Ultrasound-assisted extraction of hypolipidemic actives from lotus (Nelumbo nucifera Gaertn.) seedpod: Process optimization, phytochemical characterization, and hypolipidemic activity

Graphical abstract

Abstract

Lotus seedpod is a by-product of Lotus seed planting industry with abundant polyphenols. This study optimized the ultrasonic-assisted extraction (UAE) parameters of hypolipidemic compounds from lotus seedpod by response surface methodology guided-by lipase inhibition activity (LPIA). The major active compounds were enriched and identified, the in vitro and in vivo lipid lowering effect was explored by cell and mice models. The optimized conditions were 69% ethanol aqueous solution, ultrasonic power of 300 W and time of 62 min, under which the total polyphenol (TPC) and total flavonoid content (TFC) reached 125.41 mg GAE/g DM and 361.82 ± 2.76 mg QUE/g DM, respectively, with pancreatic lipase inhibitory activity (PLIA) of 8.38 ± 0.20 mg DM/mL. Ethyl acetate was the suitable solvent for enriching the hypolipidemic fraction in lotus seedpod polyphenol extract (LSPE). LSPE exhibited promising lipase inhibition (IC₅₀ value = 171.5 μg/mL) and significantly decreased the lipid accumulation in 3 T3-L1 cells. Flavonoids, especially for quercetin and kaempferol derivatives, were the major compounds. In vivo assays indicated that LSPE treatment reduced weight gain and serum lipid level, and repaired liver damage by alleviating oxidative stress and inflammation in obese mice. These findings highlight the potential of lotus seedpod as a natural source of hypolipidemic actives, while also demonstrating the high efficiency of UAE in extracting plant-based hypolipidemic compounds.

1. Introduction

Obesity is a chronic metabolic disease characterized by excessive accumulation of adipose tissue, and resulting to weight gain and uneven distribution of body fat[1]. As reported by World Obesity Federation in 2025, from 2010 to 2030, global adult obesity was increased from 524 million to 1.13 billion[2]. Accumulating evidence confirmed that obesity is highly linked with chronic diseases, included metabolic syndrome, cardiovascular disease, insulin resistance, and type 2 diabetes, and gave significant psychological stress on patients and great challenge to global public health systems[3], [4]. Currently, the common interventions for treating obesity includes drug and surgery therapy, while long-term intake of drugs can lead to serious side effects like palpitations, insomnia and indigestion, and surgical treatment may also cause irreversible damage for body[5]. Therefore, it is vital to explore the anti-obesity drugs or dietary supplements with high efficient and low side effect. Plant-derived polyphenolic compounds have been demonstrated to be potential source for prevention and treatment of obesity. Many studies found that phenolics exhibited anti-obesity effect by reducing lipid accumulation and enhancing lipid oxidation[6]. For example, the green tea polyphenols, resveratrol, and curcumin have been reported to reduce fat accumulation by regulating the expression level of key genes like Acetyl-CoA carboxylase alpha (ACC), fatty acid synthase (FASN), stearoyl Co-A desaturase-1 (SCD1), and sterol regulatory element-binding protein 1c (SREBP-1c)[7].

Pancreatic lipase, as a vital target for screening anti-obesity composition, is responsible for breaking down dietary triglycerides (TG), and is positively with lipid absorption and accumulation[8]. Fernandes et al.[9] found that blueberry pomace showed strong pancreatic lipase inhibitory activity with IC₅₀ value of 1.28 mg/mL, and was potential source of hypolipidemic components. Thus, it is considerable to screen anti-obesity compounds by guiding with lipase inhibitory activity.

Lotus seedpod, belonging to Nymphaeaceae family, is the mature receptacles of the lotus (Nelumbo nucifera Gaertn.). As the primary byproduct during the cultivation of lotus seeds, its production is steadily increasing. Nowadays, most of lotus seedpod are discarded directly, resulting to the waste of quality resources. Lotus seedpod is rich in various active composition, especially for phenolics like flavonoids, and exhibits antioxidant, anti-inflammatory and hypoglycemic activities[10], which is a potential resource for high value application. Shen et al.[10] reported that lotus seedpod showed good antioxidant and antiproliferative activities. Liu et al.[11] found that phenolic extracts from lotus seedpod could reduce liver lipid accumulation and lipotoxicity. Flavonoid-rich lotus seedpod extracts could regulate hepatic glucose and lipid metabolism disorders of mice through AMPK-related pathways [12]. While, the detail anti-obesity components of Lotus seedpod and its hypolipidemic activity is still unclear, which need be further explored.

While, existing methods for extracting lotus seed pod polyphenols primarily rely on traditional processes such as room-temperature maceration, reflux extraction, and solid-phase extraction. These methods commonly suffer from low extraction efficiency, high solvent and energy consumption, insufficient selectivity, and even the degradation of heat-sensitive polyphenols. Therefore, numerous innovative extraction techniques, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction, accelerated solvent extraction, and pressurized liquid extraction, have garnered considerable research attention in recent years. Among these techniques, ultrasound-assisted method displayed the advantage of high effective, low energy consumption and solvent utilization since the cavitation effect, and it is a suitable method for extracting natural bioactive compounds from plants[13], [14]. Xue et al.[15] optimized the ultrasound-assisted deep eutectic solvent extraction of peony roots based on the survival rate of PC12 cells, and found that it improved the neuroprotective effect of extract in MCAO mouse models. Bao et al.[16] employed ultrasound-assisted glycerol extraction to recover polyphenolic compounds from lotus seedpod, resulting in markedly increase in total phenolic content and antioxidant activity. Therefore, ultrasound-assisted extraction combined with response surface methodology (RSM) model optimization represents an effective strategy to develop an effective method for extracting bioactive compounds from lotus seedpod.

In this study, the ultrasound-assisted extraction parameters of polyphenols from lotus seedpod were optimized guided by pancreatic lipase inhibitory activity, and the main anti-obesity components were enriched and identified by UPLC-ESI-Q-TOF-MS/MS technology. The in vitro hypolipidemic activity of lotus seedpod extracts was further evaluated by 3 T3-L1 cell model. Finally, the anti-obesity ability of lotus seedpod polyphenols was investigated through high-fat diet induced obese mice model. This research can provide theoretical and technical guidance for the high-value utilization of lotus seedpod phenolics in anti-obesity or hyperlipidemia regulation field.

2. Material and method

2.1. Material

Lotus seedpod was obtained from Shicheng County, Jiangxi Province. 3 T3-L1 cells (Cat. CL-0006) were sourced from Procell Biotech, Inc. (Jiangsu, China). Total cholesterol (TC, Cat. A111-1–1), triglyceride (TG, Cat. A110-1–1), high density lipoprotein cholesterol (HDL-C, Cat. A112-1–1), low density lipoprotein cholesterol (LDL-C, Cat. A113-1–1), alanine aminotransferase (ALT, Cat. C009-2–1), aspartate aminotransferase (AST, Cat. C010-2–1), superoxide dismutase (SOD, Cat. A001-3), Catalase (CAT, Cat. A007-1–1), Malondialdehyde (MDA, Cat. A003-1) kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing China). TNF-α kits (Cat. PT512) was purchased from Shinbosheng Biotechnology Co., Ltd. (Shenzhen, China). Pancreatic lipase was purchased from Yuanye Biotechnology Co., Ltd (Shanghai, China)[17]. And all chemical reagents are analytical grade.

2.2. Optimization of UAE method

2.2.1. Single-factor design

Dried lotus seedpod was ground into powders. The samples were extracted by an ultrasound processor (KH-700DE, Kunshan Hechuang Ultrasonic Instruments). The lotus seedpod powders were mixed with ethanol aqueous solution at a ratio of 1:20 (g/mL, m:v). After ultrasonic extraction, the mixtures were centrifuged at 5000 r/min for 8 min to collected supernatant, following by concentration and freeze-dring to obtain crude lotus seedpod polyphenols extract. The ultrasonic extraction process was optimized guided with pancreatic lipase inhibitory activity, the parameters were as follows: the concentration of ethanol-aqueous solution was 30%, 50%, 70% and 90%; ultrasonic power was 100 W, 200 W, 300 W, 400 W and 500 W; ultrasonic time was 20 min, 40 min, 60 min and 80 min.

2.2.2. RSM based on Box–Behnken design and validation

An RSM with Box-Behnken Design (BBD) was employed to assess the optimal levels of three independent variables: Ethanol concentration (X₁), ultrasonic time(X₂) and ultrasonic power (X₃). A three-factor, three-level variable design was done following determination of the preliminary scope of extraction using single-factor tests. Table 1 lists the independent variable ranges and levels. The results of the single-factor experiment determined the independent variables and their ranges. The TPC, TFC and IC₅₀ of pancreatic lipase inhibition activity were taken as the response value.

Table 1.

Results of various factors on TPC, TFC and IC₅₀ of PLIA values.

Factors	Levels	TPC (mg GAE/g DM)	TFC (mg QUE/g DM)	IC50 of PLIA (mg DM/mL)
Ethanol concentration (%)	30	78.36 ± 9.25 ab	221.78 ± 3.58b	17.25 ± 0.32 d
	50	89.41 ± 5.56 a	276.14 ± 11.66 a	15.83 ± 0.65c
	70	83.31 ± 4.50 ab	286.9 ± 27.20 a	7.29 ± 0.30 a
	90	76.66 ± 0.89b	209.23 ± 37.25b	11.17 ± 0.76b
Uultrasonic time (min)	20	88.94 ± 7.31b	209.67 ± 2.39 a	24.83 ± 0.83 d
	40	111.22 ± 1.49 a	202.5 ± 9.56 a	19.20 ± 0.57b
	60	113.17 ± 8.04 a	216.04 ± 6.01 a	15.67 ± 0.22 a
	80	82.57 ± 3.49b	208.07 ± 9.66 a	23.96 ± 0.45c
Uultrasonic power (W)	100	98.04 ± 0.54c	238.35 ± 14.54b	14.31 ± 0.12b
	200	102.51 ± 1.32b	244.72 ± 13.16b	22.90 ± 0.37 e
	300	111.2 ± 0.86 a	280.57 ± 4.97 a	13.16 ± 0.08 a
	400	101.00 ± 1.31b	227.99 ± 7.68b	18.39 ± 0.40c
	500	100.38 ± 0.93 bc	235.16 ± 13.80b	21.50 ± 0.20 d

Note: PL: Pancreatic lipase; Different letters (a, b, c) for results of each factor indicates significant difference (p < 0.05).

Using optimized conditions (69% ethanol, 300 W ultrasonication for 62 min), the samples were extracted. The control group was prepared through maceration under the same extraction parameters (69% ethanol, 25 °C, 62 min) with ultrasound extraction, but without ultrasound treatment. The TPC, TFC and LPIA were then compared.

2.3. Enrichment of pancreatic lipase inhibitors

Then, the compounds in crude extract preparing with the optimum extraction parameters were successively partionated with petroleum ether, ethyl acetate, and n-butanol, and three fractions with individual yield of 3.65%, 17.51% and 27.58% were obtained. The pancreatic lipase inhibition activity of these fractions was evaluated to select the best fraction.

2.4. Determination of total phenolics and flavonoids content

The TPC was determined based on previous method[18], gallic acid (10–60 g/mL) was chosen as standard, and results were expressed as mg gallic acid equivalent per gram of dry matter weight (mg GAE/g DM). The TFC was evaluated by the AlCl₃-NaOH-NaNO₃ method[19], quercetin (5–35 μg/mL) was selected as standard, and results were expressed as mg rutin equivalent per gram of dry matter weight (mg QuE/g DM).

2.5. Determination of pancreatic lipase inhibition activity

Pancreatic lipase activity was measured according to the method described by Chen et al.[20] 50 μL of lotus seedpod samples were mixed with 50 μL of tris buffer (pH = 8.0) and 50 μL of pancreatic lipase solution, and incubated at 37 ℃ for 15 min. Then, 100 μL of pNPP (4-nitrophenyl palmitate) solution was added to react for another 20 min. The absorbance of mixture was monitored at 405 nm with a microplate reader (SyhergH1, BioTek). Orlistat was selected as positive control.

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{y}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\%)=\left(1-\frac{\left({\mathrm{A}}_1-{\mathrm{A}}_2\right)}{\left({\mathrm{A}}_3-{\mathrm{A}}_4\right)}\right)\times 100\%$$ (1)

where, A₁ and A₂ are the absorbance value of lotus seedpod group with and without pancreatic lipase, respectively. A₃ is the absorbance value of the group without lotus seedpod extracts, and A₄ is the absorbance value of the group without lotus seedpod extracts and pancreatic lipase.

2.6. UPLC-ESI-Q-TOF-MS/MS analysis

The main compounds in lotus seedpod extract were identified by UPLC-QTOF-MS (SCIEX, TripleTOF 5600 + ) mass spectrometry technology. The composition separation was performed with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm), column temperature, flow rate and injection volume were 45 °C, 0.8 mL/min and 10 μL, respectively.

The mobile phase included 0.1% aqueous formic acid (A) and acetonitrile (B), and the elution procedure was: 0 min, 10% B; 6 min, 15% B; 16 min, 24% B; 30 min, 32% B; 35–40 min, 95% B; 41–48 min, 10% B. The mass spectrometry conditions were: negative ion mode; ion source temperature of 550 °C, mass scan range of m/z 50–1500 Da, ion spray voltage of −4500 V. The mass spectrometry data was analyzed by Peakview 1.2 software, compounds were identified by comparing molecular weights and characteristic fragment ions with literatures and database Metlin, MassBank and Chemspider.

2.7. Determination of 3 T3-L1 cell viability and adipocyte differentiation

The 3 T3-L1 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics for 4 h at a density of 150,000 cells/well. Lotus seedpod extract with different concentrations (25, 50, 100, 200, 400 μg/mL) in new medium was added in each well, and incubated at 37 °C for 24 h. After removing the old medium, CCK-8 reagent was added in each well, and the absorbance was measured at 450 nm after 2 h of incubation.

3 T3-L1 cells were seeded in 6-well plates and incubated in differentiation medium (containing 10% fetal bovine serum, 0.5 mmol/L IBMX, 1 μmol/L DEX and 10 μg/mL insulin (MDI)) for 2–3 d. The 3 T3-L1 cells were then divided into two groups and treated with or without different concentrations of LSPE and simvastatin (SIM, 1.0 μM). After 48 h of incubation, the differentiation medium was replaced with adipocyte maintenance medium supplemented with 10% fetal bovine serum and insulin (10 μg/mL). The new adipocyte maintenance medium was replaced every 2 d until most of cells reached to adipocyte morphology. Finally, 3 T3-L1 cells were fixed overnight with 4% paraformaldehyde, stained with oil red O, and observed with DMi8 inverted microscope (Leica, Germany). The lipid droplets was quantified by ImageJ software. In addition, cells stained with oil red were extracted using isopropanol, and the OD values of all samples were measured at 510 nm by a microplate reader.

2.8. Animals and diets

5-week-old male C57BL/6J mice were purchased from Weitonglihua Experimental Animal Technology Co., Ltd. The experimental procedures were approved and conducted in accordance with the “Guidelines for the Care and Use of Laboratory Animals” of Sun Yat sen University Nanchang Research Institute (SYSUNC-IACUC-B-2025–0001). All of mice were housed at a temperature of 23 ± 3 °C, relative humidity of 40% −70% with a 12 h light/dark cycle, and was free to eat and drink. After one week of adaptive feeding, the mice were randomly divided into four groups (n = 8). The normal group was fed with standard diet, but model group, low and high dose groups were fed with high-fat diet (contained 20% protein, 20% carbohydrates and 60% fat, XTHF60, Xietong Bio Company). Low and high dose groups were supplemented with 75 and 150 mg/kg·d^-1 of lotus seedpod extract, while normal and model groups were treated with equivalent of sterile water. The weight of the mice was recorded every week. After treatment for 8 weeks, all mice were euthanized, the vein blood were collected, the serum were obtained after centrifuging at 3000 rpm for 5 min. Meanwhile, the liver of mice was gathered, and part of samples were stored at 20 °C.

2.9. Oil red O staining

Fresh liver tissues were fixed in 4% paraformaldehyde for 24 h, and dehydrated with sucrose of gradient concentration, then frozen with liquid nitrogen and cut into slices. The slices were successively rinsed, fixed and differentiation, and incubated at oil red O solution for 15 min without light. After differentiation in 60% isopropanol for 30 s, the liver samples washed with running water, and sealed by glycerin gelatin. The liver samples were observed using DMi8 inverted microscope (Leica, Germany).

2.10. Determination of biochemical indexes

The fresh liver was mixed with normal saline, prepared to 10% liver homogenate, and centrifuged to obtain the supernatant for further analysis. According to the instructions of the reagent kit (Nanjing Jiancheng Biotechnology Company, China), the serum TG, TC, HDL-C, LDL-C, ALT and AST levels were evaluated, the hepatic SOD, MDA, CAT and TNF-α contents were also measured.

2.11. Statistical analysis

The data were expressed as the mean ± SD deviation. The drawing was carried out by GraphPad Prism V10.1.2 software (San Diego, CA, USA). Statistical analysis was performed by one-way ANOVA followed by with Tukey’s test.

3. Results and discussion

3.1. Single-factor analysis of LSPE

Based on the single-factor experiments, the result showed that phenolic and flavonoid compounds are key lipid-lowering active substances, and they collectively influence the activity of pancreatic lipase. As shown in Table 1, as the ethanol concentration increased, TPC and TFC exhibited a trend of rising first and then decreasing. When the ethanol concentration was 50%, the TPC and TFC reached the maximum value of 89.41 ± 5.56 mg GAE/g DM and 276.14 ± 11.66 mg QUE/g DM, respectively, but insignificant difference was observed with that extracted by 70% ethanol (p > 0.05). In addition, 70% ethanol extract exhibits the strongest pancreatic lipase inhibitory activity, with an IC₅₀ value of 7.29 ± 0.30 mg DM/mL, significantly lower than other extracts. At low ethanol concentrations, the solvent exhibits strong polarity, which is unfavorable for the dissolution of moderately polar polyphenols. Appropriately increasing of ethanol proportion improves solvent polarity, promoting polyphenol dissolution and release. However, further increasing of ethanol concentration reduces cell wall swelling due to insufficient water content and decreases the solubility of some polar polyphenols, thereby decreasing extraction efficiency. The same phenomenon has also been observed in related studies[21]. Although the TPC and TFC of 50% and 70% ethanol extracts were comparable, but the 70% ethanol extract exhibited the strongest lipase inhibition, which may be due to the higher level of potential lipase inhibitors or the existence of compounds with stronger inhibitory activity. Therefore, 70% ethanol was selected for further optimization.

In terms of ultrasonic time, the sample extracted for 60 min showed the highest TPC (113.17 ± 8.04 mg GAE/g DM) and TFC (216.04 ± 6.01 mg QUE/g DM), as well as the strongest PLIA (IC₅₀ = 15.67 ± 0.22). When ultrasonication time was extended from 20 min to 40 or 60 min, the TPC content increased significantly. This could be that intensified cavitation enhances solvent permeability and disrupts plant cell walls, the release of intracellular phenolics and flavonoids were accelerated[22]. However, further increasing of ultrasonic time to 80 min resulted in a significant 27.04% decrease in TPC content compared to the maximum value, even lower than the level observed in the 20 min sample. This might be attributed to the heating effect and prolonged ultrasonication time, leading to structural damage and reduced extraction efficiency[23]. Therefore, 60 min was selected for further optimization.

With the variations of ultrasonis power, a same change trend was observed between TPC, TFC and PLIA, suggesting that higher TPC and TFC leading better PLIA. When the power was lower than 300 W, the TPC and TFC and PLIA were improved with the increasing of ultrasonis power, these might be due to the high-pressure cavity and disintegration effect of ultrasound[24]. Ultrasonic cavitation produces numerous bubbles that collapse violently, generating microjets and intense shear forces. These effects enhance solvent penetration and disrupt cell walls, thereby facilitating mass transfer and the release of target compounds[25]. However, when the ultrasound power was increased from 300 to 500 W, the TPC and TFC decreased by 9.73% and 16.18%, respectively. In addition, the reduction of TPC and TFC might be attributed to cavitation saturation, acoustic shielding and thermal degradation effect of UAE[26], [27]. Therefore, 300 W was selected for further experiments.

3.2. Extraction optimization using RSM

It is evident that a solitary indicator is incapable of accurately reflecting the lipid-lowering activity of lotus seedpod extract. And it is imperative to ascertain the optimal extraction conditions for LS extract in conjunction with TPC, TFC, and pancreatic lipase inhibitory activity, as these parameters must be employed in unison to ensure the accuracy and efficacy of the assessment. Therefore, based on the experimental results of single-factor optimization, RSM was employed to optimize the conditions for extracting pancreatic lipase inhibitor from LS. Results are summarized in Table 2. Specifically, the TPC ranged from 97.01 to 128.48 mg GAE/g DM, the TFC ranged from 286.66 to 359.43 mg QUE/g DM, and the IC₅₀ of pancreatic lipase inhibitory activity exhibitd a variation from 7.96 to 20.72 mg DM/mL.

Table 2.

The BBD was applied with TPC, TFC and IC₅₀ of PLIA as the dependent variable for UAE.

Run	X1: Ethanol concentration (%)	X2: Ultrasonic time (min)	X3: Ultrasonic power (W)	TPC (mg GAE/g DM)	TFC (mg QUE/g DM)	IC50 of PLIA (mg DM/mL)
1	70	60	300	128.48	358.66	9.56
2	70	70	200	118.72	336.99	13.40
3	70	50	200	110.92	316.31	17.86
4	60	60	400	113.26	310.84	18.04
5	70	50	400	117.92	324.26	13.85
6	60	60	200	112.93	314.79	16.55
7	80	50	300	101.17	302.39	18.35
8	70	60	300	127.34	359.43	8.95
9	70	60	300	126.49	355.45	8.36
10	70	60	300	125.79	357.11	9.25
11	70	60	300	123.55	357.03	7.96
12	60	50	300	111.50	333.14	14.24
13	80	70	300	104.57	323.69	13.96
14	80	60	200	97.01	298.19	19.44
15	70	70	400	123.71	315.39	17.75
16	60	70	300	121.88	339.43	13.46
17	80	60	400	112.34	286.66	20.72

3.2.1. Fitting models and statistical analysis

A response surface regression analysis was performed on the data (Table 2), yielding the following regression equations for the four variables and TPC, TFC, and pancreatic lipase inhibitory activity:

Y(TPC) = -713.916 + 18.35707X₁ + 5.6246X₂ + 0.224793X₃-0.0224X₁X₂ + 0.002493X₁X₃-0.00051X₂X₃-0.131092X₁²-0.029467X₂²-0.000559X₃².

Y(TFC) = -1363.85625 + 34.59525X₁ + 6.84137X₂ + 2.23764X₃ + 0.041825X₁X₂-0.001792X₁X₃-0.00717X₂X₃-0.268875X₁²-0.05975X₂²-0.002868X₃².

Y(PLIA) = 331.52775–5.7268X₁-1.99527X₂-0.435358X₃-0.009025X₁X₂-0.000053X₁X₃ + 0.00209X₂X₃ + 0.045795X₁² + 0.01607X₂² + 0.000529X₃².

The ANOVA results for the RSM experimental design model were summarized in Table 3. The F-values were 36.56 (Y_TPC), 147.85 (Y_TFC), and 41.24 (Y_PLIA), respectively, with P-values < 0.0001, indicating overall model significance. The regression coefficients (R2) for the fitted TPC, TFC and PLIA models all exceeded 0.95, indicating a correlation exceeding 90% between predicted and experimental data. Furthermore, the adjusted R2 (R2Adj) values for these models exceeded 0.79, indicating minimal significant deviation between predicted and adjusted values. This, in turn, validated the models' reliability. Finally, the fitted variance was non-significant (p > 0.05), confirming the models' adequacy[28].

Table 3.

Analysis of variance (ANOVA) of responses for TPC, TFC and IC₅₀ of PLIA values.

Source	TPC (mg GAE/g DM)		TFC (mg QUE/g DM)		IC50 (mg DM/mL)
	F-value	p-value	F-value	p-value	F-value	p-value
Model	36.56	< 0.0001	147.85	< 0.0001	41.24	< 0.0001
X1-EC	57.79	0.0001	147.24	< 0.0001	17.4	0.0042
X2-UT	21.88	0.0023	30.01	0.0009	5.51	0.0512
X3-UP	22.33	0.0021	16.41	0.0049	1.62	0.2432
X1X2	2.85	0.1355	8.71	0.0214	4.38	0.0747
X1X3	13.14	0.0084	2.22	0.1797	0.0148	0.9066
X2X3	0.236	0.6419	33.76	0.0007	23.47	0.0019
X12	159.72	< 0.0001	465.83	< 0.0001	118.63	< 0.0001
X22	14.27	0.0069	24.45	0.0017	14.61	0.0065
X32	21.77	0.0023	516.79	< 0.0001	158.42	< 0.0001
Lack of Fit	1.58	0.3255	4.93	0.0788	2.75	0.1765
Std. Dev.	2.07		2.54		0.8627
Mean	116.33		328.81		14.22
C.V. %	1.78		0.7733		6.07
R2	0.9792		0.9948		0.9815
Adjusted R2	0.9524		0.988		0.9577
Predicted R2	0.8041		0.9324		0.791
Adeq PrecisionR2	19.0414		36.5914		17.3497

3.2.2. Effect of independent variables on bioactive compounds and lipid-lowering activity of the LS extract

Response surfaces visually illustrate the interaction between two factors, particularly when other factors remain at zero-level constant state. Within a response surface plot, the influence degree of each factor exerts on the response value is clearly demonstrated: the steeper the three-dimensional surface, the more pronounced the effect of that factor[15]. Interactions among ethanol concentration (X₁), ultrasonic time (X₂) and ultrasonic power (X₃), as well as their effects on TPC, TFC and PLIA are graphically represented in Fig. 1.

Fig. 1.

Three-dimensional response surface plots of TPC, TFC and PLIA. The interactive effects of (A) ethanol concentration and ultrasonic time, (B) ethanol concentration and ultrasonic power, (C) ultrasonic time and ultrsonic power on TPC. The interactive effects of (D) ethanol concentration and ultrasonic time, (E) ethanol concentration and ultrasonic power, (F) ultrasonic time and ultrsonic power on TFC. The interactive effects of (G) ethanol concentration and ultrasonic time, (H) ethanol concentration and ultrasonic power, (I) ultrasonic time and ultrsonic power on PLIA.

Based on the three-dimensional response-optimized surface, the interaction between X₁ and X₂ (Fig. 1A) exerts the greatest influence on TPC. In contrast, the interaction between X₂ and X₃ (Fig. 1F, I) predominantly affects TFC and PLIA. All these interactions manifest as steeper gradients in the three-dimensional plot. Among the three variables, ethanol concentration had the greatest effect on all measured parameters compared to the other variables. The stronger effect of ethanol may be due to the principle of like dissolves like. The polarity of ethanol solution showed a great influence on enrichment of lipase inhibitors (e.g. flavonoids) from lotus seedpod, which was stronger than other factors. Oh et al.[29] also observed the same results with our study. This observation strongly corroborates the analysis results in Table 3, further validating the interactions interactions.

3.2.3. Validation of extraction conditions and fraction of pancreatic lipase inhibition actives

To validate the reliability of the predictive model, we adjusted the extraction conditions to align with the actual setup: ethanol concentration of 69%, ultrasonication time of 62 min, and ultrasonic power of 300 W. As shown in Table S1 (Shown in Supporting Materials), the TPC was 125.42 mg GAE/g DM, the TFC was 361.82 mg QUE/g DM and the PLIA was 8.24 mg DM/mL. These results closely match the predictions, confirming the model's accuracy and reliability. Additionally, the ultrasonicated group processed under the same ethanol concentration and extraction time but without ultrasonication showed TPC, TFC, and PLIA values of 82.42 mg GAE/g DM, 262.25 mg QUE/g DM, and 27.89 mg DM/mL, respectively. This demonstrates that ultrasonication significantly enhances the extraction efficiency of polyphenols from lotus seedpod. And the obtained extract was subjected to extraction enrichment using petroleum ether, ethyl acetate, and n-butanol, yielding three distinct enriched fractions. Their TPC, TFC, and PLIA results are shown in Fig. 2. The ethyl acetate fraction exhibited the highest TPC and TFC, reaching 128.74 ± 1.28 mg GAE/g E and 332.58 ± 8.26 mg QUE/g E. Additionally, it also exhibited the highest PLIA (IC₅₀ = 171.5 ± 0.5 μg/mL). The values was 3.27, 1.39, and 1.06 fold of that detected in lotus seedpod extract, the TPC was 39.35 ± 1.22 mg GAE/g E, the TFC was 239.99 ± 4.13 mg QUE/g E and the IC₅₀ of PL was 181.50 ± 0.25 μg/m, indicating excellent enriching effect of ethyl acetate. Although the lipase inhibitory of lotus seedpod extract is lower than that of Orlistat (IC₅₀ value of 0.16 μg/mL), it was significant higher than other phenolics extracts[30], [31]. Therefore, the lotus seedpod ethyl acetate polyphenol extract (LSPE) was selected for the next experimental subjects.

Fig. 2.

The TPC (A), TFC (B) and PLIA (C) of different enriched fractions of lotus seedpod extract.

3.3. Identification of individual compounds in LSPE

The major compounds in LSPE were identified by mass spectrometry technology. As shown in Fig. 3 and Table 4, by comparing the MS/MS information and molecular formula with literatures and database, a total of 31 compounds were identified from LSPE, including 3 organic acids, 2 fatty acids, 24 flavonoids, and 2 phenolic acids.

Fig. 3.

The base peak chromatogram of LSPE (A). The MS/MS fragmentation pattern of p-coumaric acid (B), procyanidin (C), gambiriin A (D), hyperoside (E), kaempferol-O-glucoside (F), and isorhamnetin-O-glucoside (G).

Table 4.

The main compounds identified from LSPE by HPLC-QTOF-MS/MS.

No.	RT (min)	Formula	MS (m/z)	Error (ppm)	Propossed compounds	MS/MS	Confidence levels
Organic acids
1	3.10	C4H6O5	133.0143	−2.2	Malic acid	115.0039, 71.0134	2
2	3.13	C6H8O7	191.0197	3.5	Citric acid	111.0164, 85.0308	2
25	22.43	C9H16O4	187.0975	0.28	Azelaic acid	125.0999	2
Flavonoids
3	3.37	C30H26O13	593.1300	0.2	Kaempferol-3-O-coumaroyl −glucuronide	425.0862, 289.0697, 177.0183, 125.0238	2
4	5.27	C30H26O12	577.1351	2.9	Procyanidin	407.0785, 289.0715, 245.0886,125.0249	2
5	6.03	C15H14O7	305.0666	0.8	Gallocatechin	305.0665	3
6	6.677	C30H26O13	593.1296	−1.2	Prodelphinidin Dimer Isomer	425.0878, 289.0698, 177.0178, 125.0257	3
7	7.144	C30H26O13	593.2334	−1.2	Prodelphinidin Dimer Isomer	423.0722, 305.0669, 287.0570, 125.0257	3
8	7.98	C30H26O12	577.2078	0.5	Procyanidin Isomer	451.1012, 407.0767, 289.0725, 125.0252	3
9	9.29	C15H14O6	289.0717	−0.13	Catechin	289.0764, 245.0832	2
10	9.70	C45H38O18	865.1985	2.44	Procyanidin C1	577.1285, 407.0922, 125.0336	2
11	10.02	C30H28O12	579.1508	1.12	Gambiriin A	289.0718, 245.0829, 205.0506, 179.038, 137.0228	2
13	13.08	C21H22O11	449.1089	−1.1	Eriodictyol-7-O-glucoside	287.0587, 151.0023	2
14	13.80	C21H20O13	479.0831	−0.28	Myricetin-3-O-glucoside	317.0335, 316.0267,	2
15	14.613	C21H20O13	479.0837	−1.3	Myricetin-3-O-hexoside(1)	317.0335,316.0243, 178.9993	3
16	14.891	C21H20O13	479.0905	0.7	Myricetin-3-O-hexoside Isomer	317.0309,316.0228, 271.0248, 178.9965	3
17	15.69	C15H12O8	319.0459	0.27	Dihydromyricetin	175.0425, 153.0160	3
18	17.05	C21H20O12	463.0882	−0.12	Isoquercitrin	300.029	2
19	17.47	C21H20O12	463.0882	0.68	Hyperoside	301.0362, 151.0027	2
20	17.47	C21H20O12	463.0882	0.68	Hyperoside isomer	301.0362, 151.0027	3
22	19.30	C21H20O11	447.0932	0.27	kaempferol-O-hexoside	285.0371, 284.0325, 255.031,227.0327	3
23	19.54	C20H18O11	433.0776	0.27	Avicularin	301.0351, 300.0276, 271.0273	2
24	20.02	C22H22O12	477.1038	0.94	Isorhamnetin-O-glucoside	314.0419, 315.0506, 243.0299	2
25	20.32	C23H24O13	507.1144	1.51	Viscidulin III-O-glucoside	345.0624, 330.0315	2
27	24.05	C15H10O8	317.0302	0.76	Myricetin	289.0367, 271.0275, 179.0006, 151.0079, 137.0277	2
28	24.83	C15H12O6	287.0561	1.33	Eriodictyol	151.0034, 135.0508, 125.0264, 107,83	2
31	32.09	C15H10O7	301.0353	2.1	Quercetin	178.9988, 151.0052, 121.0288	2
Phenolic acid
12	11.20	C7H6O3	137.025	4.2	Hydroxybenzoic acid	93.0358, 65.0397	2
21	18.34	C9H8O3	163.0400	0.23	p-Coumaric acid	119.049, 93.0457	2
Fatty acid
33	34.44	C18H32O5	327.2173	0.8	Trihydroxy-octadecadienoic acid	291.1995,229.1460, 211.1364, 171.1000	3
34	34.93	C18H34O5	329.2333	1.54	Trihydroxy-octadecenoic acid	229.1459, 211.1361, 171.1016, 139.1142	3
Unknown compound
29	25.448	C22H36O9	443.2287	2.1	Unidentified compound	397.2293, 161.0492
30	31.85	C22H40O9	447.2604	3.5	Unidentified compound	401.2530, 327.2151
32	33.988	C21H20O11	447.2600	−0.3	Unidentified compound	401.2534, 161.0469

Note: The confidence level was ensured according to the mass spectrometry identification rule proposed by Schymanski et al[62].

3.3.1. Organic acids

Compound 1 was suggested as malic acid with characteristic fragment ions at m/z 115.0039 [M−OH]^- and m/z 71.0134 [M−OH−CO₂]^-. Compound 2 showed characteristic fragment ions at m/z 111.0164 and m/z 85.0308, and was proposed as citric acid according to reference[32]. Compound 25 was identified as azelaic acid, and the fragment ion at m/z 289.0764 was corresponded to the loss of a carboxyl group and H₂O.

3.3.2. Flavonoids

Overall, most of identified flavonoids in LSPE is belong to quercetin and kaempferol derivatives. Compound 3 was suggested as kaempferol-3-O-coumaroylglucuronide, and the MS/MS fragment ion at m/z 425.0862 indicated the existence of a glucuronosyl moiety, glucuronic acid and p-coumaroyl group (Fig. 3B)[33]. Compound 4 and 8 were identified as procyanidin (Fig. 3C), and the characteristic fragment ions at m/z 407.0785, 289.0715 and 125.0249 were generated by the loss of a benzoyl group, catechin and dehydrobenzoic acid[34]. Compound 5 was identified as gallocatechin, the molecular weight of it was 16 Da higher than compound 9, and the MS/MS fragment ion at m/z 289.0764 was due to the loss of a phenolic hydroxyl group. Compound 6 and 7 were identified as prodelphinidin dimer by matching with reference[35]. Compound 9 showed a characteristic MS/MS fragment ion at m/z 245.0832, and was proposed as catechin by matching with reference[36]. Compound 10 was proposed as procyanidin C1, the fragment ions at m/z 577.1285 and 125.0336 accounted for the presence of resorcinol formaldehyde and epicatechin[34]. Compound 11 showed a characteristic fragment ion at m/z 289.0718, and was proposed as Gambiriin A (Fig. 3D) by matching with reference[37]. Compounds 18, 19, 27, 28 and 31 were respectively identified as isoquercitrin, hyperoside (Fig. 3E), eriodictyol, myricetin and quercetin by matching with standards. Compound 13 had the same MS/MS fragment ions with compound 28, and the characteristic fragment ion at m/z 287.0587 was produced by the loss of a glucoside, and was suggested as eriodictyol-7-O-glucoside. Compound 20 was assigned as hyperoside isomer since the same molecular formula and characteristic fragment ion. Compound 14 was proposed as myricetin-3-O-glucoside, the MS/MS fragment ion at m/z 317.0335 accounted for the presence of glucoside. Compounds 15 and 16 were identified as myricetin-3-O-hexoside, the MS/MS fragment ion at m/z 317.0335 represented the presence of myricetin and loss of a hexose glycoside[35]. Compound 17 with MS/MS fragment ions at m/z 175.0425 and 153.0160 was identified as dihydromyricetin by compared with literature, and was the hydrogenation of myricetin. The MS ion at 285 and molecular formula of C₁₅H₁₀O₆ was correspond with kaempferol, and compound 22 was identified as kaempferol-O-hexoside (Fig. 3F) due to the MS/MS ion at m/z 284.0371 [M−H−C₆H₁₀O₅]-[38]. The MS ion of quercetin was 301 [C₁₅H₁₀O₇-H]^-. Compound 23 was hypothesized to be avicularin, and the characteristic fragment at m/z 301.0351 was generated by losing a pentose [M−C₅H₈O₄]^-[39]. Compound 24 was assigned as isorhamnetin-O-glucoside (Fig. 3G), and the MS/MS fragment ions at m/z 314.0506 [M−H−C₆H₁₀O₅]^- and 243.0299 [M−H]^- was produced by isorhamnetin[40]. Compound 25 was proposed as viscidulin III-O-glucoside, the MS/MS ions at m/z 345.0624 and 330.0315 was corresponded to loss a glucoside and methyl group[41].

3.3.3. Phenolic acids

Compound 12 and 21 was suggested as hydroxybenzoic acid and p-coumaric acid by matching with standards.

3.3.4. Fatty acids

Compound 33 and 34 were identified as trihydroxy-octadecadienoic acid and trihydroxy-octadecenoic acid by matching with reference[19].

Polyphenols and flavonoids were the main anti-obesity compounds in LSPE. Many studies have reported that ethyl acetate were the suitable solvent for extracting phenolics due to the similar polarity[42], and was consistent with our research. Furthermore, 31 compounds were respectively identified from LSPE. Quercetin and kaempferol derivatives were the major ingredients in LSPE. Catechin, quercetin and myricetin showed a high response value in LSPE, and have been reported to be the potential pancreatic lipase inhibitors, which may contribute to the anti-obesity of lotus seedpod[43], [44], [45].

3.4. LSPE reduced lipid accumulation in 3 T3-L1 cells

Since pancreatic lipase was related with TG catabolism, the anti-obesity activity of LSPE was also measured by analyzing the lipid levels in 3 T3-L1 cell. As listed in Fig. 4A, the simvastatin cell viability of 3 T3-1 cells was significantly declined when the concentration of LSPE was over 100 μg/mL, and the sample concentration ranged from 0-25 μg/mL was chosen for next experiment. According to Fig. 4B-C, compared with Mod group, the TC and TG levels in 3 T3-L1 cells was decreased by 34.4% − 43.0% after supplement with LSPE, and exhibited dose effect. In addition, Oil red O staining can reflect the formation of lipid accumulation, and the number lipid droplets can be detected by UV spectrophotometer. As shown in Fig. 4D-E, the darker red was found in Mod group, and the color in LSPE was nearly to Nor group, which was accorded with the results of OD value. Additionally, lotus leaves and seeds also exhibited significant lipid-lowering effect on 3 T3-L1 cell, while the decrease of TG content was less than that of LSPE[46], [47]. The identified composition in LSPE like myricetin and quercetin have demonstrated to lower TG level and inhibit lipid accumulation in 3 T3-L1 cell, which may make a great contribution[48], [49]. Above results suggest that LSPE could inhibit the lipid accumulation in 3 T3-L1 cells, and displayed the potential of anti-obesity activity.

Fig. 4.

The lipid-lowering effect of LSPE on 3 T3-L1 cells. A: Cell viability; B: TC level; C: TG level; D: Oil red O staining area; E: Oil red O staining. *: p < 0.05; **: p < 0.01 ***: p < 0.001 compared with Mod group.

3.5. LSPE alleviate weight gain and fat index of obese mice

The mice model was further used to investigate the in vivo anti-obesity effect of LSPE. As given in Fig. 5A and 5B, in comparison with Mod group, the weight gain of mice in low (L-LSPE) and high (H-LSPE) dose of LSPE group was decreased by 43.6% and 46.1% after treatment for 11 weeks. The impact of LSPE on body weight of mice was positively related with fat accumulation. From Fig. 5C and Fig. 5D, the epididymal fat, subcutaneous and perirenal adipose tissue indexes in Mod group was inidividually enhanced by 113.5%, 130.6% and 237.0% than that in Nor group, while the phenomenon was reversed after consumption of LSPE, and exhibited a dosage-dependent effect. Notably, there was an obvious increase (16.2%) in the brown adipose tissue (BAT) index of H-LSPE treated mice with high-fat diet.

Fig. 5.

Effect of LSPE on body weight and adiposity indexes of obese mice. A: Body weight; B: Body weight gain; C: Epididymal index; D: Subcutaneous fat index; E: Perirenak fat index; F: Interscapular brown index. *: p < 0.05; **: p < 0.01 ***: p < 0.0001 compared with Mod group.

WAT is the main tissue for storing body energy, and is closely with the increasing of weight[50]. BAT is involving in energy metabolism and thermogenesis, and also can control lipid level by enhancing the clearance capacity of lipids[51]. The increase of BAT mass was resulted form the increment of mitochondrial content and tissue thermogenesis with the rising of energy expenditure[52]. LSPE intervention effectively alleviated weight gain and WAT accumulation, while promoted the generation of BAT in obese mice. In addition, the serum lipid content like TG and TC in obese mice also reversed with the supplement of LSPE, and the results was accorded with pancreatic lipase inhibition activity and 3 T3-L1 cell model. Flavonoids such as catechin, hyperoside and isoquercitrin, as proposed compounds in LSPE, have been observed to display hypolipidemic activity[53]. The above results indicate that LSPE can effectively suppress weight and fat deposition of obese mice.

3.6. LSPE alleviated serum lipid level of obese mice

As displayed in Fig. 6A-D, compared with the Nor group, the TG, TC, and LDL-C levels in Mod group were respectively elevated by 35.1%, 35.7% and 32.7%, while the HDL-C level was reduced by 42.2%. After consumption with LSPE, the content of TC, TG and HDL-C in obese mice was reserved. In comparison with Mod group, the TC and TG levels in high dose of LSPE group were reduced by 12.48% and 23.87%, respectively, while the HDL-C level was increased 16.36%. The LDL-C level in different groups of obese mice had no significant difference (p < 0.05). The results display that LSPE treatment could improve the dyslipidemia of mice caused by obese, and showed good in vivo anti-obesity ability.

Fig. 6.

Effect of LSPE on serum and hepatic biochemical indicators in mice. A: Serum TG; B: Serum TC; C: Serum HDL-C; D: Serum LDL-C; E: Hepatic AST; E: Hepatic ALT; G: Hepatic MDA; H: Hepatic CAT; I: Hepatic SOD; J: Hepatic TNF-α; K: Oil red O staining of liver. *: p < 0.05; **: p < 0.01 ***: p < 0.0001 compared with Mod group.

3.7. LSPE repaired liver damage of obese mice

Liver is the target organ of lipid metabolism, AST and ALT activities are the marker of liver fucntion. According to Fig. 6E-J, the AST, ALT, MDA, TNF-α levels in Mod group were remarkably higher than that in Nor group, and the CAT and SOD levels exhibited a significant decline. The AST and ALT content in LSPE group was reached to Nor group. Meanwhile, after the administration of H-LSPE, the MDA, TNF-α levels were reduced by 34.7% and 31.1%, while the CAT contents was rised by 8.9%. In addition, Oil Red O staining was carried out to explore the effect of LSPE on fat deposition of obese mice, and the result was described in Fig. 6K. In contrast to Nor group, the number and size of lipid droplets in liver was significantly larger, and the cell nucleus displayed a slight deformation, which showed a fat deposition phenomenon. The morphology of liver cells was improved in H-LSPE group, and showed a smaller size of lipid droplet and clear of cell nucleus. The results were accorded with the basic indicators of liver. Hence, these foundings suggest that LSPE treatment can repair liver injury and steatosis, ameliorate hepatic oxidative stress and inflammation of obese mice, and display a dose effect.

Liver is a crucial organ for lipid metabolism, and the hepatic indicators such as ALT, AST and TNF-α levels can reflect the degree of liver injury in obese mice[54]. The AST and ALT content of obese mice was restored to normal level after consumption of LSPE, indicating the excellent repair effect of LSPE on liver damage. The excessive fat accumulation in liver may cause the aggravation of oxidative stress and inflammation. SOD is an antioxidant enzyme for eliminating superoxide radicals, and MDA level was highly linked to hepatic lipid peroxidation[55]. The inflammatory cytokines such as TNF-α can exacerbate liver damage by promoting oxidative stress and inflammatory responses[56]. LSPE treatment enhanced the antioxidant and anti-inflammatory abilities of obese mice by alleviating SOD, CAT and TNF-α levels. Quercetin and kaempferol derivatives like quercetin, hyperoside, myricetin-3-O-glucoside, kaempferol-O-hexoside and myricetin displayed strong antioxidant activity[57], while catechin, gallocatechin, procyanidin and procyanidin C1 have been reported to show favorable anti-inflammatory effect[58]. The identified compounds like myricetin, quercetin and catechin were observed to significantly reduce TNF-α levels in mice, and may play a important role for the anti-inflammatory effect of LSPE[59], [60], [61]. These compounds may play a vital role to reversed liver damage of obese mice. Oil Red O staining data also observed that the fat deposition in liver was improved by LSPE treatment. Taken together, LSPE can ameliorate liver damage caused by obesity, and liver maybe the target for LSPE to regulate lipid metabolism.

4. Conclusion

In summary, the optimized ultrasonic method for extracting anti-obesity ingredients from lotus seedpod was 69% ethanol aqueous solution, ultrasonic power of 300 W, and ultrasonic time of 61 min, ethanol concentration showed the highest effect. Ethyl acetate was the best solvent to screen lipase inhibitors from lotus seedpod, and its IC50 value was 171.5 μg/mL. LSPE supplement also exhibited an 12.48% and 23.87% reduction in TG and TC content in 3 T3-L1 cells. These results indicating that LSPE showed significant in vitro lipid-lowering effect. A total of 31 compounds were identified from LSPE, quercetin and kaempferol derivatives were the potential anti-obesity compounds. In vivo experiment further proved that LSPE exhibit obvious hypolipidemic effect by reducing weight, fat deposition, serum lipid and hepatic TNF-α levels as well as enhancing hepatic CAT and SOD content. This study could provide scientific basis for the application of lotus seedpod in the field of obesity. The anti-obesity mechanism of LSPE will be investigated base on genomics, metabolomics and microbiologyin technologies in our next work.

CRediT authorship contribution statement

Xinpeng Cheng: Writing – original draft, Validation, Investigation, Formal analysis. Xing Xie: Writing – review & editing, Validation, Supervision, Methodology. Quanyuan Xie: Writing – review & editing, Software, Formal analysis. Peixin Wang: Software, Investigation, Formal analysis. Qiao Ding: Supervision, Software, Funding acquisition, Formal analysis. Zhangyuan Zuo: Investigation. Lu Zhang: Writing – review & editing, Supervision, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

No data was used for the research described in the article.