Phytochemical Analysis,Antioxidant,and Antineurodegenerative Properties of Gentiana lutea L. Root Extract and Derived Fractions

Abstract

Objectives

Yellow gentian (Gentiana lutea L., Gentianaceae) has long been valued in traditional medicine primarily as a bitter drug. While recent studies have confirmed its pleiotropic properties, the antineurodegenerative potential of its bioactive compounds remains insufficiently studied. This study aimed to evaluate the chemical composition, in vitro antioxidant and neuroprotective activity of the primary 70% ethanol extract of G. lutea roots and its fractions obtained by liquid-liquid extraction.

Methods

Total phenolic content, dominant secoiridoid gentiopicroside and the main xanthone isogentisin were quantified, as well as the correlation among investigated compounds and activities of samples. The antioxidant activity was tested through DPPH (2,2-diphenyl-1-picrylhydrazyl radical), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), FRAP (ferric-reducing ability of plasma), and β-carotene bleaching assays, while neuroprotective activity was investigated through testing against acetylcholinesterase (AChE) and tyrosinase (Tyr) activity.

Results

The ethyl acetate fraction exhibited significantly higher total phenolic and isogentisin contents (4.3 and 17.6 times higher than the primary extract, respectively), while the n-butanol fraction contained the highest amount of gentiopicroside (2.9 times higher than the primary extract). Antioxidant assays indicated that the ethyl acetate fraction had the strongest activity. The primary ethanolic extract demonstrated the highest acetylcholinesterase and tyrosinase inhibitory activities. Notably, the primary ethanolic extract showed promising tyrosinase inhibition (50.85% inhibition), comparable to kojic acid used as a positive control (51.81% inhibition) at the same concentration (100 μg/mL).

Conclusions

Therefore, G. lutea roots extract possesses significant potential for further investigation and possible utilization as a component of pharmaceutical or food products with antioxidant and anti-neurodegenerative features.

Keywords

Yellow gentian neuroprotection isogentisin gentiopicroside tyrosinase acetylcholinesterase

Introduction

Gentiana lutea L. (yellow gentian) is a perennial plant that belongs to the genus Gentiana (Gentianaceae). The underground parts of G. lutea (Gentianae radix) are well-known sources of healing agents, traditionally employed as a stomachic to enhance appetite and digestion, as well as for the treatment of hepatic and gallbladder disorders.¹ In recent years, G. lutea has garnered significant attention, confirming its numerous pharmacological activities and establishing its pleiotropic properties.² Its principal pharmacologically active compounds include bitter secoiridoids (gentiopicroside, sweroside, swertiamarin, amarogentin), xanthones (isogentisin, gentioside, gentisin), and flavones (isovitexin, isosaponarin, isoorientin).^1,2 Although the potential of G. lutea phytochemicals in neurodegenerative diseases has been recognized,² the underlying mechanism of their neuroprotective action remains poorly understood. Moreover, the ingredients of G. lutea extracts are potent monoamine oxidase inhibitors, which indicates that yellow gentian may be useful in the prevention and treatment of anxiety, depression, Alzheimer's, and Parkinson's disease.³

Parkinson's and Alzheimer's disease are complex neurodegenerative disorders of unknown etiology, with multi-faceted pathogenesis characterized by selective, slow, and progressive deterioration and loss of function of certain brain regions. Oxidative stress, which is associated with neuroinflammation, plays a key role in the cascade of pathogenesis.⁴ Brain tissue is quite sensitive to oxidative stress because it has high oxygen requirements due to intensive metabolism, while on the other hand it is relatively deficient in antioxidant enzymes and contains a high percentage of unsaturated fatty acids that are susceptible to peroxidation.^4,5 In addition to oxidative stress, the target for the development of a new drug may be enzymes whose increased activity is associated with disease progressions, such as acetylcholinesterase in Alzheimer's disease or tyrosinase in Parkinson's disease.⁶ The current treatment is symptomatic and does not stop the further progression of the disease.⁷ An innovative approach to the treatment of multifactorial diseases is the concept of “multi-target directed therapy”. In this context, concomitant antioxidant activity and inhibition of enzymes involved in disease progression may be a valuable strategy for achieving neuroprotection.⁸ It is known that some natural polyphenols, including xanthones such as mangiferin, morin, and bellidifolin can achieve neuroprotective activity.^9–11 Moreover, recent research indicates that some secoiridoid drugs and isolated secoiridoids (eg oleuropein and oleuropein aglycone from olive oil) show a promising neuroprotective effect in cellular and animal models.⁴

The neuroprotective effects of G. lutea extract were evaluated using various in vitro models. In rat pheochromocytoma PC-12 cells, 25 μg/mL of the extract promoted neurite outgrowth and increased cell length after 5 days, confirmed by neurofilament staining.¹² Extract (200 and 400 μg/mL) also protected SH-SY5Y neuroblastoma cells from apoptosis induced by vinblastine, enhancing cell viability and preventing Bcl-2 phosphorylation. Additionally, it reversed the down-regulation of Sirt-1, a protein crucial for cells’ survival and protection against radiation or oxidative stress, caused by vinblastine. Furthermore, G. lutea (200 μg/mL) extract reduced TNF-α levels in LPS-stimulated RAW264.7 macrophages, showing significant anti-inflammatory activity with long-term exposure.¹³ Furthermore, G. lutea compounds were found to competitively inhibit MAO-B, suggesting potential for treating Parkinson's and Alzheimer's diseases. The extract also inhibited E-NTPDase activity, with individual compounds like amarogentin, gentiopicroside, and isovitexin showing greater inhibition than mixtures. These findings highlight G. lutea extract and its constituents as promising candidates for treating neurodegenerative diseases.² However, the tyrosinase and acetylcholinesterase inhibitory activities have not been previously investigated.

Considering previous studies suggesting that G. lutea extract and taxonomically related species with a similar phytochemical profile may be safe for treating diseases of the central nervous system,² we hypothesized that one of the potential mechanisms underlying its antineurodegenerative activity could involve the inhibition of acetylcholinesterase and tyrosinase, coupled with antioxidant activity. To investigate this hypothesis, the study aimed to evaluate the in vitro antioxidant and antineurodegenerative activities of G. lutea root extract and its fractions, focusing on their ability to inhibit acetylcholinesterase and tyrosinase. Additionally, the crude extract was fractionated, and the chemical composition of the obtained fractions was analyzed to explore the correlation between chemical composition and bioactivity and to identify compounds potentially responsible for the observed effects.

Materials and Methods

Chemicals

Gallic acid, ascorbic acid, 2(3)-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4 hydroxytoluene (BHT), 2,2-dyphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), potassium-persulfate (K₂S₂O₈), dimethylsulfoxide (DMSO), sodium carbonate anhydrous (Na₂CO₃), sodium acetate (C₂H₃NaO₂), iron (III) chloride (FeCl₃), iron (II) sulfate heptahydrate (FeSO₄ × 7 H₂O), β-carotene, Folin-Ciocalteu reagent, sodium phosphate monobasic (NaH₂PO₄), sodium phosphate dibasic (Na₂HPO₄), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), acetylcholinesterase from Electrophorus electricus (AChE), acetylcholine iodide, galanthamine hydrobromide from Lycoris sp., kojic acid, tyrosinase from mushroom, and 3,4-dihydroxy-L-phenylalanine (L-DOPA) were purchased from Sigma Aldrich (USA), while Tween 40 and linoleic acid were purchased from Acros Organics (Belgium). Acetonitrile (Merck, Germany) was of HPLC grade and ultra-pure water was prepared using a Milli-Q purification system (Millipore, France). All other chemicals such as methanol, ethanol, glacial acetic acid, hydrochloric acid, dichloromethane, chloroform and ethyl acetate were technical grade and purchased from Zorka Pharma, Šabac (Serbia).

Plant Material

Dried roots of G. lutea were purchased from the Institute for Medicinal Plants Research “Dr Josif Pančić“, Belgrade (product serial number 11190520). The plant material was ground using an industrial mill and sieved through a series of five sieves with pore sizes standardized according to the Yugoslav Pharmacopoeia (Ph. Jug. V, 2000). For further experiments, the particle size fraction of 0.75–2 mm was used. Throughout the study, the material was stored in paper bags at room temperature.

Extraction Procedure and Preparation of Fractions

Extraction and preparation of fractions were performed according to the previously described procedure.¹⁴ Briefly, 200 g of powdered underground parts were extracted with 70% ethanol (solid-to-liquid ratio of 1:10, w/v). The selection of 70% ethanol as the primary extraction solvent was based on its traditional use in the extraction of plant materials, as well as its widespread application in the food and pharmaceutical industries. Furthermore, 70% ethanol is commonly used as an extraction solvent according to the European Medicines Agency (EMA) guidelines for the preparation of Gentian root tincture.¹⁵ Fractionation was carried out by liquid–liquid extraction using a series of selected organic solvents, commonly used for the separation of bioactive compounds from plant extracts, applied in increasing polarity order, as previously described.^6,14 Briefly, the extract was evaporated to dryness (extraction yield 76.3 g), and dispersed in distilled water for successive re-extraction with petroleum ether, ethyl acetate, and n-butanol. After the evaporation, yields of dry petroleum ether, ethyl acetate, n-butanol, and water fractions were 0.57, 2.67, 6.06, and 58.9 g, respectively.

Regarding sample preparation for bioassays, the extract and its fractions were dissolved in suitable solvents to achieve a stock concentration of 1000 µg/mL. The stock solutions were additionally diluted to concentrations of 100, 200 and 500 µg/mL. The antioxidant, AChE and tyrosinase inhibition assays were performed for three concentrations to test the dose dependence, while the total phenolic content was only determined for the sample concentration of 500 µg/mL. To exclude the influence of sample color, the absorbance values of the reaction mixture for each sample at each concentration tested were corrected by the absorbance values of the color control (containing the sample and a suitable solvent in the appropriate ratio). All measurements in the analyses described in the following sections were performed in triplicate.

Determination of Total Phenolic Content

The total phenolic content of samples was determined by the spectrophotometric method.¹⁶ The total phenolics content was expressed as milligrams of gallic acid equivalents per gram of extract/fractions dry weight (mg GAE/g dw).

HPLC Analysis

HPLC analysis was performed following the previously established procedures.¹⁷ An Agilent 1200 RR HPLC instrument (Agilent, Waldbronn, Germany) was used on a reverse-phase Zorbax SB-C18 (Agilent) analytical column (150 mm × 4.6 mm i.d.; 5 µm particle size). Quantification of gentiopicroside and isogentisin was performed using calibration curves of the standards, and the content was expressed as milligrams per gram of extract/fractions dry weight (mg/g dw).

DPPH Assay

The radical scavenging ability of plant extract and fractions were screened by DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assay according procedure described by Blois (1958) with slight modifications.^14,18 Butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), and ascorbic acid were used as positive controls while methanol was used as blank. Absorbance was measured at 517 nm by Perkin Elmer Lambda Bio UV/VIS. Results were expressed as a percentage of DPPH radical inhibition (mean ± standard deviation).

ABTS Assay

The ABTS (2,2′-azino-bis (3-ethyl-benzothiazoline-6- sulfonic acid)) assay, which was used to assess the antioxidant capacity of plant extract and its fractions, was performed according to a previously reported method¹⁹ with slight modifications.¹⁴ BHT, BHA and ascorbic acid were used as positive controls while distilled water was used as blank. The absorbance of the mixture was measured at 734 nm by Perkin Elmer Lambda Bio UV/VIS. Results were expressed as a percentage of ABTS radical inhibition (mean ± standard deviation).

Ferric-Reducing Ability of Plasma (FRAP) Assay

The ferric-reducing ability of plasma (FRAP) assay was performed according to the procedures described by Benzie and Strain (1996) with slight modifications.^14,20 Absorbance was measured at 593 nm by Perkin Elmer Lambda Bio UV/VIS. Total antioxidant activity is expressed as μmol FeSO₄ × 7H₂O/g dry weight of extract/fractions (mean ± standard deviation).

β-Carotene Bleaching Assay

The β-carotene bleaching assay was conducted according to the previously described procedure²¹ with slight modifications.¹⁴ The β-carotene bleaching assay is designed to measure the ability of gentian root extract and its fractions to reduce the oxidative loss of β-carotene in an emulsion with linoleic acid. Absorbance was measured at 490 nm by Perkin Elmer Lambda Bio UV/VIS. The antioxidant capacity of the samples was expressed as a percentage of β-carotene bleaching inhibition (mean ± standard deviation).

Acetylacholinesterase (AChE) Inhibitory Activity Assay

Inhibition of AChE was determined according to the previously described spectrophotometric method.^22,23 The assay was performed in 96-well plates and absorbance was measured at 412 nm by using Tecan Sunrise SN microplate reader equipped by XFluor4 software. The alkaloid galanthamine was used as a reference substance. The inhibitory activity of the extract and its fractions are expressed as percentage inhibition of enzyme activity (mean ± standard deviation).

Tyrosinase Inhibitory Activity Assay

Tyrosinase inhibitory activity assay was performed based on a previously described method²⁴ with slight modifications.¹⁴ The assay was performed in 96-well plates and absorbance was measured at 475 nm by using Tecan Sunrise SN microplate reader equipped by XFluor4 software. Kojic acid was used as a reference substance. The inhibitory activity of the extract and its fractions are expressed as percentage inhibition of enzyme activity (mean ± standard deviation).

Statistical Analysis

Statistical evaluation was performed by one-way analysis of variance (ANOVA) with post hoc analysis of differences between means carried out by Tukey's multiple range tests. Results were considered statistically significant for a p-value of less than .05. The degree of linear dependence between the content of bioactive compounds and activity of investigated extracts was calculated by Pearson's correlation (r), considering p < .05 as significant. All data were statistically analyzed using SPSS statistical computer software package (SPSS, trial version, Dublin, Ireland).

Results

Chemical Analysis

In the present study, total phenolic content as well as the amount of dominant secoiridoid gentiopicroside and the main xanthone compound isogentisin were quantified in G. lutea 70% ethanolic extract and its fractions. The ethanolic extract contains 23.08 mg GAE/g dw of total phenolics, and after fractionation, total phenolic content varied between 11.72 and 99.28 mg GAE/g dw (Table 1). The highest phenolic level was determined in the ethyl acetate fraction while in the water fraction phenolic content was the lowest. The concentration range of gentiopicroside in all investigated samples was between 8.08 and 89.79 mg/g dw, and the highest content was determined in the n-butanol fraction. The content of isogentisin, the most abundant xanthone in G. lutea roots, was highest in the ethyl acetate fraction (269.57 mg/g dw), followed by the petroleum ether fraction (49.37 mg/g dw) and the ethanol extract (15.27 mg/g dw). The lowest amount of isogentisin was found in the water fraction (0.93 mg/g dw).

Table 1.

Content of Total Phenolics, Gentiopicroside, and Isogentisin in 70% Ethanolic Extract of Gentiana lutea Roots and its Fractions.

Sample	Total phenolics (mg GAE/g dw)	Gentiopicroside (mg/g dw)	Isogentisin (mg/g dw)
70% ethanolic extract	23.08 ± 2.45^b	31.21 ± 1.11^c	15.27 ± 0.62^a
Petroleum ether fraction	24.45 ± 0.20^b	Traces^a	49.37 ± 3.46^b
Ethyl acetate fraction	99.28 ± 1.53^d	8.08 ± 1.39^b	269.57 ± 18.33^c
n-Butanol fraction	54.98 ± 2.64^c	89.79 ± 5.39^d	6.45 ± 0.39^a
Water fraction	11.72 ± 1.44^a	12.01 ± 0.86^b	0.93 ± 0.08^a

GAE/g dw ‒ gallic acid equivalents/g dw.

The values presented as a means ± standard deviation (n = 3) marked by different superscript letters (a–d) differ significantly according to Tukey's multiple range test at p < .05.

Antioxidant Activity

The antioxidant activity of G. lutea root extract and its fractions was evaluated by four in vitro assays (Table 2). Ethyl acetate fraction was characterized by the highest radical scavenging activity, according to the results of DPPH, ABTS and β-carotene assays. Furthermore, it was shown that this fraction manifested strong activity in the FRAP test, along with the n-butanol fraction. The water fraction exhibited the weakest activity in all assays, which might be due to the lowest amount of total phenolics and isogentisin in this fraction. Although the antioxidant activity of G. lutea fractions is lower in comparison with the same dose of positive controls, BHT and BHA, stronger activity of fractions in comparison with vitamin C has been achieved in β-carotene test.

Table 2.

Antioxidant Activity of 70% Ethanolic Extract of Gentiana lutea Roots and its Fractions.

Sample	Concentration (μg/mL)	DPPH (% of inhibition)			ABTS (% of inhibition)			FRAP (μmol FeSO₄ × 7H₂O/g dw)			β-carotene (% of inhibition)
70% ethanolic extract	100	10.06 ± 0.48	a	b	1.45 ± 0.89	a	a	41.93 ± 5.03	a	b	20.72 ± 1.18	a	b
	200	13.37 ± 0.70	b		2.91 ± 0.30	a		72.64 ± 6.27	b		29.60 ± 2.19	b
	500	24.36 ± 0.75	c		11.92 ± 0.89	b		112.91 ± 3.80	c		33.98 ± 1.90	b
Petroleum ether fraction	100	9.77 ± 0.38	a	b	3.34 ± 0.47	a	b	47.32 ± 7.47	a	b	22.78 ± 1.82	a	b
	200	16.62 ± 0.33	b		8.09 ± 0.67	b		71.39 ± 8.75	b		47.36 ± 0.81	b
	500	28.53 ± 0.32	c		16.23 ± 0.29	c		124.53 ± 6.23	c		57.92 ± 0.97	c
Ethyl acetate fraction	100	20.19 ± 0.86	a	c	19.53 ± 0.44	a	d	99.21 ± 7.61	a	c	25.23 ± 2.04	a	b
	200	33.20 ± 0.61	b		32.82 ± 0.67	b		246.16 ± 8.10	b		43.37 ± 2.35	b
	500	62.45 ± 0.67	c		55.50 ± 0.58	c		430.47 ± 8.75	c		74.78 ± 1.35	c
n-Butanol fraction	100	18.90 ± 0.60	a	c	9.94 ± 0.51	a	c	124.95 ± 4.37	a	d	6.18 ± 1.60	a	a
	200	31.21 ± 0.70	b		20.89 ± 0.52	b		244.50 ± 7.08	b		40.93 ± 2.33	b
	500	49.64 ± 0.54	c		43.09 ± 0.44	c		427.56 ± 5.18	c		67.31 ± 1.93	c
Water fraction	100	8.10 ± 0.39	a	a	2.13 ± 0.58	a	ab	22.00 ± 4.37	a	a	3.47 ± 1.82	a	a
	200	8.70 ± 0.38	a		4.31 ± 0.95	b		17.43 ± 6.23	a		21.88 ± 0.59	b
	500	13.41 ± 0.79	b		7.80 ± 0.52	c		62.68 ± 3.80	b		31.27 ± 0.98	c
BHA	100	43.33 ± 0.87		e	64.95 ± 0.63		f	572.85 ± 5.71		f	57.70 ± 1.91		c
BHT	100	34.31 ± 0.43		d	55.11 ± 0.44		e	413.03 ± 3.13		e	56.29 ± 1.44		c
Vit C	100	91.65 ± 0.21		f	55.70 ± 0.69		e	576.17 ± 7.61		f	2.99 ± 2.13		a

DPPH ‒ 2,2-diphenyl-1-picrylhydrazyl; ABTS ‒ 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid); FRAP ‒ ferric reducing antioxidant power; BHA ‒ 2(3)-t-butyl-4-hydroxyanisole; BHT ‒ 3.5-di-tert-butyl-4 hydroxytoluene; Vit C – vitamin c (ascorbic acid).

Results are expressed as mean values ± standard deviation (n = 3). Significant differences between the activities of tested concentrations of the same sample are indicated by different letters in contiguous cells (a–c), while different letters in the next column (a–f) denote significant differences between the activities of the lowest tested concentrations (100 μg/mL) of different samples, as determined by Tukey's multiple range test at p < .05.

The correlation between total phenolics, gentiopicroside and isogentisin content, and antioxidant activity was determined by calculating Pearson's correlation coefficient (Table 3). A good correlation between total phenolic content (r > 0.7) and antioxidant capacity (DPPH, ABTS, and FRAP test) was observed for all samples. Isogentisin content was significantly correlated with antioxidant capacity in the ABTS test (r > 0.8), and moderately in DPPH and β-carotene tests (r > 0.6). On the other hand, no correlation was found between gentiopicroside and antiradical activity in the DPPH, ABTS, and β-carotene tests, whereas a moderate correlation was observed in the FRAP assay (r > 0.6).

Table 3.

Pearson's Correlation Coefficients (r) Between the Content of Phytocompounds and in Vitro Bioactivity of 70% Ethanolic Extracts of Gentiana lutea Roots and its Fractions.

	Total phenolics	Gentiopicroside	Isogentisin
DPPH	0.933**	0.452	0.640*
ABTS	0.989**	0.088	0.868**
FRAP	0.779**	0.658**	0.390
β-carotene	0.402	−0.509	0.635*
AChE	−0.375	0.655**	−0.632*
Tyr	−0.333	0.604*	−0.567*

DPPH ‒ 2,2-diphenyl-1-picrylhydrazyl; ABTS ‒ 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid); FRAP ‒ ferric reducing antioxidant power; AChE – acetylcholinesterase; Tyr – tyrosinase.

Values followed by “*” are significant at p < .05; “**” are significant at p < .001.

Acetylcholinesterase (AChE) Inhibitory Activity

According to the results presented in Table 4, ethanolic extract of G. lutea was the most active (24.64-39.16%) among investigated samples, and AchE was inhibited in a dose-dependent manner. Considering fractions, the n-butanol fraction showed moderate activity (19.53-24.17%) and was more active than other fractions. The standard galantamine (57.11%) exhibited higher activity than all samples.

Table 4.

Antineurodegenerative Activity of 70% Ethanolic Extracts of Gentiana lutea Roots and its Fractions.

Sample	Concentration (μg/mL)	AChE (% of inhibition)			Tyr (% of inhibition)
70% ethanolic extract	100	24.64 ± 1.42	a	c	50.85 ± 2.12	a	d
	200	35.84 ± 3.77	b		49.65 ± 2.61	a
	500	39.16 ± 0.78	b		52.14 ± 1.08	a
Petrol ether fraction	100	10.35 ± 1.14	a	ab	13.31 ± 3.37	b	ab
	200	13.07 ± 2.83	a		21.76 ± 1.11	c
	500	13.87 ± 3.01	a		3.03 ± 1.10	a
Ethyl acetate fraction	100	8.84 ± 1.45	a	a	8.54 ± 2.20	a	a
	200	12.44 ± 1.88	a		27.64 ± 3.36	b
	500	10.16 ± 2.14	a		40.13 ± 2.30	c
n-Butanol fraction	100	21.00 ± 3.47	a	c	36.46 ± 1.68	a	c
	200	24.17 ± 4.92	a		30.58 ± 2.91	a
	500	19.53 ± 2.32	a		38.66 ± 4.59	a
Water fraction	100	15.15 ± 1.13	a	b	17.72 ± 3.54	a	b
	200	12.20 ± 2.83	a		19.56 ± 1.10	ab
	500	12.84 ± 3.01	a		25.80 ± 3.87	a
Galanthamine	100	57.11 ± 1.68		d	—
Kojic acid	100	—			51.81 ± 2.55		d

AChE – acetylcholinesterase; Tyr – tyrosinase.

Results are expressed as mean values ± standard deviation (n = 3). Significant differences between the activities of tested concentrations of the same sample are indicated by different letters in contiguous cells (a–c), while different letters in the next column (a–d) denote significant differences between the activities of the lowest tested concentrations (100 μg/mL) of different samples, as determined by Tukey's multiple range test at p < .05.

Correlation between total phenolics, gentiopicroside and isogentisin content in G. lutea extracts and AChE inhibition was presented in Table 3. No correlation between total phenolic content and AChE inhibitory activity was observed, whereas gentiopicroside and isogentisin showed a moderate correlation (r > 0.6).

Tyrosinase (Tyr) Inhibitory Activity

The capability of G. lutea roots ethanolic extract and its fractions to inhibit the activity of Tyr was tested and compared with the well-known Tyr inhibitor kojic acid (Table 4). According to our results, the activity of G. lutea (3.03-52.14%) samples was significantly influenced by the type of solvent that was used for fractionation. Primary ethanol extract was more potent than obtained fractions, with activity comparable to the activity of kojic acid at the same concentration of 100 μg/mL (50.85 and 51.81%, respectively). Among fractions, the n-butanol fraction showed higher activity (30.58-38.66%) than ethyl acetate (8.54-40.13%) and water (17.72-25.80%) fractions. According to the results presented in Table 3, a moderate positive correlation was found between gentiopicroside content and tyrosinase inhibitory activity. In contrast, a moderate negative correlation was found between isogentisine content and tyrosinase inhibitory activity.

Discussion

Both variability in data and batch effects can affect the quantification of secondary metabolites in plant extracts due to biological factors, technical procedures, and instrumentation. Addressing these challenges, the experimental design was carefully planned, with previously standardized protocols and statistical tools. On the other hand, plant material of G. lutea is known to be highly variable, particularly in chemical composition,²⁵ and it is expected that batches may vary in both scientific research and manufacturing processes. Therefore, the appropriate selection of marker phytocompounds in chemical analyses is essential. Chemical analysis by LC-ESI TOF MS of the ethanolic extract of G. lutea root has shown that the most abundant compounds identified were secoiridoids, with gentiopicroside as the dominant one. Among polyphenols, the xanthone isogentisin had the highest content.¹ This affirmed gentiopicroside and isogentisin as marker compounds of the two major classes of secondary metabolites in the root of G. lutea. As expected, fractionation led to a significant separation of the monitored bioactive compounds across the fractions compared to the initial crude extract. The choice of solvent for fractionation plays a crucial role in concentrating bioactive compounds, thereby influencing their biological activity. This effect has also been observed in studies on fractionated extracts of Gentiana species.^26–29 A distribution pattern similar to that observed in the present study was recorded during the fractionation of the G. lutea root extract obtained by ultrasound-assisted extraction, with gentiopicroside being most concentrated in the n-butanol fraction and isogentisin in the ethyl acetate fraction.³⁰ The lowest quantity of isogentisin reported in the water fraction is not surprising, as it is a xanthone aglycone and a water-insoluble compound. This is consistent with the literature reports that xanthones are mainly present in non-polar and medium-polar extracts.³¹ It was shown that a mixture of isogentisin and gentisin has been isolated from an ethyl acetate fraction of G. lutea root methanol extract.³² Pure isogentisin was obtained from the ether fraction of G. lutea root extract and was identified as the most effective in the prevention of smoking-caused endothelial injury.³³ The observed significant concentration of gentiopicroside in the n-butanol fraction is consistent with the fractionation of G. asclepiadea root extract, where the secoiridoids gentiopicroside, swertiamarin, and sweroside were most abundant in the n-butanol fraction.³⁴

The highest radical scavenging activity recorded in the ethyl acetate fraction could be explained by its highest content of phenolics and the concentration of isogentisin. Consistent with our results, among the chloroform, ethyl acetate, and n-butanol fractions of G. asclepiadea root, the ethyl acetate fraction, which concentrated the highest content of phenolic compounds (including total phenolics, flavonoids, flavonols, and gallotannins), exhibited the strongest antioxidant activity.³⁴ Previous studies have been carried out on several Gentiana species, including G. lutea, and their antioxidant effects were also attributed to the presence of phenolic compounds.^35,36 This can be explained by a phenomenon known as “polar paradox” due to a presence of polar phenolic compounds. This theory that illustrates the paradoxical behavior of antioxidants in different media and rationalizes the fact that polar antioxidants are more effective in less polar media, while nonpolar antioxidants are more effective in relatively more polar media.^37,38 Consequently, the β-carotene test has shown that G. lutea fractions obtained by using solvents of lower polarity were more active, while the water fraction was characterized by the lowest activity. This result is in accordance with previous findings where xanthones are considered as promising antioxidants due to their chemical structure.^39,40 It has been reported that secoiridoids, including gentiopicroside, were very weak free radical scavengers.^41,42 It has been suggested that secoiridoids did not show direct scavenging activity on free radicals, but their activity could be related to the activation of the antioxidant enzymes.^41,43 Similarly, in an earlier study on the antioxidant activity of a taxonomically related species, Blackstonia perfoliata (L.) Huds., Gentianaceae, it was emphasized that gentiopicroside-rich extracts exhibited stronger inhibitory effects on lipid peroxidation than on radical scavenging activity.⁴⁴ Higher antioxidant efficiency in lipid systems was also observed for extracts of Gentiana species containing higher amounts of secoiridoids than phenolic compounds, including G. asclepiadea,⁴⁵ G. cruciata,⁴⁶ G. decumbens, G. algida, G. macrophylla, and G. triflora.⁴⁷

Inhibition of AChE enzyme is a vital target in the treatment of Alzheimer's disease.⁴⁸ Our results are in accordance with literature data where some Gentiana species and their fractions have shown mild to moderate cholinesterase inhibitory activity.³⁶ There are several reports of the activity of plant extracts from Gentianaceae family and individual compounds against cholinesterase enzymes, but published results are inconsistent. Some studies have reported that bellidifolin, xanthone isolated from Gentiana comperestris leaves, has exhibited anti-AChE activity similar to galanthamine.¹⁰ On the other hand, in another study by the same authors, bellidifolin isolated from Gentianella amarella was a weak inhibitor,⁴⁹ while other studies showed that bellidifolin from Swertia chirata exhibited moderate activity.⁵⁰ Previous studies reported that four xanthones from the Gentiana species exhibit significant AChE inhibitory activity, while it was suggested that the methoxy group (MeO) at C(3) could enhance this activity.⁵¹ The role of xanthones from Gentiana species in AChE inhibition is further supported by the finding that mangiferin and rhodanthenone D, xanthones isolated from the whole plant of G. rhodantha, exhibited 13.4% and 18.4% AChE inhibitory effects, respectively, at a concentration of 10⁻⁴ M.⁵² Secoiridoids swertiamarin and gentiopicroside, along with the flavones isoorientin and isovitexin, were found to be inactive,³⁶ and very low activity of gentiopicroside was also reported.⁵³ However, it has been shown that isoorientin and isovitexin were promising AChE inhibitors.⁵⁴ Additionally, swertiamarin has been demonstrated to possess AChE inhibition potential and modulate cholinergic transmission at the transcriptomic level.⁴³ Furthermore, loganin, an iridoid glycoside and secoiridoid precursor, improved scopolamine-induced memory impairment in mice and significantly inhibited AChE activity in the hippocampus and frontal cortex.⁵⁵ Taking into account both results from our study and published data, it can be concluded that ethanolic gentian extracts showed moderate activity against AChE and this inhibitory activity cannot be attributed to specific compounds but rather to possible synergistic effect of the components present in extract.

Tyrosinase is a key enzyme involved in the biosynthesis of mammalian melanin pigments. This enzyme also catalyzes oxidation of dopamine and causes the formation of reactive oxygen species in cytosol and mitochondria, thus contributing to dopaminergic neurons loss in Parkinson's disease.⁵⁶ Our study is the first report of the anti-tyrosinase activity of G. lutea root extract and its fractions. Literature data about tyrosinase inhibitory activity of gentian species is very scarce, only suppression by G. scabra and G. veitchiorum flower water extracts has been reported so far.^57,58 Aqueous methanol (50%) extract of taxonomically related species Swertia chirata showed higher tyrosinase-inhibitory activity than acetone or water extracts,⁵⁹ which is consistent with our results where ethanol (70%) was the most efficient solvent system for extracting metabolites with tyrosinase-inhibition potential. There are several attempts to identify compounds which might be responsible for the anti-tyrosinase activity of extracts. This includes performing experimental studies, as well as molecular docking studies in order to clarify possible mechanisms of action. Among secoiridoid glucosides, marker compounds of Gentiana lutea,⁶⁰ sweroside showed good tyrosinase inhibitory activity.⁶¹ Molecular docking study revealed that sweroside has good binding affinity with the enzyme, which occurs mainly through hydrogen bonds and van der Waals interactions. Another two secoiridoids, gentiopicroside and swertiamarin, showed similar binding affinity towards tyrosinase as sweroside,⁶² and all three compounds have low binding affinity with AChE. Furthermore, it has been reported that sweroside decreased melanin production through down-regulation of tyrosinase expression.⁶³ Flavone isovitexin showed high inhibitory activity,⁶⁴ and suppression of melanin synthesis by this compound was also via down-regulation of tyrosinase activity.⁶⁵ Overall, published results suggest that the tyrosinase-inhibitory activity of gentian root extract may be attributed to its various components. Therefore, similar to the previously estimated anti-AChE activity, it is plausible that the significant inhibitory activity of G. lutea is related to the synergistic effects of these bioactive compounds.

Although the conducted in vitro assays provide valuable insights into the potential neuroprotective mechanisms of bioactive compounds from G. lutea roots, extrapolating the results to more complex biological systems should be approached with caution, as in vitro studies cannot fully replace animal and clinical research. In vitro studies serve as a starting point for investigating the molecular targets and cellular of plant metabolites, providing insights into their pharmacological potential before clinical research. Understanding their mechanisms of action at this stage helps guide the development of effective phytomedicines. Indeed, findings obtained from in vitro assays do not always correlate with in vivo efficacy, primarily due to the complex pharmacokinetic factors and the physiological and pathophysiological conditions that are critical for the efficiency and safety of bioactive compounds. Therefore, further in vivo studies are necessary to validate these findings and assess the pharmacokinetics, safety, and therapeutic potential of the identified bioactive compounds for tretment of neurodegenerative diseases.

Conclusion

In the present study, the 70% ethanolic extract of G. lutea roots and its fractions have shown antioxidant activity, as well as inhibitory activity against the acetylcholinesterase (AChE) and tyrosinase (Tyr) enzymes in vitro. Results indicated that the type of solvent which was used for fractionation had a critical role in the extraction and concentration of bioactive compounds which further affected the activity of tested samples. The ethyl acetate fraction was the richest in total phenolics and isogentisin, and this fraction was characterized by the strongest antioxidant activity in DPPH, ABTS, and β-carotene tests. On the other hand, primary ethanolic extract has shown the highest AChE and Tyr inhibitory activity, with activity towards Tyr being more pronounced and comparable with control. This is the first report of the inhibitory activity of G. lutea roots against these enzymes, and the connection between inhibitory effect and constituents of this species may be interesting for further research. The results presented in the current study, confirming significant inhibition of enzymes relevant to neurodegenerative disorders coupled with antioxidant effects, provide new insights into the underlying mechanisms of the previously recognized neuritogenic activity of bioactive compounds from G. lutea. Finally, given that data from in vitro studies cannot be simply extrapolated to a living organism, further studies, including in vivo experiments and clinical trials, are required to validate the antineurodegenerative potential of G. lutea root extract demonstrated in this study.

Footnotes

Acknowledgements

The authors are thankful to the Serbian Ministry of Science, Technological Development and Innovation.

ORCID iD

Miloš S. Jovanović

Ethical Approval

This article does not contain any studies with human or animal participants.

Informed Consent

Not applicable.

Author Contributions/CRediT

J.M.: investigation, data curation, formal analysis, software, writing - original draft. T.J.: conceptualization, investigation, methodology, writing - review and editing. A.A.: conceptualization, investigation, methodology. M.S.J.: investigation, data curation, formal analysis, visualization, writing - original draft, writing - review and editing. G.Z.: conceptualization, methodology, writing - review and editing, validation. J.Ž.: conceptualization, writing - review and editing, validation. K.Š.: conceptualization, writing - review and editing, resources, project administration, funding acquisition, validation.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Science, Technological Development and Innovation of Republic of Serbia, (grant number 451-03-136/2025-03/200003, 451-03-137/2025-03/200113).

Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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, et al. Antioxidant and antimelanogenic activities of compounds isolated from the aerial parts of Achillea alpina L. Chem Biodivers. 2019;16(7):e1900033. doi:https://doi.org/10.1002/cbdv.201900033