and leaf extracts, respectively. In addition, the NB-stamen extract (EC₅₀ of 36.29 ± 5.61 µg/ml) showed stronger anti-tyrosinase activity than the other extracts. These results indicated that the stamen, petal, and leaf extracts of water lilies in this study could be a good source of phytochemicals and had high anti-oxidative, anti-α-glucosidase, and anti-tyrosinase activities.

Keywords: Nymphaea nouchali, Nymphaea ‘Blue Sakura’, Nymphaea ‘Chularath’, Phytochemicals, Biological activities

Funding: The authors are grateful for the research funding provided the research grant from the Office of Projects under the Royal Initiative, Rajamangala University of Technology Tawan-ok, fiscal year 2022.

Citation:  Narkpiban, K., Ratanacoon, M., Aree, M., Jaisamut, S., Promsomboon, S., Nubpetchploy, S., and Jaisamut, O. 2026. Phytochemical screening and biological activities of ethanolic extracts from new tropical water lilies Nymphaea ‘Blue Sakura’ and Nymphaea ‘Chularath’. Natural and Life Sciences Communications. 25(2): e2026033.

Graphical Abstract:

INTRODUCTION

Nymphaea nouchali Burm. or water lily, is an aquatic plant belonging to Nymphaeaceae family. This water lily has a bright blue color petal with yellow color at the center. It can be found in nature as a wild type and as an ornamental hybrid strain. New hybrid water lilies, Nymphaea ‘Chularath’ and Nymphaea ‘Blue Sakura’, are hybrid strains that were developed by Assoc. Prof. Dr. N. Nopchai Chansilpa, Director of Waterlily Institute of Rajamangala Tawan-ok, Rajamangala University of Technology Tawan-ok, Chonburi, Thailand. These hybrid water lilies have a sweet smell like cherry blossom flower smell. Water lilies have been used as food and medicine. For example, N. nouchali (syn. Nymphaea stellata Willd.) or blue water lily is a prominent herb in Ayurveda and Siddha formulations for treatment of several diseases such as diabetes mellitus, hepatic diseases, fever, problems in kidney, eyes, heart and liver (Swapna et al., 2011; Kiranmai et al., 2023). This water lily was reported to have several pharmacological activities (Kiranmai et al., 2023) such as anti-diabetic (Dhanabal et al., 2007; Parimala and Shoba, 2014; Priyanka et al., 2016), anti-oxidative (Parimala and Shoba, 2013), anti-microbial (Vasu and Singaracharya, 2008), anti-inflammatory (Rajagopal et al., 2008), anti-hepatotoxic (Rao Nadendla et al., 2017), antipyretic (Sarwar et al., 2016), anti-tumor (Al-Harbi et al., 2020), anti-ulcer (Verma et al., 2012) and immunomodulatory (Sikder et al., 2013). Moreover, species within the Nymphaea genus have been widely recognized for their considerable phytochemical diversity. N. pubescens has been reported to contain substantial amounts of phenolic acids and flavonoids, reflecting its strong antioxidant potential (Thongdonphum et al., 2023). In contrast, N. lotus exhibits an even broader spectrum of secondary metabolites, encompassing alkaloids, flavonoids, terpenoids, tannins, glycosides, and steroids (Tungmunnithum et al., 2020; Puma et al., 2024). Additionally, extracts obtained from the stamens and petals of N. antares have been identified as particularly rich in flavonoids, further underscoring the genus’s capacity to accumulate bioactive compounds (Mohd Zin et al., 2021). Collectively, these findings highlight Nymphaea species as promising sources of chemically diverse constituents with potential pharmacological relevance.

Therefore, water lilies can be a good candidate for natural products containing health benefit effects. However, there was no scientific information about phytochemical composition and the biological activities of these new hybrid water lily stains. The aims of the present study are to determine the phytochemicals of stamen, petal, and leaf extracts from three water lily strains including Nymphaea ‘Blue Sakura’ (NB), Nymphaea ‘Chularath’ (NC), and Nymphaea nouchali Burm. (NN), to elucidate the chemical structure of isolated pure compound, and to investigate their biological activities including anti-oxidative, anti-α-glucosidase, anti-α-amylase, and anti-tyrosinase activities.

MATERIALS AND METHODS

Materials and isolation

The water lily strains including Nymphaea nouchali Burm. f. (NN), Nymphaea ‘Chularath’ (NC) and Nymphaea ‘Blue Sakura’ (NB) (Figure 1) were cleaned using tap water. Their stamen, petal, and leaves were separated, cut, and dried at 45°C using hot air oven. The dried pieces of each part were separately macerated with 95% ethanol at room temperature for 7 days. The macerated liquid was filtered and concentrated using vacuum rotary evaporator. The concentrated extracts were dried using freeze dryer and the yield of the extracts were calculated. 

The NC-leaf extract (25.41 g) was separated by quick column chromatography over silica gel 60H using the eluents of hexane, ethyl acetate, acetone, and methanol, respectively, to give 10 fractions (C1 to C10). Fraction C6 (241.5 mg) was recrystallized with dichloromethane to give compound 1 (180.3 mg).

Figure 1. Morphologies of stamen, petals, and leaves of (A) Nymphaea nouchali Burm. (NN), (B) Nymphaea ‘Chularath’ (NC), and (C) Nymphaea ‘Blue Sakura’ (NB).

Phytochemical screening

NN, NC, and NB extracts were screened for their phytochemicals using qualitative identification tests, Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), and Nuclear magnetic resonance (NMR).

Qualitative identification tests       

The qualitative identification of the secondary metabolites including alkaloids, flavonoids, anthraquinone, coumarin, saponin, tannin, phlobatannin, terpenoids, steroids, and cardiac glycosides in the extracts were determined following the previous study (Dangnoi, 2016).

Thin layer chromatography (TLC)

Each extract (20 mg) was dissolved with methanol (200 µl), sonicated for 20 min, and then filtrated using Whatman No. 1 filter paper. The standard chemicals (1 mg) including catechin, rutin, cinchonine, gallic acid, hydroquinone, and caffeic acid were dissolved in methanol (100 µl). The samples and standard chemicals (10 µl) were spotted on TLC plate (10 × 10 cm) as a stationary plate. The plate was put in a developing tank containing a developing solvent as a mobile phase. The mobile phase was the solvent mixture of ethyl acetate: hexane: acetic acid (60 : 3.9 : 0.7). The developed plate was visualized under 254 and 360 nm UV lamps. The plate was also sprayed with chemical reagents including anisaldehyde-H₂SO₄, 30% H₂SO₄ in ethanol, and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The plates were photographed.

High performance liquid chromatography (HPLC)

Each extract (10 mg) was dissolved with methanol (1.0 x 10⁴ µl), sonicated for 15 min, centrifuged, and then filtered using syringe filter. The standard chemicals (2 mg) including gallic acid, tannic acid, vanillic acid, vanillin, epicatechin, p-coumaric acid, and chlorogenic acid were dissolved in methanol (1.0 x 10⁴ µl) (Sripanidkulchai and Junlatat, 2014). The HPLC analysis system was Agilent Technology 1260 infinity. The chromatography assay was performed on a C18 Agilent ZORBAX column (150 mm × 4.6 mm) reversed phase matrix (5 µm). The elution was carried out using mobile phase of acetonitrile (7): 1% acetic acid (93) mixture with the flow rate of 1.1 ml/min. The diode array detector was set at 272 nm with 16 nm band width. The sample volume of injection was 20 µl.

Nuclear magnetic resonance (NMR) spectroscopy analysis

The extract samples (30 mg) were dissolved in deuterated methanol (CD₃OD) (0.5 ml) and then transferred into the NMR tubes. The sample tubes were analyzed for ¹H NMR using BRUKER 500 spectrometer (400 MHz).

To elucidate the structure of the isolated pure compound, the compound 1 was dissolved in acetone-D₆ and then determined its structure using ¹H NMR and ¹³C NMR on BRUKER 500 spectrometer (400 MHz for ¹H NMR and 100 MHz for ¹³C NMR).

Determination of anti-oxidative activities

DPPH radical scavenging assay

The extract samples were dissolved in methanol at the concentration of 5 mg/ml. The standard ascorbic acid was dissolved in methanol at the concentration of 0.02 mg/ml. The sample or ascorbic acid (0.2 ml) was mixed with 1.8 ml of 0.05 mM DPPH in methanol. The mixture was incubated in the dark at room temperature for 30 min. The solution was measured at 517 nm using UV-Vis spectrophotometer (Braca et al., 2002). The experiment was performed in triplicate. The results were calculated for % inhibition and expressed as half maximal effective concentration (EC₅₀).  

ABTS assay

The Trolox was used as a standard antioxidant in ABTS assay (Sridhar and Charless, 2019). The extract samples were dissolved in dimethyl sulfoxide (DMSO) and 2-fold serial diluted to the 8 concentrations in the range of 2.5 to 0.001 mg/ml. Each concentration of sample or Trolox (100 µl) was added with ABTS^•+ solution (100 µl). The mixture was incubated at room temperature for 6 min. The solution was measured at 734 nm using UV-Vis spectrophotometer. The experiment was performed in triplicate. The results were calculated for % inhibition and expressed as EC₅₀.

Determination of anti-α-glucosidase activity

The anti-α-glucosidase activity was determined using p-nitrophenol colorimetric method. Acarbose was used as a standard chemical. The 20 µl of 20 mg/ml extract sample or 20 mg/ml acarbose was added in 96-well plate. The 50 mM sodium phosphate buffer (pH 6.8) (100 µl), and 10 U/ml α-glucosidase in sodium phosphate buffer (pH 6.8) (20 µl) were added into the samples and acarbose wells. The mixture was incubated at room temperature for 10 min. The sample and acarbose wells were then added with 2 mM p-nitrophenyl-α-D-glucopyranoside (PNP-G) (20 µl). The mixture was incubated at room temperature for 5 min. The sample and acarbose wells were then added with 1 mM sodium bicarbonate (40 µl). The solution was measured at 405 nm using microplate reader. The experiment was performed in triplicate. The results were calculated for % inhibition and expressed as EC₅₀ (Matsui et al., 1996). 

Determination of anti-α-amylase activity

The anti-α-amylase activity was determined using p-nitrophenol colorimetric method and the acarbose was used as a standard chemical. The 20 µl of 20 mg/ml extract sample or 20 mg/ml acarbose was added in 96-well plate. The 50 mM sodium phosphate buffer (pH 6.8) (100 µl), and 10 mg/ml α-amylase in sodium phosphate buffer (pH 6.8) (20 µl) were added into the samples and acarbose wells. The mixture was incubated at room temperature for 10 min. The sample and acarbose wells were then added with 2 mM 2-chloro-4-nitro-phynyl-α-D-maltotrioside (CNPG₃) (20 µl). The mixture was incubated at room temperature for 5 min. The sample and acarbose wells were then added with 1 mM sodium bicarbonate (40 µl). The solution was measured at 405 nm using microplate reader. The experiment was performed in triplicate. The results were calculated for % inhibition and expressed as EC₅₀ (Gella et al., 1997).

Determination of anti-tyrosinase activity

The method as described by Momtaz et al. (2008) was slightly modified. Briefly, the extract was dissolved in DMSO, then diluted with 50 mM phosphate buffer (pH 6.5) to different concentrations. Ascorbic acid and tannic acid were used as a positive control. 70 μl of test sample was added to a 96-well micro plate containing 100 μl of a substrate (2 mM L-tyrosine), then 30 μl of mushroom tyrosinase (167 unit/mL) was added and mixed well. After incubation at room temperature for 30 min, the absorbance was determined at 492 nm with the microplate reader. The experiment was performed in triplicate. The results were calculated for % tyrosinase inhibition and expressed as EC₅₀.

Statistical analysis

Results in this study were expressed as mean   ±   S.D. Significant difference was analyzed using SPSS using One-Way ANOVA and multiple comparison (LSD) with P-value < 0.05.

RESULTS

Phytochemicals of water lily extracts

The extracts were screened for phytochemicals using qualitative identification methods. Tannins, terpenoids, and cardiac glycosides were detected in all parts of all water lily extracts (Table 1). Steroids were present in both the petal and leaf parts of all water lily extracts, whereas alkaloids were found only in the stamen extracts of NC and NB. All parts and all water lily extracts, except for the leaf tissues of NC and NB, were identified as containing flavonoids. Additionally, coumarin was detected in the leaf part of NN, the stamen and petal parts of NC, and all parts of NB. Saponin was detected in the leaf of all water lily extracts and was also detected in the petals of NN and NC. Phlobatannin was present exclusively in the petals of NN and NB, while anthraquinones were not detected in all parts of all water lily extracts.

Table 1. Yield and phytochemical identification screening of the extracts.

Notes: + is detectable, - is undetectable.

From the TLC fingerprints of water lily extracts (Figure 2), there were no bands that correlated with the standard compounds in this study including catechin, rutin, cinchonine, hydroquinone and caffeic acid, except gallic acid. When spraying with DPPH solution, the bands from all extracts showed anti-oxidative activity (Figure 2G and 2H). The phytochemicals of extracts were also analyzed using HPLC and the results showed that gallic acid were found in all parts and all water lily extracts, except petal extracts of NC and NB (Table 2). In addition, only stamen and petal extracts of NC were found the vanillin content.

Table 2. Phytochemical analysis of the extracts by using HPLC.

Note: - is undetectable.

The ¹H NMR spectra from all extracts are shown in Figure 3. The proton signaling between 3-5 ppm correlated with oxymathine and oxymethylene, and 6-8 ppm correlated with the aromatic proton indicating that all extracts contained similar major compounds and most compounds were in phenolic compound group. These proton results were correlated with TLC and HPLC results.

Figure 2. TLC fingerprint of the extracts. A) 254 nm, B) 360 nm, C) Anisaldehyde-sulfuric acid, D) Anisaldehyde-sulfuric acid under 360 nm, E) 30% Sulfuric acid, F) 30% Sulfuric acid under 360 nm, G) DPPH, and H) DPPH under 360 nm. 1) Catechin, 2) Rutin, 3) Cinchonine, 4) NC-stamen extract, 5) NC-petal extract, 6) NC-leaf extract, 7) NB-stamen extract, 8) NB-petal extract, 9) NB-leaf extract, 10) NN-stamen extract, 11) NN-petal extract, 12) NN-leaf extract, 13) Gallic acid, 14) Hydroquinone, and 15) Caffeic acid.

Figure 3. ¹H NMR spectra (400 MHz) of the extracts in CD₃OD. A) NN-stamen extract, B) NC-stamen extract, C) NB-stamen extract, D) NN-petal extract, E) NC-petal extract, F) NB-petal extract, G) NN-leaf extract, H) NC-leaf extract, and I) NB-leaf extract.

Compound 1 was isolated from NC extract and determined its structure. In Figure 4, the ¹³C NMR spectral data of compound 1 exhibited 7 carbons including a carbonyl at δ 167.5 (C-7), oxyaromatic carbons at δ 145.0 (2C-5, 3) and 137.7 (C-4), a quternary aromatic carbon at δ 121.2 (C-1), and the aromatic carbon at δ 109.2 (2C-2, 6). The ¹H NMR spectrum of compound 1 showed the aromatic singlet signals at δ 7.02 (2H). This proton also showed HMBC correlations to the carbons at δ 109.2 (2C-2, 6), 137.7 (C-4), 145.0 (2C-5, 3) and 167.5 (COOH-7). These data suggested a carbonyl group at C-1 and an OH group at C-3, C-4 and C-5. In addition, the ¹H and ¹³C NMR spectroscopic data of compound 1 (¹H NMR 400 MHz and ¹³C NMR 100 MHz in acetone-D6) were compared with a gallic acid (¹H NMR 500 MHz and ¹³C NMR 125 MHz in CD₃OD) (López-Martínez et al., 2015) and the results revealed that the compound 1 was gallic acid.

Anti-oxidative activity of water lily extracts

All extracts from water lilies in this study showed high anti-oxidative activity as shown in Table 3. In NN extracts, the NN-stamen extract showed significantly higher anti-oxidative activity than that of NN-petal and NN-leaf extracts. Similarly, NC-stamen extract also showed significantly higher anti-oxidative activity than that of NC-petal and NC-leaf extracts. For NB, the NB-petal extract showed significantly higher anti-oxidative activity than that of NB-stamen and NB-leaf extracts. When compared between strains, the stamen extract from NN showed higher anti-oxidative activity than that of all parts of all strains. The high anti-oxidative activity of stamen extracts in this study might be the effect of flavonoid containing stamen.

Figure 4. NMR spectra of compound 1 in acetone-D6. A) ¹³C NMR (100 Hz), B) ¹H NMR (400 Hz), and C) HMBC correlation (δ in ppm, multiplicities).

Table 3. Anti-oxidative activities of the extracts.

Note: ND is not determined. a, b and c represent significant differences between stamen, petal and leaf extracts in each strain. A, B, C and D represent significant differences between each part extract from each strain and positive controls. P-value is ≤ 0.05.

Biological activities of water lily extracts

For anti-α-glucosidase activity of extracts, the NN-stamen extract showed stronger activity than its petal and leaf parts, and stronger than all parts from the NC and NB extracts (Table 4). Interestingly, the stamen extracts from each strain showed higher anti-α-glucosidase activity than that of their petal and leaf extracts. For anti-α-amylase activity, the NC-stamen extract showed higher activity than that of NC-petal and NC-leaf extracts. In addition, the NC-stamen extract also showed higher activity than all extracts from NN and NB. Similarly with anti-α-glucosidase activity, the stamen extracts from each strain showed higher anti-α-amylase activity than that of their petal and leaf extracts.  

Table 4. Anti-α-glucosidase, anti-α-amylase, and anti-tyrosinase activities of the extracts.

Note: ND is not determined. a, b and c represent significant differences between stamen, petal and leaf extracts in each strain. A, B, C, D and E represent significant differences between each part extracted from each strain and positive controls. P-value is ≤ 0.05.

For anti-tyrosinase activity, the NB-stamen extract showed stronger anti-tyrosinase activity than NB-petal and NB-leaf extracts and the other extracts from NN and NC (Table 4). Interestingly, these results indicated that the stamen extracts from each strain had higher anti-oxidative, anti-α-glucosidase, anti-α-amylase and anti-tyrosinase activities than their petal and leaf extracts.

DISCUSSION

Hybridization can restructure the regulation of secondary-metabolite biosynthetic pathways, causing hybrids to accumulate phenolics, flavonoids, and other metabolites at levels different from their parental species. These changes may arise from altered gene expression and enzyme activity, together with heterosis that enhances overall metabolic capacity. Thus, the distinct phytochemical patterns observed in the hybrid Nymphaea cultivars in this study are consistent with hybridization-driven chemical diversification (Caseys et al., 2015).

Gallic acid was found in leaf parts of all water lily and NC- and NB- stamen extracts which a major compound that can be used as a chemical marker for quality control of water lily extracts in this study. This result correlated with the ethanolic extracts from N. pubescens leaves that contained gallic acid as a major compound (Thongdonphum et al., 2023). 

Nymphaea species have been used as a food and as a traditional medicine for diabetic treatment (Prodhan and Mridu, 2023). The ethanolic extracts from N. pubescens (pink water lily) leaves from Thanyaburi, Thailand, contained high phenolic acid and flavonoids (Thongdonphum et al., 2023). Alkaloids, flavonoid, terpenoid, tannins, glycoside and steroid, except sterol, were found in an aqueous extract of N. lotus leaves from Nigeria (Tungmunnithum et al., 2020; Puma et al., 2024). Leaf extract of N. lotus from Zaria was reported to contain tannins (4.16%), alkaloid (2.42%), glycoside (1.80%), flavonoid (0.68%) and saponins (0.47%) (Yusuf et al., 2023), findings that are consistent with the present study, in which tannins, terpenoids, and cardiac glycosides were detected in all parts of all water lily extracts. Additionally, flavonoids were observed in nearly all extracts, with the exception of the leaf extracts from the NC and NB. In addition, the phytochemical study of water lily petal extracts including purple, red, blue and yellow petal colors from Beijing, China, was reported that 34 flavonoids were found among 35 tropical cultivars and among them several anthocyanins and flavonols were identified, whereas no anthocyanins were detected within white and yellow petal color (Zhu et al., 2012). It is possible that flavonoids from water lily petals in this study might contribute to petal color formation. Furthermore, phytochemical analysis of the extracts using HPLC revealed that gallic acid was detected in nearly all samples examined in this study, with concentrations ranging from 2.68 to 15.17 µg/mL. This finding is in agreement with previous reports on N. pubescens, in which the gallic acid was found the most dominant (0.600–3.21% w/w), sinapic acid (0.37–0.83% w/w), catechin (0.02–1.08% w/w) and rutin (0.002–0.03% w/w) (Thongdonphum et al., 2023). The stamen and petal extracts of water lily (Nymphaeaceae antares) from Kuala Terengganu, Malaysia was reported to contain several flavonoids such as quercetin, kaempferol, rutin, gallic acid, catechin, epigallocatechin, p-coumaric acid and myricetin (Mohd Zin et al., 2021). The petal extract contained catechin (6.5 mg/100 ml), rutin (4.0 mg/100 ml), gallic acid (1.5 mg/100 ml) and epigallocatechin gallate (1.5 mg/100 ml) (Mohd Zin et al., 2021).

Seven species of water lilies were reported to have anti-diabetic properties including N. stellata, N. pubescens, N. lotus, N. alba, N. nouchali, N. rubra, and N. odorata. The antidiabetic properties of these water lilies were mediated through antihyperglycemic, antihyperlipidemic, pancreatic β cell-regenerating, insulin secretion and sensitivity promoting, glucose uptake and metabolizing protein-expressing, intestinal glucose metabolizing enzyme inhibiting, hepatoprotective, cardiovascular protective, nephroprotective, antioxidant, and anti-inflammatory activities (Prodhan and Mridu, 2023). Methanolic extract of N. stellata leaves (200 mg/kg) as well as metformin showed significant restoration of glucose and insulin levels when compared to the diabetic control group in STZ-NAD-induced rat model (Raja et al., 2017). This extract was postulated to exhibit anti-diabetic activity through its synergistic multi-effects such as anti-hyperglycemic, anti-dyslipidemic and anti-oxidative activities of β-carotene and/or gallic acid that were present in extract (Raja et al., 2017). Moreover, the flower extract of N. stellata showed glucose lowering effect on alloxan-induced diabetic rats (Rajagopal and Sasikala, 2008). This anti-hyperglycaemic effect might be linked to many mechanisms including the stimulation of β-cells, insulin releasing and activation of insulin receptors resulting in an increased level of insulin in diabetic rats (Rajagopal and Sasikala, 2008). The N. pubescens tuber extract showed preventive effect of diabetes that could be mainly attributed to its anti-oxidative properties and enhancement of antioxidant defense systems in pancreatic tissue (Shajeela et al., 2012). The flavonoids and phenolic compounds, the main active ingredients, in the extract might play anti-diabetic effect through their potent anti-oxidative activities (Shajeela et al., 2012; Tungmunnithum et al., 2021; Nutho and Tungmunnithum, 2024). The N. nouchali seed extract showed blood sugar lowering effect in streptozotocin-induced diabetic rats through extrapancreatic mechanisms (Parimala et al., 2015) and promoted adipocyte differentiation and glucose consumption by inducing the activation of PPARγ that plays an important role in glucose and lipid homeostasis and subsequently induced insulin target tissues (Parimala and Shoba, 2014). This extract contained high phenolic and flavonoid contents and showed strong anti-oxidative activity (Parimala and Shoba, 2013) that might be attributed to its anti-diabetic properties. Phenolics and flavonoids are reported to be a potent anti-diabetic constituent in improving the pathogenesis of diabetes by regulation of glucose metabolism, hepatic enzymes activities, and a lipid profile (AL-Ishaq et al., 2019; Deka et al., 2022; Khanaree et al., 2025; Musa et al., 2025). The extracts in this present study showed anti-α-glucosidase and anti-α-amylase activities that might be associated with their phenolic and flavonoid contents. 

The N. lotus stamens and perianth extracts were reported to contain two most prominent flavonoids, quercetin-3-O-rhamnoside (Que-3-Rha) and kaempferol-3-O-galactoside (Kae-3-Gal), that exhibited moderate to good inhibitory activity toward skin-aging enzymes including collagenase, elastase, and tyrosinase (Nutho and Tungmunnithum, 2024). In addition, N. nouchali flower extract was studied its effect on melanogenesis process (Alam et al., 2018). The extract significantly suppressed cellular tyrosinase activity and melanin synthesis in vitro using melan-a cells and
in vivo using HRM2 hairless mice by several mechanisms such as reducing melanogenesis, inhibiting mushroom tyrosinase activity, interfering with the transcription factors and common signaling pathways implicated in melanin synthesis, as well as downregulating MITF expression via suppression of cAMP (Alam et al., 2018). The phenolic and flavonoid contents in water lily extracts in this present study might play a role in their anti-tyrosinase activity through the binding of their hydroxy groups of flavonoids to form hydrogen bond with the amino acid residues of tyrosinase (Fan et al., 2019). This corresponds with our findings, wherein all extracts exhibited strong tyrosinase-inhibitory effects, suggesting their promising potential for further development as cosmetic or cosmeceutical agents.

CONCLUSION

In conclusion, the stamen, petal and leaf extracts from the new hybrid water lilies, NB and NC as well as from the Nymphaea nouchali Burm. (NN), exhibited varying degree of anti-oxidative, anti-α-glucosidase, anti-α-amylase, and anti-tyrosinase activities. Among them, the stamen extracts demonstrated stronger effects than the petal and leaf extracts, which may be attributed to their phenolic and flavonoid contents. Moreover, gallic acid could be used as a marker for quality control. Therefore, extracts from both hybrid and Nymphaea nouchali Burm. in this study can be a good candidate for the development of cosmeceutical and/or functional health products. The mechanisms of action of their anti-diabetic activities in vitro and in vivo will be further studied.

ACKNOWLEDGEMENTS

This work was supported by the research grant from the Office of Projects under the Royal Initiative, Rajamangala University of Technology Tawan-ok, fiscal year 2022. We would like to thank Faculty of Science and Technology, Institute of Research and Development, Rajamangala University of Technology Tawan-ok, Chonburi Province, Thailand, for their participation and facility supports. The authors wish to thank Assoc. Prof. Dr. N. Nopchai Chansilpa, Director of Waterlily Institute of Rajamangala Tawan-ok, Rajamangala University of Technology Tawan-ok, Chonburi, Thailand, for providing waterlily samples and some waterlily pictures used in this study.  

AUTHOR CONTRIBUTIONS

Koranat Narkpiban: Conceptualization (Lead), Methodology (Lead), Funding Acquisition (Lead); Manoch Ratanacoon: Conceptualization (Lead), Writing – Review & Editing (Lead); Manut Aree: Resources (Lead); Sunan Jaisamut: Investigation (Lead), Writing – Original Draft (Lead), Methodology (Equal); Sutunya Promsomboon: Supervision (Lead); Sinsup Nubpetchploy: Formal analysis (Lead), Software (Lead); Orapun Jaisamut: Conceptualization (Lead), Methodology (Lead), Data Curation (Lead), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Visualization (Equal). All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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