Determination of the Phytochemical Profile and Antioxidant Activity of Some Alcoholic Extracts of Levisticum officinale with Pharmaceutical and Cosmetic Applications

Abstract

Levisticum officinale (lovage) is an aromatic and medicinal plant traditionally used for its antioxidant, anti-inflammatory and antimicrobial properties. The aim of this study was to evaluate the phytochemical composition and antioxidant activity of hydroalcoholic extracts obtained from leaves, roots and the whole plant, as well as to develop hydrogels with pharmaceutical potential. The hydroalcoholic extracts (70% ethanol) were characterized by spectrophotometric and HPLC-DAD methods to determine the total content of phenolic compounds, phenolic acids, flavonoids and condensed tannins. The antioxidant activity was evaluated by DPPH and ABTS methods. The extracts were included in 2% carbopol-based hydrogels and tested for stability and antioxidant efficacy. The hydroalcoholic extract of the leaves showed the highest content of total phenolic compounds (20.84 ± 2.18 mg GAE/g), total flavones (11.39 ± 2.48 mg QE/g) and condensed tannins (1.98 ± 1.55 mg CE/g), and was also the richest in quercetin (3.32 ± 1.25 mg/g), kaempferol (1.84 ± 1.63 mg/g), luteolin (2.12 ± 1.19 mg/g), rutin (4.38 ± 1.84 mg/g) and apigenin (1.91 ± 1.44 mg/g). The root extract had the highest content of phenolic acids, including ferulic acid (3.86 ± 1.37 mg/g), vanillic acid (2.53 ± 1.76 mg/g) and caffeic acid (3.28 ± 1.28 mg/g). The antioxidant activity was highest in the leaves extract, with values of 276.2 ± 3.4 µmol TE/g (ABTS) and 246.4 ± 3.6 µmol TE/g (DPPH). The whole-plant extracts showed intermediate values, offering a balance between flavonoids and phenolic acids. Hydrogels formulated with 5% extracts demonstrated stability and sustained antioxidant activity over time. Leaf extracts, due to their high flavonoid content, are recommended for formulations with antioxidant and photoprotective effects, while root extracts are suitable for anti-inflammatory and antimicrobial applications. Hydrogels obtained based on 2% carbopol represent a promising delivery system for dermato-cosmetic and pharmaceutical applications because they exhibited significant antioxidant action.

1. Introduction

Levisticum officinale, traditionally known as lovage, is an aromatic and medicinal plant used extensively in both gastronomy and traditional European and Asian medicine. It is valued not only for its distinctive aroma, but also for its multiple therapeutic properties, documented in numerous scientific studies [1,2]. The phytochemical composition of lovage is complex, including flavonoids, phenolic acids, coumarins, phthalides and essential oils, compounds with antioxidant, anti-inflammatory, antimicrobial and diuretic effects [2,3].

Recent research on Levisticum officinale has highlighted its therapeutic potential through phytochemical, pharmacological and clinical studies, in addition to strengthening the position of lovage as a source of antioxidant substances, essential for reducing oxidative stress, a factor involved in numerous chronic diseases [4,5,6,7].

Studies have shown that lovage essential oil contains ligustilide (up to 70% of its composition), a compound responsible for its antispasmodic and neuroprotective effects. This compound is particularly important in the treatment of colic and functional digestive disorders, being used in supplements based on lovage root extracts [3,8,9]. In addition to essential oils, flavonoids represent an important class of bioactive compounds identified in the leaves and stems of the plant. Quercetin, a flavonol present in concentrations of 170 mg/100 g in dried leaves, is recognized for its ability to inhibit lipid peroxidation and protect cells against oxidative stress [3,10,11,12,13]. Kaempferol, another flavonol, has been investigated for its anticancer and anti-inflammatory effects, having been shown to reduce the expression of genes involved in inflammation, such as TNF-α and IL-6 [14,15].

Another important class of bioactive compounds are phenolic acids, including chlorogenic acid, ferulic acid and caffeic acid, substances with cardioprotective and antioxidant effects [16]. Studies have shown that lovage leaf extracts have an antioxidant capacity comparable to that of sage and rosemary extracts, two of the most powerful medicinal plants with antioxidant activity [5,17].

Coumarins, such as apterin, xanthotoxin and falcarinol, are predominantly present in the roots of the plant, where their concentration varies between 1.7–2.9 mg/g in the fresh state and 15–24 mg/g in the dried form [18]. Studies have shown that dried roots stored at room temperature retain a superior stability in terms of coumarin content, compared to fresh ones [19].

At the clinical level, Levisticum officinale extracts have been investigated for their anti-inflammatory, antimicrobial, diuretic and neuroprotective effects [5,19,20]. Studies have shown that hydroalcoholic extracts of the plant reduce the production of inflammatory mediators, such as prostaglandins and leukotrienes, and exhibit antibacterial activity against some species of Staphylococcus aureus and Escherichia coli [5,18]. In addition, diuretic effects have been correlated with the presence of coumarins and flavonoids, being useful in the management of renal and cardiovascular diseases [21,22]. Recent research also suggests a potential neuroprotective role of lovage extracts, by reducing oxidative stress and protecting neurons against degeneration associated with neurodegenerative diseases [20].

In addition to its use in the form of extracts, capsules or teas, lovage is of particular interest to the pharmaceutical and cosmetic industries due to its high content of bioactive compounds. In particular, the formulation of carbopol-based hydrogels represents a modern strategy for the valorization of hydroalcoholic extracts from leaves, roots and the whole plant. Hydrogels are colloidal systems with superior rheological properties, capable of incorporating plant extracts and gradually releasing them, maximizing the bioavailability of active compounds.

For example, recent studies on carbopol-based hydrogels have shown that they can improve the stability and antioxidant activity of plant extracts [23,24]. By integrating lovage extracts, these hydrogels could be used in the following ways:

-

As dermato-cosmetic products for skin protection against oxidative stress, reducing inflammation and preventing premature aging;

-

As pharmaceutical formulations for wound treatment due to the antimicrobial and anti-inflammatory properties of flavonoids and phenolic acids;

-

As controlled release systems for topical administration of extracts with therapeutic action on skin or joint conditions.

The present study contributes to the expansion of knowledge of Levisticum officinale through a detailed investigation of alcoholic extracts and their integration into carbopol-based hydrogels with therapeutic and cosmetic potential. The novelty of the research consists in the following approaches:

-

Comparison of the phytochemical profile of leaf, root and whole-plant extracts, providing comparative data on the distribution of bioactive compounds;

-

Use of hydrogels as a release matrix for lovage extracts, optimizing their bioavailability and opening new directions for pharmaceutical and cosmetic applicability;

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Correlation of phytochemical composition with antioxidant activity, highlighting the potential of lovage as a source of new active ingredients for the pharmaceutical and food supplement industries.

Through these innovative approaches, the proposed study will contribute to the optimization of the use of Levisticum officinale in pharmaceutical and cosmetic products, opening new perspectives for the development of effective formulations with extended therapeutic applicability.

2. Materials and Methods

The present study aims to compare the hydroalcoholic extracts (70% ethanol) obtained from the root, leaves and whole plant of Levisticum officinale by determining the total content of phenolic compounds using the Folin–Ciocâlteu method, characterizing the polyphenolic profile by HPLC-DAD and evaluating the antioxidant activity by ABTS and DPPH methods. In addition, the extracts obtained were incorporated into carbopol-based hydrogels, which were characterized from a rheological and physicochemical point of view, and their antioxidant activity was evaluated to determine their stability and efficiency as potential pharmaceutical or dermato-cosmetic forms with antioxidant action.

2.1. Preparation of Plant Extracts

The working samples included the root, stem and leaves of the species Levisticum officinale L., harvested from the Lunca Mare area, Prahova County (coordinates: 45°11′57″ N 25°44′41″ E), Romania (Figure 1).

The identification of Levisticum officinale L. was carried out with the support of the Herbarium of the Discipline of Pharmacognosy, Faculty of Pharmacy, “Ovidius” University Constanța. A reference specimen was deposited as an identification voucher in the Department of Analysis and Quality Control of Medicines within the same faculty, ensuring botanical accuracy. The collection of the plant material took place in July 2024. Additionally, several specimens of the harvested species were preserved in the Exiccata collection of the Discipline of Pharmacognosy, serving as a permanent record of the collected material.

Fresh plant material was cleaned by washing under cold running water, sorted, blotted with absorbent paper to remove moisture and then air-dried in well-ventilated areas at temperatures of 20–30 °C to prevent the degradation of volatile substances. The dried samples were ground by trituration in a mortar.

Choice of solvent: Seventy percent ethyl alcohol (v/v) was used, which is an effective solvent for the extraction of phenolic compounds, flavonoids, alkaloids and other bioactive substances from plants. Alcoholic extracts (70% ethanol) are widely used in phytotherapy and research due to their efficiency in the extraction of bioactive compounds from plants, including flavonoids, phenolic acids and coumarins. The choice of 70% ethanol as a solvent was based on its ability to extract both polar and semi-polar compounds, having an optimal polarity for most secondary metabolites of pharmaceutical interest.

Calculation of the solvent volume: To prepare a 15% (w/v) alcoholic extract, meaning 15 g of plant material per 100 mL of solvent, the required amount of alcohol was determined accordingly. The plant materials used in this study (leaves, roots and the whole Levisticum officinale plant) were processed, and the appropriate quantities of plant material and solvent were measured to obtain 15% extracts using 70° ethanol.

The shredded plant material was left to macerate for 14 days, in a cool place and away from light, according to the method in the Romanian Pharmacopoeia, 10th Edition [25]. During this period, the samples were shaken daily for 30 min to ensure optimal extraction of the bioactive compounds. After 14 days, the hydroalcoholic extracts were filtered at normal pressure, using Whatman quantitative filter paper with a blue band, to remove impurities and obtain clear solutions (Figure 2).

2.2. Determination of Total Phenolic Content (Folin–Ciocâlteu)

Reagents and materials: Folin–Ciocâlteu reagent purchased from Sigma-Aldrich (St. Louis, MO, USA), Na₂CO₃ 7.5% (m/v).

Reference standard: gallic acid.

UV-Vis spectrophotometer V-630 Jasco, Heckmondwike, UK.

Analytical procedure: A volume of 100 µL of extract was pipetted into an Eppendorf tube. Then, 500 µL of Folin–Ciocâlteu reagent, previously diluted 1:10 with distilled water, was added. After 5 min, 400 µL of 7.5% Na₂CO₃ was introduced. The mixture was incubated for 30 min at 25 °C in the dark. The absorbance was then measured at 765 nm against a control solution prepared with solvent (70% ethanol) and Folin–Ciocâlteu reagent without extract. A standard solution of gallic acid in ethanol at a known concentration (100 mg/L) was prepared and used for the construction of the calibration curve. Calibration curve equation: y = 0.0191x + 0.1265, R² = 0.9922. The total content of phenolic compounds is expressed in mg of gallic acid equivalents/g extract (mg GAE/g) [26].

2.3. Determination of Phenolic Acid Content (Folin–Ciocâlteu)

Preparation of solutions for analysis:

A dilute solution of lovage alcoholic extract (1:10) in 70% ethanol was prepared to obtain a concentration suitable for absorbance measurement. The Folin–Ciocâlteu solution (20%) was prepared by diluting the commercially available Folin–Ciocâlteu reagent (typically supplied at a concentration of 1:1 or 1:2) with distilled water. A 20% sodium carbonate solution was prepared by dissolving 20 g of sodium carbonate in 100 mL of distilled water.

Analytical procedure: A volume of 1 mL of the diluted extract was mixed with 5 mL of 20% Folin–Ciocâlteu reagent and allowed to react for 3–5 min. Then, 4 mL of 20% sodium carbonate solution was added, mixed thoroughly, and left in the dark for 30 min. The absorbance was measured at 765 nm against a control solution prepared with solvent (70% ethanol) and Folin–Ciocâlteu reagent without extract. To determine the concentration of phenolic acids in the lovage extract, a standard calibration curve was generated using a gallic acid standard. Gallic acid solutions with varying concentrations (10–100 µg/mL) were prepared, and their absorbance was measured at 765 nm. Calibration curve equation: y = 0.155 x + 0.0019, R² = 0.9982.

2.4. Determination of Total Flavonoids by the Colorimetric Method with AlCl₃

This colorimetric method is used to quantify flavonoids in alcoholic extracts of Levisticum officinale (loving plant) by forming colored complexes between flavonoids and aluminum chloride (AlCl₃). The complex formed has a maximum absorbance in the region of 415 nm, which can be measured spectrophotometrically to determine the concentration of flavonoids [27].

Preparation of solutions for analysis:

A dilute solution of alcoholic extract of the lovage plant (1:10) was prepared in 70% ethanol to obtain a concentration suitable for measuring the absorbance.

Aluminum chloride solution (AlCl₃): a 2% solution of AlCl₃ was prepared in 70% ethanol.

Analytical procedure: A total of 1 mL of each of the two solutions was mixed, allowed to react for 10–15 min at room temperature, and then 2 mL of 70% ethanol was added. The absorbance at 415 nm was measured against a solvent-prepared control (2% AlCl₃ solution in 70% ethanol). The flavonoid concentration of the lovage extract was determined based on a quercetin standard. Quercetin solutions with known concentrations (10–100 µg/mL) were used to determine the calibration curve equation. Calibration curve equation: y = 0.0236x + 0.1426, R² = 0.9994.

2.5. Determination of Total Condensed Tannins Content—Vanillin-HCl Method

Condensed tannins (also known as proanthocyanidins) are polyphenols that can be quantified by spectrophotometric methods, using the vanillin-HCl reagent or the butanol-HCl method. These methods are based on the interaction of tannins with specific chemical reagents, resulting in the formation of spectrophotometrically measurable colored complexes. In this study, the vanillin-HCl method was used, as it provides accurate results and is widely used for the analysis of condensed tannins in plant materials [28].

Preparation of solutions for analysis:

A 1% vanillin solution was prepared by dissolving 500 mg of vanillin in 50 mL of methanol. An 8% HCl solution was obtained by diluting 8 mL of concentrated HCl to 100 mL with methanol. The catechin standard solution was prepared by dissolving catechin in methanol to obtain a stock solution of 1 mg/mL, which was further diluted to concentrations ranging between 50 and 500 µg/mL for the calibration curve.

Analytical procedure: A volume of 0.5 mL of the alcoholic extract was pipetted into a test tube, followed by the addition of 2.5 mL of 1% vanillin solution and 2.5 mL of 8% HCl solution. The mixture was homogenized and left to react at room temperature for 20 min in the dark. The absorbance was then measured at 500 nm using a UV-Vis spectrophotometer against a blank consisting of extract without vanillin-HCl. The calibration curve was constructed using standard catechin solutions, and the concentration of condensed tannins was expressed as mg catechin equivalents (CE) per gram of extract (mg CE/g). Calibration curve equation: y = 0.265 x + 0.0046, R² = 0.9988.

2.6. Polyphenol Profile Analysis by HPLC-DAD

Reagents and solvents:

MS grade HPLC solvents (acetonitrile, acetic acid 0.1%).

Reference standard: quercetin, kaempferol, apigenin, luteolin, rutin, chlorogenic acid, ferulic acid, caffeic acid and vanillic acid.

Chromatographic conditions:

Apparatus: Agilent 1200 HPLC, with quaternary pump, DAD, thermostat, degassing system and autosampler (Agilent Technologies, Santa Clara, CA, USA).

Column: C18, 150 mm L. × 4.6 mm i.d. × 5 μm d.p.

Mobile phase: solvent A: 0.1% acetic acid solution; solvent B: 99.9% acetonitrile.

Eluent gradient: 0–5 min → 5% B; 5–15 min → 5–30% B; 15–25 min → 30–50% B; 25–30 min → 50–70% B; 30–35 min → 70% B.

Flow rate: a total of 1 mL/min; temperature: 31 °C; detection: DAD at 280 nm and 315 nm.

Injection volume: a total of 10 µL; analysis time: 40 min.

Table 1. Retention times for the reference substances.

No. Ctr.	Phenolic Compound	Retention Time ± SD
1.	Quercetin	17.566 ± 0.017
2.	Kaempferol	31.826 ± 0.008
3.	Apigenin	34.346 ± 0.015
4.	Luteolin	23.621 ± 0.036
5.	Rutin	13.325 ± 0.051
6.	Chlorogenic acid	3.503 ± 0.015
7.	Ferulic acid	8.106 ± 0.058
8.	Caffeic acid	4.771 ± 0.036
9.	Vanillic acid	9.732 ± 0.051

SD—standard deviation.

shows the retention times for the reference substances.

The identification and quantitative determination of the active principles in the solutions to be analyzed was performed by comparing the retention times and purity of the peaks of the standard compounds used with the corresponding ones on the chromatograms of the solutions to be analyzed. The reproducibility of the method was assessed by the square of the correlation coefficients (

Table 2. Regression equations of reference standard compounds.

Reference Chemical Compound	n a	Regression Equation b	Working Range (μg/mL)	Regression Coefficient R	Correlation Coefficient R2	LOD	LOQ
Quercetin	7	y = 24.221x − 17.85	2–200	0.9997	0.9995	0.2667	1.3025
Kaempferol	7	y = 17.882x + 13.561	2–200	0.9998	0.9996	0.3226	1.9741
Apigenin	7	y = 23.229x + 7.703	2–200	0.9997	0.9997	1.0733	3.3024
Luteolin	7	y = 4.335x − 1.782	2–200	0.9997	0.9999	0.4899	0.8972
Rutin	7	y = 5.279x – 3.336	2–200	0.9995	0.9997	0.5292	1.7503
Chlorogenic acid	7	y = 8.744x − 3.811	2–200	0.9995	0.9996	0.4292	1.2944
Ferulic acid	7	y = 6.773x − 0.890	2–200	0.9997	0.9995	0.8478	1.4779
Caffeic acid	7	y = 11.676x + 4.226	2–200	0.9996	0.9995	0.5865	1.2766
Vanillic acid	7	y = 17.433x − 2.178	2–200	0.9996	0.9989	0.7824	1.2989

^a n: number of measurements. ^b y: peak area; x: concentration of standard or reference compound (μg/mL).

). The equations of the calibration lines of the standard compounds are presented in Table 2.

2.7. Determination of Antioxidant Activity by ABTS

A stock solution of ABTS (7 mM) was prepared by dissolving 38.4 mg ABTS in 10 mL of double-distilled water. A solution of potassium persulfate (K₂S₂O₈, 2.45 mM) was prepared by dissolving 6.6 mg K₂S₂O₈ in 10 mL of double-distilled water. The ABTS and K₂S₂O₈ solutions were mixed in a 1:1 ratio and left in the dark, at room temperature, for 12–16 h for the formation of the ABTS•⁺ radical. The ABTS•⁺ radical solution was diluted with phosphate buffer (pH 7.4) until an absorbance of 0.70 ± 0.02 at 734 nm (measured spectrophotometrically) was obtained [29].

Trolox (antioxidant standard) was used in varying concentrations (0–500 µM). Trolox was dissolved in methanol to obtain solutions with precise concentrations. A Trolox calibration curve was constructed by measuring the absorbance at 734 nm. Calibration curve equation: y = 77.572x + 21.319, R² = 0.9965.

Appropriate dilutions of the alcoholic extracts (1:10) were prepared. A volume of 200 µL of the diluted extract was mixed with 2 mL of ABTS•⁺ solution. The absorbance was measured at 734 nm after 6 min. The results were expressed in µmol Trolox equivalents per gram of extract (TEAC).

2.8. Determination of Antioxidant Activity by DPPH

A solution of DPPH in methanol was prepared to achieve an absorbance of approximately 1.00 at 517 nm. Trolox was used as an antioxidant standard, with varying concentrations ranging from 10 to 500 µM. Trolox was dissolved in methanol to obtain precise solutions. A calibration curve was constructed by measuring the absorbance at 517 nm [29]. Calibration curve equation: y = 54.691x + 1.744, R² = 0.9977.

Appropriate dilutions of the alcoholic extracts (1:10) were prepared. A volume of 100 µL of the diluted extract was mixed with 3.9 mL of DPPH solution. The absorbance was measured at 517 nm after 30 min. The IC₅₀ value, representing the concentration required to reduce the DPPH radical by 50%, was calculated.

2.9. Formulation and Characterization of Hydrogels with Hydroalcoholic Extracts of Lovage

Three hydrogel formulas based on 2% Carbopol 940 were made, in which the hydroalcoholic extracts were included in a concentration of 5%: Formula 1 with hydroalcoholic extract of the roots, Formula 2 with hydroalcoholic extract of the whole plant and Formula 3 with hydroalcoholic extract of the leaves.

Investigations carried out on hydrogels formulated with hydroalcoholic extracts from Levisticum officinale included the evaluation of essential parameters such as stability, antioxidant activity and rheological characteristics.

Stability assessment: The stability of the hydrogels was monitored over a period of time (60 days), assessing changes in texture and visual appearance. Thermal and pH stability tests were performed to ensure that the gel maintained its characteristics over time, without undergoing significant changes. The pH was analyzed using a multiparameter pH meter from Hanna Instruments (Hanna Instruments, Woonsocket, RI, USA).

Antioxidant activity evaluation: the hydrogels were tested for their antioxidant capacity using the ABTS and DPPH methods, to evaluate the potential to neutralize free radicals.

Rheological characteristics analysis: The rheological properties of the hydrogels were determined by viscosity measurements, which indicated the consistency and ability of the gel to spread on the skin or other surfaces. These tests are essential to understand the behavior of the gel in dermatological applications. The rheological properties of the hydrogels were determined by measuring the viscosity at different rotational speeds (ω) ranging from 4 to 200 rpm. The evaluations were performed using rotational viscometer ST-2020 R, manufactured by Laboquimia (Logroño, La Rioja, Spain), which allows viscosity measurements at 10 s intervals for each determination. To ensure the accuracy of the measurements according to the viscosity range of the analyzed samples, R5 and R6 type needles were used. These instruments allow the calculation of the shear rate (D) in relation to the rotational speed (ω), thus obtaining a detailed profile of the rheological behavior of the hydrogels under variable conditions of mechanical stress. Rheological evaluations are essential for understanding the behavior of the material in pharmaceutical applications, such as application to the skin and control of the release of bioactive substances. The stretchability was evaluated using the Ojeda Arbussa method [30].

All chemical reagents used in the experiments were of analytical grade and were purchased from Merck KGaA (Darmstadt, Germany), thus ensuring the accuracy and reproducibility of the analyses. The analytical determinations were performed under controlled conditions, and the results obtained were expressed as mean values, calculated based on measurements performed in triplicate, to ensure the precision and reliability of the experimental data.

2.10. Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software version 9.5.1. (GraphPad Software, San Diego, CA, USA). Significant differences between samples were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, with a significance threshold set at p < 0.05 [31,32].

3. Results

3.1. Total Content of Polyphenolic Compounds

Figure 3 illustrates the polyphenolic compound content in the analyzed hydroalcoholic extracts. The results indicate that the extract from lovage leaves exhibited the highest concentration of polyphenols (20.84 ± 1.18 mg GAE/g), suggesting a richer presence of flavonoids and phenolic acids. In contrast, the extract from the whole plant contained the lowest amount of polyphenols (18.26 ± 1.61 mg GAE/g), highlighting the variation in bioactive compound distribution within different plant parts. These findings emphasize the superior antioxidant potential of leaf extracts and their possible applications in pharmaceutical and cosmetic formulations.

3.2. Phenolic Acid Content

Analysis of the phenolic acid content revealed the highest content in the hydroalcoholic extract of the whole plant (18.26 ± 1.65 mg GAE/g) and the lowest content in the alcoholic extract of the leaves (8.08 ± 0.33 mg GAE/g) (Figure 4).

3.3. Total Flavone Content

The highest flavone content was recorded in the hydroalcoholic extract of lovage leaves (11.39 ± 1.48 mg QE/g), and the lowest in the alcoholic extract of the root (6.6 ± 0.25 mg QE/g) (Figure 5).

3.4. Condensed Tannin Content

The highest content of condensed tannins (1.98 ± 0.55 mg CE/g) was presented in the hydroalcoholic extract of lovage leaves and the lowest in the alcoholic extract of the root (0.92 ± 0.12 mg CE/g) (Figure 6).

3.5. Phenolic Profile of Hydroalcoholic Lovage Extracts

Figure 7, Figure 8 and Figure 9 provide a detailed overview of the flavonoid and phenolic acid composition in hydroalcoholic lovage extracts. The analysis highlights a higher concentration of flavonoids such as quercetin, kaempferol and rutin in the leaves, while the roots exhibit elevated levels of phenolic acids, including ferulic, caffeic and vanillic acids (

Table 3. Bioactive compounds analyzed by HPLC from hydroalcoholic extracts.

Bioactive Compounds	Hydroalcoholic Extract from Leaves (mg/g)	Hydroalcoholic Extract from Root (mg/g)	Hydroalcoholic Extract from Whole Plant (mg/g)
Quercetin	3.32 ± 0.25	1.89 ± 0.66	2.85 ± 0.18
Kaempferol	1.84 ± 0.63	1.05 ± 0.28	1.56 ± 0.44
Apigenin	1.91 ± 0.44	1.13 ± 0.12	1.62 ± 0.48
Luteolin	2.12 ± 0.19	1.38 ± 0.72	1.73 ± 0.55
Rutin	4.38 ± 0.84	2.87 ± 0.26	3.02 ± 0.66
Chlorogenic acid	2.81 ± 0.26	2.35 ± 0.25	2.12 ± 0.12
Ferulic acid	1.28 ± 0.25	3.86 ± 0.37	2.54 ± 0.48
Caffeic acid	1.91 ± 0.42	3.28 ± 0.28	2.37 ± 0.14
Vanillic acid	1.08 ± 0.33	2.53 ± 0.76	1.71 ± 0.32

). These findings emphasize the distinct phytochemical profiles of different plant organs, suggesting their potential for targeted pharmaceutical or nutraceutical applications.

3.6. Antioxidant Activity of Hydroalcoholic Extracts of Lovage

The determination of the antioxidant activity of hydroalcoholic extracts of lovage by the ABTS and DPPH methods revealed significant variations depending on the part of the plant used (

Table 4. Antioxidant activity of hydroalcoholic extracts of lovage by the ABTS method.

Sample	ABTS Inhibition at 100 µg/mL	IC50	Antioxidant Activity
Hydroalcoholic extract from leaves	84.1 ± 1.5%	0.064 mg/mL	276.2 ± 3.4 µmol TE/g
Hydroalcoholic extract from the root	68.8 ± 2.0%	0.098 mg/mL	182.5 ± 4.1 µmol TE/g
Hydroalcoholic extract from the whole plant	76.7 ± 1.8%	0.083 mg/mL	228.7 ± 4.3 µmol TE/g

and

Table 5. Antioxidant activity of hydroalcoholic extracts of lovage by the DPPH method.

Sample	DPPH Inhibition at 100 µg/mL	IC50	Antioxidant Activity
Hydroalcoholic extract from leaves	78.2 ± 1.3%	0.075 mg/mL	246.4 ± 3.6 µmol TE/g
Hydroalcoholic extract from the root	65.5 ± 2.1%	0.112 mg/mL	159.8 ± 4.8 µmol TE/g
Hydroalcoholic extract from the whole plant	69.8 ± 1.7%	0.098 mg/mL	187.3 ± 4.1 µmol TE/g

).

The hydroalcoholic extract of the leaves presented the highest antioxidant activity, with values of 276.2 ± 3.4 µmol TE/g by the ABTS method (Table 4) and 246.4 ± 3.6 µmol TE/g by the DPPH method (Table 5). These results can be correlated with the high content of flavonoids and phenolic acids identified in the extract, especially quercetin, rutin, luteolin and chlorogenic acid, known for their ability to donate electrons and neutralize reactive oxygen species.

The correlations between individual phenolic compounds and antioxidant activity (evaluated using the ABTS and DPPH methods) are presented in Figure 10 (for leaves), Figure 11 (for roots) and Figure 12 (for whole plant) in the form of color-coded heatmaps. These visual representations allow the identification of significant associations between various bioactive compounds and the overall antioxidant capacity.

For the leaves, significant correlations were observed between total phenolic compounds and ABTS antioxidant activity (r = 0.90, p < 0.01) and total phenolic compounds and DPPH antioxidant activity (r = 0.88, p < 0.01), confirming the strong contribution of polyphenols to antioxidant capacity. Among individual compounds, quercetin (r = 0.96, p < 0.001), rutin (r = 0.89, p < 0.001) and chlorogenic acid (r = 0.82, p < 0.01) showed strong correlations with antioxidant activity.

For the root extract, correlations were lower than in leaves, but still significant. Total phenolic compounds were correlated with ABTS antioxidant activity (r = 0.75, p < 0.05) and with DPPH antioxidant activity (r = 0.72, p < 0.05). The main contributors to antioxidant activity were ferulic acid (r = 0.80, p < 0.01), caffeic acid (r = 0.85, p < 0.01) and vanillic acid (r = 0.75, p < 0.05).

For the whole plant, a global correlation analysis showed that Total Phenolic Compounds had a strong positive correlation with antioxidant activity ABTS (r = 0.94, p < 0.001) and antioxidant activity DPPH (r = 0.93, p < 0.001), reinforcing the role of polyphenols in antioxidant capacity. Additionally, chlorogenic acid (r = 0.90, p < 0.001), rutin (r = 0.88, p < 0.001), and quercetin (r = 0.95, p < 0.001) exhibited strong relationships with antioxidant activity, confirming their role as key bioactive compounds.

3.7. Characteristics of Hydrogels with Hydroalcoholic Extracts of Lovage

Hydrogels with alcoholic extracts were prepared according to the formulas presented in

Table 6. Formulas of hydrogels with hydroalcoholic extracts of lovage.

Ingredients	Formula 1	Formula 2	Formula 3
Carbopol 940	2 g	2 g	2 g
Glycerin	5 g	5 g	5 g
Triethanolamine	q.s.	q.s.	q.s.
Hydroalcoholic extract of leaves	-	-	5 g
Hydroalcoholic extract of roots	5 g	-	-
Hydroalcoholic extract of the whole plant	-	5 g	-
Distilled water	until 100 g	until 100 g	until 100 g

q.s. = quantum satis.

.

The hydrogels (Figure 13) with hydroalcoholic extract of Levisticum officinale were prepared by dispersing 2 g of Carbopol 940 in distilled water and glycerin. The mixture was stirred vigorously and allowed to hydrate for 30–60 min. Subsequently, 5% hydroalcoholic extracts of the lovage plant were added to the Carbopol suspension, with continuous stirring to ensure uniform distribution. Once a homogeneous mixture was obtained, the pH of the gel was adjusted to a value of 5.0–5.5 using a triethanolamine (TEA) solution. Following preparation, the gel was stored under refrigeration for 24 h to allow stabilization and ensure optimal development of its properties.

The results of the control tests carried out on hydrogels with hydroalcoholic extracts of lovage are presented in

Table 7. Characteristics of hydrogels with hydroalcoholic extracts of lovage.

Characteristics	Formula 1	Formula 2	Formula 3
Initial organoleptic evaluation	appearance: homogeneous; colour: pale yellow; smell: specific	appearance: homogeneous; colour: chartreuse; smell: specific	appearance: homogeneous; colour: green; smell: specific
Organoleptic evaluation after 30 days	maintenance of the original characteristics unchanged	maintenance of the original characteristics unchanged	maintenance of the original characteristics unchanged
Organoleptic evaluation after 60 days	maintenance of the original characteristics unchanged	maintenance of the original characteristics unchanged	maintenance of the original characteristics unchanged
pH—initial	4.5–5.0	4.7–5.2	5.0–5.5
pH—after 30 days	4.5–5.0	4.7–5.2	5.0–5.5
pH—after 60 days	5.0–5.2	5.3–5.5	5.5–5.8
Viscosity—initial	824.14 ± 0.35 Pa·s	812.18 ± 0.25 Pa·s	780.35 ± 0.33 Pa·s
Viscosity—after 30 days	808.26 ± 0.65 Pa·s	796.33 ± 0.66 Pa·s	766.08 ± 0.45 Pa·s
Viscosity—after 60 days	782.32 ± 0.53 Pa·s	774.24 ± 0.50 Pa·s	758.11 ± 0.25 Pa·s

.

The viscosity of the hydrogels remained constant throughout the testing, indicating an adequate interaction between the polymer used (Carbopol 940) and the hydroalcoholic extracts, without signs of phase separation or syneresis. This property is essential for maintaining optimal rheological characteristics, influencing the applicability of the product and its ability to retain active substances.

In Figure 14, Figure 15 and Figure 16, the evolutions of the hydrogels’ surface area as a function of the applied weight are represented—an essential parameter for evaluating the mechanical response and deformation capacity. It is observed that the hydrogel with hydroalcoholic extract of lovage leaves presents the highest surface area, which can be correlated with a higher content of flavonoids and condensed tannins, which are compounds that can influence the structure of the polymer matrix and its interaction with water.

In contrast, the hydrogel with root extract presented the lowest surface area, which can be associated with a higher content of phenolic acids, which can influence the hydrogel network through interactions with the polymer, reducing its flexibility. The hydrogel with whole-plant extract recorded intermediate values, confirming that this formulation offers a balance between rigidity and extensibility.

These data suggest that the phytochemical profile of the extracts directly influences the mechanical properties of the hydrogels, which may be an essential aspect in choosing the optimal formula for pharmaceutical or dermato-cosmetic applications.

The rheograms and flow curves for hydrogels with alcoholic extracts of lovage are presented in Figure 17, Figure 18 and Figure 19. The rheograms and flow curves obtained for hydrogels with alcoholic extracts of lovage provide essential information about the rheological behavior of the analyzed formulations. The results indicate a non-Newtonian behavior of pseudoplastic type, characterized by a decrease in viscosity with increasing shear force. This phenomenon is typical for Carbopol 940-based hydrogels and is favorable for topical application, as it allows easy spreading on the skin and a return to the initial structure after the cessation of the deformation force.

The results presented in

Table 8. Antioxidant activity of hydrogels with hydroalcoholic extracts of lovage.

Sample	DPPH Inhibition at 100 µg/mL	ABTS Inhibition at 100 µg/mL
Hydrogel with hydroalcoholic extract from leaves	77.6 ± 2.4%	69.4 ± 1.6%
Hydrogel with hydroalcoholic extract from roots	63.8 ± 1.2%	61.2 ± 2.2%
Hydrogel with hydroalcoholic extract from the whole plant	69.2 ± 1.6%	65.8 ± 1.4%

highlight the antioxidant activity of hydrogels formulated with alcoholic extracts of lovage, analyzed by the DPPH and ABTS methods. It is noted that the hydrogel with lovage leaf extract presents the highest free radical neutralization capacities, both in the case of the ABTS method (69.4%) and the DPPH method (77.6%). These results are consistent with the high content of flavonoids identified in the leaf extract, especially quercetin, rutin and apigenin, which are compounds recognized for their strong antioxidant effects.

In contrast, the hydrogel with alcoholic extract of the root presents the lowest antioxidant activity. This result can be correlated with the phytochemical profile of the root, which, although rich in phenolic acids (ferulic acid, caffeic acid, vanillic acid), does not contain the same high levels of flavonoids as the leaf.

The hydrogel formulated with an extract from the whole plant exhibits intermediate values of antioxidant activity, reflecting a balance between flavonoids and phenolic acids in its composition.

These results confirm that the antioxidant activity of hydrogels is directly influenced by the phytochemical composition of the extracts used. Formulations with leaf extract are the most promising for protection against oxidative stress, being suitable for dermatological applications with antioxidant and anti-aging roles, while hydrogels with root extract may be more suitable for anti-inflammatory and antimicrobial effects.

4. Discussion

The results indicate a differentiated distribution of polyphenolic compounds depending on the part of the plant used for extraction (Figure 3), which can be correlated with the metabolic variability of Levisticum officinale. Lovage leaves have a significantly higher content of phenolic compounds compared to the roots, which can be explained by the physiological role of these secondary metabolites in protection against oxidative stress and environmental factors, such as UV radiation or pathogens [7]. These aspects are supported by data from the specialized literature, which indicate a preferential distribution of phenolic compounds in the aerial organs of many species of the Apiaceae family, including Foeniculum vulgare (fennel) and Petroselinum crispum (parsley) [33,34].

In addition, the chemical composition of the extracts is also influenced by the differential solubility of these metabolites in the solvent used, with 70% ethanol being known for its efficiency in the extraction of flavonoids and phenolic acids, but also for its variable selectivity towards the different classes of polyphenols. This observation may also explain the significant differences between the extracts analyzed, suggesting that the leaves represent the optimal source for obtaining extracts with high antioxidant potential [35].

Experimental data indicate a differentiated distribution of phenolic acids depending on the part of the plant used for extraction, highlighting a majority accumulation of them in the root (Figure 4). This observation is consistent with the specialized literature, which suggests that, in many species of the Apiaceae family, the roots constitute an important reservoir for secondary metabolites with a role in the plant’s defense against pathogens and abiotic stress [36]. For example, Angelica archangelica and Daucus carota have been reported to accumulate phenolic acids, coumarins and polyacetylenes in their root systems, which are compounds known for their antimicrobial and antifungal properties [37].

Phenolic acids are compounds with an important antioxidant, anti-inflammatory and antimicrobial role [38], and their significant presence in the root can be correlated with protective functions against soil microorganisms and other subterranean stress factors. For example, caffeic, ferulic and chlorogenic acids, frequently found in the roots of species in this family, are involved in the plant’s defense mechanisms by inhibiting the development of pathogenic microorganisms and by strengthening the structure of the cell wall [39].

On the other hand, the lower concentration of phenolic acids in extracts obtained from leaves can be explained by their preferential distribution towards other classes of metabolites, such as flavonoids, which have been previously identified in higher concentrations in this part of the plant. At the same time, the presence of a lower content of phenolic acids in leaves can also be influenced by the metabolic rate of these compounds, given that in plant leaves, many of these substances are transformed into more complex derivatives and adapted for protection against UV radiation [40].

These results suggest that lovage root can be considered a valuable source of phenolic acids, with potential for use in the development of pharmaceutical or cosmetic products intended to prevent oxidative stress and inflammation. In addition, the differences observed between the analyzed extracts highlight the importance of choosing the appropriate plant part for obtaining specific extracts with well-defined therapeutic applications.

The data obtained for the flavone content highlight a predominant accumulation of these compounds in lovage leaves, which is consistent with data reported in the literature for other species of the Apiaceae family. For instance, Apium graveolens (celery) and Coriandrum sativum (coriander) have been shown to accumulate significant amounts of flavones, particularly apigenin and luteolin derivatives, in their leaves, contributing to their antioxidant properties [41]. Similarly, studies on Foeniculum vulgare (fennel) have reported high levels of flavonoids in aerial parts, reinforcing the preferential distribution of these bioactive compounds in Apiaceae leaves [34]. Flavones, including compounds such as apigenin and luteolin, are secondary metabolites involved in plant defense mechanisms against UV radiation and pathogen attack, thus explaining their high concentration in leaves, where they are directly exposed to environmental factors [7,42].

Previous studies have demonstrated that flavones exhibit a wide range of biological activities, including antioxidant, anti-inflammatory and neuroprotective effects [43,44]. The high concentration of flavones in the leaf extracts supports their potential for use in phytotherapeutic or cosmetic products with antioxidant and anti-aging roles. In addition, apigenin and luteolin are recognized for their ability to interact with various inflammatory mediators, inhibiting pro-inflammatory signaling pathways and contributing to cellular protection [45,46].

The lower content of flavones in the hydroalcoholic extract of the root suggests a differentiated distribution of polyphenolic compounds in the plant, with the root accumulating mainly other classes of metabolites, such as coumarins and phenolic acids. This aspect can be explained by physiological differences between the plant organs, with the root having an essential role in nutrient absorption and protection against soil pathogens, which determines its distinct chemical composition [47].

The results obtained regarding the content of condensed tannins indicate a significantly higher accumulation of these compounds in the hydroalcoholic extract of lovage leaves, compared to the extracts obtained from the root and the whole plant. This distribution is justified by the ecological role of tannins, which act as defense agents against herbivores and pathogens, being present in higher quantities in exposed organs, such as the leaves [48].

Condensed tannins, also known as proanthocyanidins, are oligomers and polymers of flavan-3-ols, with high antioxidant potential and an ability to interact with proteins, thus influencing the bioavailability and biological activity of other compounds. These properties give them great importance in the pharmaceutical and nutritional fields, with tannins being associated with beneficial effects on cardiovascular, digestive and metabolic health [49].

The low concentration of condensed tannins in root extracts suggests a differentiated distribution of phenolic compounds in the plant, with the root being richer in phenolic acids, according to previous results. This aspect is also supported by the specialized literature, which highlights a greater accumulation of tannins in the aerial parts of plants, where they contribute to the regulation of physiological processes and protection against abiotic stress [50].

The presence of a high content of condensed tannins in leaf extracts suggests an important potential for their use in food supplements and products with antioxidant and gastroprotective properties [48]. These extracts can also be used in the formulation of cosmetic or pharmaceutical products intended for skin protection, due to the ability of tannins to stabilize collagen and reduce oxidative stress at the cellular level [51].

Distinctively, the leaf hydroalcoholic extract presented the highest concentrations of essential flavonoids (Table 3), such as quercetin, kaempferol, apigenin, luteolin and rutin. These flavonoids are well known for their antioxidant, anti-inflammatory and neuroprotective activity, having an important role in neutralizing reactive oxygen species and protecting cells against oxidative stress [52]. The high content of these compounds in the leaves confirms that the aerial parts of the plant are rich in secondary metabolites with significant biological activity, which makes them valuable in the pharmaceutical and food fields.

Also, the hydroalcoholic extract of the leaves recorded a high concentration of chlorogenic acid, a phenolic compound known for its antioxidant, antimicrobial and modulatory effects on glucose metabolism. This observation is consistent with the specialized literature, which reports the accumulation of these phenolic acids in the leaves of plants with medicinal properties [2,5].

On the other hand, the hydroalcoholic extract of the root presented the highest concentrations of ferulic acid, caffeic acid and vanillic acid, which suggests a differentiated distribution of phenolic compounds in the plant. Phenolic acids in the root are recognized for their antioxidant activity and beneficial effects on cardiovascular and liver health [7]. Ferulic acid, for example, plays an important role in stabilizing cell membranes and protecting against oxidative stress, while caffeic acid and vanillic acid are involved in anti-inflammatory and neuroprotective processes [53,54].

These results highlight the importance of selecting the right plant organ to obtain extracts with specific biological activity. Thus, leaf extracts may be preferred for antioxidant and anti-inflammatory applications, while root extracts may be exploited for their cardiovascular and metabolic protective effects. The high antioxidant activity of the leaf extract supports its use in formulations intended for protection against oxidative stress, including in cosmetic, pharmaceutical or food applications.

In contrast, the hydroalcoholic root extract recorded the lowest antioxidant activity values by both methods, which can be attributed to a lower flavonoid content and the predominant presence of phenolic acids such as ferulic acid, caffeic acid and vanillic acid, which, although possessing antioxidant activity, are less efficient than flavonoids in free radical scavenging reactions.

The differences observed between the antioxidant activity of the extracts can also be explained by the chemical structure of the majority compounds in each sample, with leaf flavonoids having a higher number of free hydroxyl groups, which are essential in neutralization of radicals. These data confirm the importance of choosing the appropriate plant organ depending on the desired application, with leaf extracts being more suitable for formulations with a strong antioxidant effect, while root extracts can be used for other biological properties, such as anti-inflammatory or hepatoprotective effects.

The hydroalcoholic extract obtained from the whole plant showed intermediate values of antioxidant activity, lying between those of the leaf and root extracts, indicating a combined contribution of bioactive compounds from all parts of the plant.

This result reflects a balance between flavonoids and phenolic acids present in the leaves and roots, providing a moderate antioxidant profile. Its total phenolic content, higher than that of the root extract but lower than that of the leaves, suggests that the whole-plant extract may be an optimal option for formulations requiring a broad spectrum of biological activities.

Also, the use of the whole plant in the extraction process may be advantageous from an economic and technological point of view, reducing the need to separate the different plant parts and allowing the obtaining of a product with a diversified biochemical profile. This type of extract could be useful in pharmaceutical and nutritional applications that aim for a balanced antioxidant effect, combining the benefits of flavonoids from the leaves with those of phenolic acids from the root.

The choice of a 2% carbopol base and a 5% concentration of lovage extracts for the formulation of hydrogels is justified by the desire to obtain a stable, effective and safe product for topical application. Carbopol provides texture and stability [55], while lovage extracts at the mentioned concentration provide optimal antioxidant and anti-inflammatory activity, suitable for skin care and for cosmetic or pharmaceutical treatments aimed at protecting the skin against oxidative stress and inflammation.

The results of the control tests confirm that the formulated hydrogels exhibit good physicochemical stability, maintaining their essential properties throughout the testing period (Table 7). The pH parameter was stable in the range of 4.5–5.5, which makes them suitable for topical application, being compatible with the physiological pH of the skin. The lack of significant variations suggests that the bioactive extracts did not suffer major degradation and that the hydrogel matrix ensured adequate stability.

Comparing the three types of hydrogels, it is observed that the hydrogel with lovage leaf extract exhibits the lowest initial viscosity, which can be attributed to the interaction of flavonoids and tannins with the hydrogel structure, modifying the polymer network and increasing its flexibility. This characteristic may contribute to a more uniform application and a controlled release of bioactive compounds.

On the other hand, the hydrogel with root extract exhibits the highest viscosity, which can be explained by the higher content of phenolic acids, which can interact with the polymer, forming additional bonds that stiffen the gel structure. This property may influence the retention time on the skin surface, which may be an advantage in slow absorption applications.

The hydrogel with the whole-plant extract exhibits an intermediate behavior, which suggests a balance between flexibility and stability, making it suitable for various applications.

In conclusion, rheological analysis demonstrates that the phytochemical profile of the extracts directly influences the flow behavior of the hydrogels, which can guide the choice of the optimal formula depending on the desired application (e.g., rapid absorption vs. long-term retention).

Table 8 highlights the antioxidant capacity of lovage extract hydrogels, with the leaf extract formulation showing the highest free radical neutralization (69.4% ABTS, 77.6% DPPH), correlating with its high flavonoid content. The root extract hydrogel exhibited the lowest activity, likely due to its lower flavonoid concentration despite being rich in phenolic acids. The whole-plant extract hydrogel displayed intermediate antioxidant effects. These findings suggest that leaf extract hydrogels are best suited for antioxidant and anti-aging applications, while root extract formulations may be more beneficial for anti-inflammatory and antimicrobial uses.

5. Conclusions

This study provides an original contribution by comparatively analyzing the chemical composition and antioxidant activity of hydroalcoholic extracts (70% ethanol) obtained from different parts of Levisticum officinale (roots, leaves, and the whole plant). A distinctive aspect is the integration of these extracts into carbopol-based hydrogels, followed by their physicochemical and rheological characterization, alongside an evaluation of their antioxidant activity. This interdisciplinary approach supports the identification of novel pharmaceutical and dermato-cosmetic applications, emphasizing the stability and efficacy of lovage extracts as bioactive agents with therapeutic potential.

The extract from lovage leaves exhibited the highest concentrations of flavonoids, particularly quercetin, kaempferol, rutin and apigenin, conferring strong antioxidant activity. Additionally, it contained the highest levels of condensed tannins, reflecting their protective role against UV radiation and pathogens. In contrast, the root extract was richer in phenolic acids (ferulic acid, caffeic acid, vanillic acid), enhancing its anti-inflammatory, antimicrobial and antispasmodic properties. The whole-plant extract displayed a balanced phytochemical profile, combining flavonoids and phenolic acids.

These findings suggest that hydroalcoholic extracts from different parts of Levisticum officinale can be selectively utilized in pharmaceutical formulations based on desired therapeutic effects. The leaf extract, due to its flavonoid-rich composition, is suitable for antioxidant and photoprotective applications, including anti-aging cosmetics, cardiovascular supplements and skin health products. The root extract, with its high phenolic acid content, is ideal for anti-inflammatory, antimicrobial and antispasmodic formulations, supporting digestive health and providing calming effects. The whole-plant extract, offering a synergistic blend of bioactive compounds, is indicated for formulations targeting immune support and the prevention of oxidative stress-related chronic diseases. Furthermore, incorporating these extracts into 2% carbopol-based hydrogels enhances topical delivery, making them viable for dermato-cosmetic applications, anti-inflammatory treatments and oxidative stress protection solutions.