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Article

Impact of Daily and Seasonal Variation on the Phytochemical Profile of Larrea cuneifolia in Northwestern Argentina

by
María Celeste Barrera
1,
Mariana Daniela Rosa
1,2,*,
Iris Catiana Zampini
1,2 and
María Inés Isla
1,2,*
1
Instituto de Bioprospección y Fisiología Vegetal (INBIOFIV-CONICET-UNT), San Martín 1545, San Miguel de Tucumán T4000CBG, Argentina
2
Facultad de Ciencias Naturales, Instituto Miguel Lillo, Miguel Lillo 205, San Miguel de Tucumán T4000CBG, Argentina
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(21), 3332; https://doi.org/10.3390/plants14213332 (registering DOI)
Submission received: 17 September 2025 / Revised: 26 October 2025 / Accepted: 28 October 2025 / Published: 31 October 2025
(This article belongs to the Special Issue Chemical Characterizations and Biological Activities of Plant Products and By-Products and Their Applications, 2nd Edition)
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Versions Notes

Abstract

Larrea cuneifolia Cav. (common name: jarilla macho) is an endemic Argentinian medicinal shrub that has traditionally been used by the Diaguita-Calchaquí communities in the Monte Desert region in northwestern Argentina. The aim of the present study was to analyze the phytochemical profile and biological activity of the aerial parts of jarilla collected in different places throughout the year, in different seasons and times of day, to determine the optimal harvesting conditions for promoting its medicinal use. The aerial parts were collected three times a day over the course of four seasons in eight L. cuneifolia populations. The total phenolic compounds (TPCs), total flavonoid (TF) content, total lignans (TL), sugars (S) and soluble protein (SP) content were quantified by using spectrophotometric methods and HPLC-DAD. Antioxidant activity was determined by using ABTS scavenging. Significant seasonal, diurnal and spatial variations in the accumulation of TPC (52.61 to 113.52 mg GAE/g), TF (3.71 to 17.92 mg QE/g), TL (283 to 582 μg NDHGAE/g); S (5.73 to 15.17 mg GE/g) and SP (36.75 to 103.10 mg BSAE/g) in aerial parts of L. cuneifolia were revealed. The highest concentrations of TPC and TF were recorded in spring mornings. Maximum accumulation of nordihydroguaiaretic acid (291.8 ± 2.8 μg NDHGAE/mg dry weight) and other lignans were also observed in spring. Heat map analyses pinpoint Ampimpa (Site 1) as a site for jarilla sustainable harvesting, balancing high metabolite content with population abundance, especially in spring, when the highest antioxidant activity (SC50 = 1.560 ± 0.021 μg GAE/mL) coincides with increased phenol levels. These studies highlight the importance of integrating ecological and phytochemical data to define harvesting strategies; collecting during spring mornings optimizes the yield of bioactive compounds, simultaneously minimizing ecological pressure. This study demonstrates how seasonal bioprospecting can inform pharmacological research and local development while safeguarding the endemic plant population.

1. Introduction

A semiarid ecosystem, with relatively low humidity, temperature fluctuations and intense solar radiation [1], predominates in the eco-regions “Monte de Sierras y Bolsones” (between 1800 and 2700 masl) in north-western Argentina [1]. These regions, well-known for their great ecological, social, and cultural importance, are home to medicinal plant species that have been used since ancient times. Such is the case of Larrea cuneifolia Cav, a native shrub popularly known as jarilla, jarilla norte-sur or jarilla macho, which belongs to the family Zygophyllaceae (Figure 1) [2].
Due to limited access to urban areas and health centers, as well as the high cost of medicines, the indigenous community of the Calchaquí Valleys keeps a long-standing traditional practice of using L. cuneifolia as a remedy, especially when dealing with respiratory diseases, as well as musculoskeletal and skin disorders [1,3]. L. cuneifolia is recognized for its anti-inflammatory, antirheumatic, hypotensive, rubefacient, diaphoretic, febrifuge, oxytocic, emmenagogue, odontalgic and antitussive properties, as well as its antifungal properties [1,2,4,5]. In this sense, several studies have documented its anmicrobial activity against multi-resistant Gram-positive and Gram-negative bacteria [6], antifungal activity against phytopathogenic filamentous fungi and yeasts [7,8,9,10], larvicidal activity [11], antioxidant, antiinflammatory [3,12,13,14] and antitumoral [15] properties. Phytochemical studies revealed that this species accumulates bioactive polyphenol compounds [3,6,7,8,15]. It is important to note that, in addition to the traditional use that local communities make of L. divaricata, it is exploited directly from the environment by wild harvesting for the development of cosmetic commercial products for dandruff and hair loss [16,17]. These extractive practices can have negative effects on population abundance, so it is necessary to consider designing conservation methods aimed at maintaining the population abundance of the species. Furthermore, L. cuneifolia is on the red list of endemic plants in Argentina’s Monte Desert due to the intensive extraction from its natural habitat.
Biosynthesis and accumulation of metabolites in plants are regulated by multifactorial interactions between genetic and external environmental factors, i.e., temperature, light, humidity and precipitation, as well as circadian rhythms [18,19,20,21,22,23,24]. Control of primary and secondary metabolism ensures that phytochemicals are in tune with the demands of the environment and the available resources. Consequently, phytochemical composition varies throughout the day and year (seasonal variation) [18,19,20,21,22].
At present, both an increase in temperature and a progressive rainfall decrease have been observed in areas such as the Monte Desert, a fact which is starting to have a negative impact on the habitat of L. cuneifolia [23]. As metabolite production depends on environmental factors, these variations could also influence the phytochemical profile of L. cuneifolia and consequently affect its therapeutic value. A previous report described the distribution and population abundance of L. cuneifolia in the Calchaquies Valleys [1]. To promote sustainable harvesting of this species in this region, it is essential to conduct studies to identify not only the population but also the most appropriate season and time of day. It is the aim of the present study to determine the optimal conditions for harvesting this species for medicinal use.

2. Results and Discussion

The Valley Calchaquies shrub lands are arid regions with daily and seasonal temperature variations and with seasonal rainfalls not exceeding 50 mm per year, generally concentrated in spring The aerial parts of L. cuneifolia were collected at three different times of the day in summer, winter, spring, and autumn (Figure 1). Then, they were dried and milled and 96 ethanolic extracts from the aerial parts were prepared. Both the yield of ethanol-soluble principles and the content of primary and specialized metabolites in L. cuneifolia, as well as the antioxidant activity, were determined (Figure 2).

2.1. Phytochemical Characterization

The yield of the soluble principles ranged from 0.1837 to 0.3974 g DW (dry weight of soluble principle)/g of dry plant material while the TPC content ranged from 52.61 to 113.52 mg GAE/g of dry plant material. TF content varied from 3.71 to 17.92 mg QE/g dry plant material depending on the time of day, season, and collection site. Variation was also observed in the content of primary metabolites, including reducing sugars (5.73 to 15.17 mg GE/g dry plant material) and soluble proteins (36.75 to 103.10 mg BSAE/g dry plant material). Seasonal and daily fluctuations of the metabolite content of L. cuneifolia collected in different sites are shown in Supplementary Material Tables S1–S5.
To integrate all data, i.e., location and collection site, time of year and the most appropriate time for collection, a heat map was drawn. The rows depict different locations and collection sites, seasons and times of day for harvesting and the columns depict the chemical composition in each one. The results were conveyed by using a color scale, where red indicates an increase in phytochemical concentration and blue indicates a decrease.
Cluster analyses based on Euclidean distance facilitated grouping of extracts according to their phytochemical composition, revealing distinct patterns within the clusters. Figure S1, shows two primary clusters. Cluster 1 includes extracts with high concentrations of TPC, TF, reducing sugars and soluble proteins (warm colors) and corresponds mostly to L. cuneifolia samples from Fuerte Quemado and Los Poleos, obtained in autumn and winter in the morning and the afternoon. Cluster 2 comprises samples with low and medium concentrations of TPC, TF, reducing sugars, and soluble proteins (cool colors). This group mainly comprises samples from Tío Punco and Ampimpa, obtained in summer and spring at midday.
The heatmap clearly illustrates variation in the phytochemical composition of L. cuneifolia extracts.
A principal component analysis (PCA) was performed because it provides valuable insights into the impact of individual variables on overall variability, which a heatmap alone cannot offer (Figure 3).
This analysis shows that two components explain a total cumulative variance of 86.88%. The first component (PC1) explains over 64% of the cumulative variance and is positively correlated with TPC and TF while the second component (PC2) explains 22.53% of the overall variance and is influenced by proteins and reducing sugar content. Points closest to TPC and TF correspond to FQ and LP sites with higher concentrations of TPC and TF (green and light blue points). TPC and TF content are the variables most strongly correlated with PC1, indicating that they play a key role in explaining overall variability among the samples. This suggests that extracts with higher concentrations of phenolic compounds and flavonoids will cluster on the positive side of PC1.
Although the plant material collected at Fuerte Quemado and Los Poleos showed the highest concentration of phytochemicals, previous studies have shown that these sites have a very low population abundance [1] making sustainable collection of plant material from these sites unviable. Conversely, a high biodiversity index value was found for L. cuneifolia at Ampimpa Site 2 [1], but this area is very likely to be affected by urbanistic development in the near future. Consequently, a heat map was made (Figure 4) that excluded these sites considering only samples from Los Poleos Sites 1 and 2, due to their high phytochemical content; the same criterion was used with Tío Punco Site 1 and Ampimpa Site 1, due to their higher population abundance [1].
Thus, three clusters were observed: Cluster 1 presents the warmest tones (red orange) and depicts samples with the highest TPC and TF concentrations collected at Los Poleos site 1 and 2 in autumn and winter, and at Ampimpa site 1 in spring. Cluster 2 corresponds to the coolest colors (green blue) and depicts the samples with the lowest concentration of TPC and FT, collected at the same spot in winter. Cluster 3 corresponds to L. cuneifolia extracts with average TPC and TF values of samples collected at Ampimpa site 1 during winter, autumn, and summer.
Based on these results, we conclude that Ampimpa Site 1 could be selected for sustainable harvesting, given the abundance of its population and the levels of TPC and TF present in plant tissue throughout the year, particularly in spring (Cluster 1).
To confirm this, a new heat map was drawn, this time considering only Ampimpa site 1 in all four seasons and at different times of day. Figure 5 shows this new heat map, which reveals two clusters. Cluster 1 includes samples with high phytochemical content from the morning, midday, and afternoon collections in spring, autumn, and winter, while Cluster 2 consists of extracts with medium or low phytochemical contents from the remaining seasons and periods. The analysis of the results in the present work indicates spring is the most appropriate season for collecting plant material, regardless of the time of day. Furthermore, the concentration of specialized metabolites observed in the morning has intermediate values in autumn and winter.
The variation in accumulation levels of phytochemicals in the plants sampled at different times of day and year can directly be affected by changes in light quality, temperature, and water availability both throughout the day and year [18,19,20]. Following are two examples of how light quality is closely related to those levels. The blue light and other shorter wavelengths are strongly affected by Rayleigh scattering; thus, a lower abundance of these wavelengths in winter as well as at dawn and dusk leads to an increase in the relative amount of the longer, red wavelengths. A second instance is L. cuneifolia paraheliotropism; linked to solar movements, it orients its leaves reversibly, thus allowing for greater use of solar energy in the early hours of the day [24,25]. In the Monte Desert in the Calchaquí Valleys, winter and autumn are dry seasons, rainfall < 50 mm, with shorter days and, consequently, fewer hours of sunlight [26]. Accordingly, our results show a decrease in the content of reducing sugars in winter and autumn evenings. This decrease in the levels of precursor molecules of specialized metabolites probably explains, in part, the observed decrease in TPC and TF contents observed for the said seasons and times (Tables S2–S4). Conversely, Larrea divaricata, another Larrea species that grows in Patagonia in the South of Argentina, shows a higher production of TPC and TF in autumn and a lower one in winter and spring [27]. The authors attribute this increase to mechanisms of resistance to water stress.
As previous studies have reported that this species primarily accumulates bioactive lignans [3,6,7,8,15], these compounds were quantified by using spectrophotometric methods, and HPLC-DAD on the extracts obtained from samples collected at Ampimpa S1. This was performed during the spring three times a day, and during the autumn and winter, in the morning, to determine the most suitable season to obtain lignan-rich extracts.
Total lignan quantification indicates that levels remain at around 497–582 µg NDHGAE/g dry weight of extract, except in L. cuneifolia samples collected in autumn and winter afternoons, when concentrations decrease by around 47% and 25%, respectively (Table 1). The expression of genes involved in the lignans biosynthesis pathway as well as in other phenolic compounds, is frequently altered by abiotic stress, light type and photoperiod regimes [18,19,20,28]. At the same time, lignans participate in the synthesis of other chemical components, resulting in a gradual decrease in their content in two seasons (autumn and winter) [29]. The lignans can contribute to the plant’s defense by being incorporated into cell walls, making them slightly more resistant to degradation, and reinforcing the plant’s structural barriers against pathogens and herbivores.
HPLC-DAD profiles (Figure 6, Table 2) revealed the presence of seven lignans, including NDHGA, epoxylignans, and cyclolignans. NDHGA was more abundant in spring regardless of the time of day (254–292 μg/mg DW). However, a maximum accumulation of other lignans, such as 3,4,3′,4′-tetrahydroxy-6,7′ cyclolignan and 4-[4-(4-hydroxyphenyl)-2,3 dimethyl butyl [benzene-1,2-diol] was also observed in the morning. Based on these results, the most suitable conditions for a sustainable harvest of L. cuneifolia appear to be in spring, preferably in the morning.
The presence of high levels of NDGA and its derivatives confirms the medicinal importance of this species, as it exhibits chemopreventive and antimicrobial activities, effects on fertility and reproduction, as well as hypoglycemic activity, larvicidal activity and activity against Botrytis cinerea [12,30,31,32,33,34,35,36]. Antifungal and antioxidant activity has been previously described for ethanolic extracts of L. cuneifolia [7,12,13,14].

2.2. Biological Activity

The antioxidant activity of ethanolic extracts of L. cuneifolia from the Ampimpa site 1 location was found to have scavenging concentration values of 50% ABTS (SC50 values) ranging from 1.56 to 2.32 µg GAE/mL in samples collected during summer months and spring morning. In contrast, during winter and autumn, the SC50 values ranged from 3.07 to 4.9 µg GAE/mL (Table 3). The higher antioxidant capacity was recorded in spring (morning and midday). These results coincide with a higher NDHGA content and surpass the values reported by Moreno Et Al. (2018) [7].

3. Materials and Methods

3.1. Chemicals, Reagents and Materials

Ethanol was purchased from Cicarelli (Santa Fé, Argentina). Folin–Ciocalteau reagent, AlCl3, gallic acid, quercetin, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), glucose, bovine serum albumin and HPLC grade solvent and standards were obtained from Sigma Aldrich, St. Louis, MO, USA.

3.2. Plant Material

Aerial parts of L. cuneifolia (Figure 1) were collected in the summer, autumn, winter and spring of 2022 and 2023, in the morning (8 a.m.), midday (1 p.m.) and afternoon (6 p.m.) at different locations: Ampimpa Site 1 (26°35′33″ S 65°55′00″ W; 1960 m a.s.l), Ampimpa Site 2 (26°35′23″ S 65°53′14″ W; 2109 m a.s.l), Tío Punco Site 1 (26°31′58″ S 65°57′53″ W; 1823 m a.s.l), Tío Punco Site 2 (26°31′34″ S 65°57′41″ W; 1816 m a.s.l), Fuerte Quemado Site 1 (26°34′31″ S 66°03′00″ W; 1855 m a.s.l), Fuerte Quemado Sitio2 (26°34′46″ S 66°03′27″ W; 1866 m a.s.l), Los Poleos Sitios1 (26°37′33″ S 65°57′33″ W; 1954 m a.s.l) and Los Poleos Sitio 2 (26°37′52″ S 65°57′52″ W; 1949 m a.s.l), Tucumán, Argentina (Figure 7). A field identification was performed by the authors using fresh material and native flora guide [37]. The determinations were subsequently confirmed by experts from the Phanerogamic Herbarium of the Miguel Lillo Foundation (LIL), San Miguel de Tucumán, Argentina [1].
Five plots were defined at each site, and three mid-canopy samples, which were subsequently pooled for processing, were taken from individuals of similar size in each plot. Collecting was performed in summer, autumn, winter and spring three times a day (morning, midday and afternoon). This resulted in three samples from each season on each site (3 times during the day × 4 seasons × 8 sites). The samples were 96 altogether.
The samples were dried until constant weight was reached in a forced air oven (Dhacel, FD101) at 40 °C, after which they were ground in a helical mill (Numak, F 100 Power ½ hp-0.75 KW, Brusque, Brazil). The powder was vacuum-sealed and stored at room temperature to protect the plant material from moisture, as well as physical and microbial contamination. Control specimens were deposited in the herbarium of the Miguel Lillo Foundation, Tucumán, Argentina.

3.3. Extract Preparation

Ethanolic extracts were prepared by using 1 g of plant material (96 samples) in 20 mL of 60° ethanol. The mixture was then macerated in an ultrasonic bath (Ultrasonic Washer Arcano Model PS-10A, Shandong Arcano Ultrasonic Technology Co., Ltd., Jinan, China) for 30 min at 40 °C. The extracts were then vacuum-filtered and dried in a vacuum oven (Arcano DF2-6020, Cientifica Dominguez, San Miguel de Tucumán, Argentina), at 40 °C until they reached a constant weight. The dried extracts were stored at −20 °C until use (g DW or g dry weight of extract). All extracts were obtained in triplicate.

3.4. Phytochemical Analysis

3.4.1. Quantification of Total Phenolic Compounds

The total phenolic compound (TPC) content of each sample (96 extracts) was determined by using the Folin–Ciocalteau reagent [38]. Samples (1 mL of different dilutions of each extract) were oxidized with 0.1 mL of Folin–Ciocalteau reagent (Sigma-Aldrich), and then the reaction was neutralized with 0.4 mL of 15.9% sodium carbonate and kept at room temperature for 20 min. Measurements were performed by using a UV-visible spectrophotometer (Jasco V-630 Thermo Fisher Scientific, Tokyo, Japan) at 765 nm. Gallic acid was used as the reference compound.
Measurements were performed in triplicate on all extracts, and the results were expressed as milligrams of gallic acid equivalent (mg GAE) per gram of dry plant material (mg GAE/g PM) (Sigma Aldrich, St. Louis, MO, USA). The chemical stability of the plant material was assessed throughout a three-year period.

3.4.2. Quantification of Total Flavonoids

The total flavonoid (TF) content of each extract (96 extracts) was determined according to the AlCl3 method [39]. Samples (1 mL of different dilutions of each extract) were added with 1.5 mL of ethanol 96° and 0.05 mL of AlCl3 5%. The reaction was kept at room temperature for 30 min. Measurements were performed using a UV-visible spectrophotometer (Jasco V-630 Thermo Fisher Scientific, Tokyo, Japan) at 425 nm. Quercetin was used as the reference compound (Sigma Aldrich, St. Louis, MO, USA). Measurements were performed in triplicate on all extracts, and the results were expressed as milligrams of quercetin equivalent (mg QE) per gram of dry plant material (mg QE/g PM). The chemical stability of the plant material was assessed throughout a three-year period.

3.4.3. Quantification of Total Lignans

To determine total lignan (TL) content, appropriate dilutions of 2 mL of each L. cuneifolia extract were performed, and their absorbance was read at 282 nm by using a UV-visible spectrophotometer (Jasco V-630 Thermo Fisher Scientific, Tokyo, Japan). A calibration curve of nordihydroguaiaretic acid (NDGA) (Sigma Aldrich, St. Louis, MO, USA) was drawn. The results were expressed as milligrams of nordihydroguaiaretic acid equivalents per gram of dry plant material (µg NDGAE/g PM). The chemical stability of the plant material was assessed throughout a three-year period.

3.5. Chromatographic Profile, HPLC-DAD

L. cuneifolia extracts were analyzed by means of high-performance liquid chromatography (HPLC), using a system including a Waters 1525 binary pump and a manual injection valve (Rheodyne Inc., Cotati, CA, USA) with a 20 μL loop. The separation was performed on a Waters C18, 5 μ, 4.6 × 250 mm column, kept at 25 °C. Analyses were performed by using a linear gradient composed of 1% formic acid in water (A) and acetonitrile (B) as follows: 30% to 40% B over 35 min, increasing to 45% B at 50 min, 70% B at 70 min, 70% to 100% B from 70 to 80 min, 100% to 30% B from 80 to 85 min, and keeping the 30% until the end at 95 min. The flow rate was 0.5 mL/min and the injected volume was 20 µL. Compounds were monitored at 280 nm. Compounds present in the extracts were identified by comparing retention times and UV-visible profiles with those of commercial reference compounds. Quantification of the different compounds was performed in NDGA equivalent to 280 nm. The chemical stability of the plant material was assessed throughout a three-year period.

3.6. Macronutrient Quantification

3.6.1. Quantification of Reducing Sugars

Reducing the sugar content of each extract (96 extracts) was quantified by using the Somogyi-Nelson method [40,41]. Aliquots of each sample in a final volume of 500 µL were added to 500 µL of Somogyi’s reagent (1 part of 10% CuSO4.5H2O plus 9 parts of Na+ and K+ tartrate solution). The mixture was incubated at 100 °C for 15 min. Then, 500 µL of Nelson’s reagent (a solution of ammonium molybdate and disodium arsenate in sulfuric acid) and 1000 µL of distilled water were added. The absorbance was measured in a UV-Visible spectrophotometer (Jasco V-630 Thermo Fisher Scientific, Tokyo, Japan) at 520 nm. The reference standard curve was prepared with different concentrations of 1 mM glucose. Measurements were performed in triplicate and expressed as milligram glucose equivalent (GE) per gram of dry plant material (mg GE/g PM). The chemical stability of the plant material was assessed throughout a three-year period.

3.6.2. Quantification of Soluble Proteins

Proteins were quantified by using the Bradford method [42]. Appropriate volumes of each extract were diluted in distilled water up to 800 µL. Then, 200 µL of Bradford’s reagent was added and incubated for 10 min at room temperature. Measurements were performed with a UV-visible spectrophotometer (Jasco V-630 Thermo Fisher Scientific, Tokyo, Japan) at 595 nm. Bovine serum albumin (BSA) was used as the reference compound. Measurements were performed in triplicate on all extracts (96 extracts) and expressed as milligrams of BSA equivalent per gram of dry plant material (mg BSAE/g PM). The chemical stability of the plant material was assessed throughout a three-year period.

3.7. Determination of Antioxidant Activity

The antioxidant activity of the ethanolic extracts of L. cuneifolia was assessed by using the ABTS (2,2-azinobis-(3-ethylbenzothioazolin-6-sulfonic acid) scavenging method (Sigma Aldrich, St. Louis, MO, USA) according to Re et al. (1999) [43]. The ABTS•+ radical is generated by reacting 7 mM ABTS (2,2′-azinobis (3-ethylbenzothiazolinone-6-sulfonic acid) with 2.45 mM potassium persulfate and incubating at room temperature in the dark for 16 h. The ABTS•+ solution is diluted with 95% ethanol until an absorbance of 0.90 at 734 nm is reached.
For the reaction mixture, 100 μL of different dilutions of extracts were added to 200 μL of the working solution and mixed by shaking. The reaction was carried out at room temperature. Absorbance was recorded at 764 nm after 6 min (Thermo Scientific Multiskan GO, Vanta, Finland). Results are expressed as SC50 values (µg GAE/mL), defined as the concentration of phenolic compounds required to eliminate 50% of the ABTS free radicals. Quercetin was used as the reference compound.

3.8. Statistical Analysis

A one-way analysis of variance (ANOVA) was used to assess the significance of the influence of each independent variable separately (season and time of day), followed by Tukey’s post hoc test. All results were expressed as mean ± standard deviation. Differences between samples were considered statistically significant at p ≤ 0.05. Statistical analyses were performed by using InfoStat V1.1 [44]. A heatmap was drawn with Euclidean distance and Ward’s clustering algorithm on the standardized dataset, performed in R software 3.0.2 [45]. Principal component analysis (PCA) was also performed. The score plot was performed based on the first and second principal components (PCs).

4. Conclusions

Based on the results obtained, we can conclude that harvesting the aerial parts of L. cuneifolia in spring and especially during the early morning ensures the highest efficiency when extracting phytochemicals, particularly lignans, compounds recognized for their biological activity. Our results emphasize the importance of combining ecological and phytochemical data when devising sustainable harvesting strategies in fragile environments such as the Monte Desert in the Calchaquies Valleys.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14213332/s1, Table S1. Content of soluble principles in ethanol. Table S2. Phenolic compound content in L. cuneifolia samples collected in different seasons and at different times of the day. Table S3. Flavonoid content in L. cuneifolia samples collected in different seasons and at different times of the day. Table S4. Content of reducing sugars in samples of L. cuneifolia collected in different seasons and at different times of the day. Table S5. Protein content in L. cuneifolia samples collected in different seasons and at different times of the day. Figure S1. Heat map of the phytochemical composition of L. cuneifolia aerial parts collected at different sites. locations. seasons and times of day.

Author Contributions

Conceptualization, M.C.B., M.D.R., I.C.Z. and M.I.I.; methodology, M.C.B., M.D.R., I.C.Z. and M.I.I.; validation, M.I.I. and M.D.R.; formal analysis, experiments performed: M.C.B., M.D.R., I.C.Z. and M.I.I.; analyzed and interpreted the M.C.B., M.D.R., I.C.Z. and M.I.I.; resources, M.I.I. and I.C.Z.; data curation, M.C.B. and M.I.I.; writing—original draft preparation, M.C.B. and M.I.I.; writing—review and editing, M.C.B., M.D.R., I.C.Z. and M.I.I.; supervision, M.I.I. and M.D.R.; project administration, M.I.I.; funding acquisition, M.I.I. and I.C.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by Universidad Nacional de Tucumán (PIUNT 2022-G727 Project), Agencia Nacional de Promoción Científica y Técnica (ANPCyT PICT 2020-3619; PICT-2021-CAT-II-00132), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-PUE 2018-0011), Fundación Williams 2023. Fondos complementarios para proyectos de investigación con impacto en el territorio argentino and Biolates network “P320RT0186—Sustainable use of Ibero-American vegetable biomass resources in cosmetics” (CYTED).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to thank Universidad Nacional de Tucumán, Consejo Nacional de Investigaciones Científicas y Técnicas, Ministerio de Ciencia y Técnica, CYTED network “P320RT0186— Sustainable use of Ibero-American vegetable biomass” (BIOLATES) and Fundación Williams 2023. The authors wish to express their gratitude to the INBIOFIV fellows and researchers and to Norma Cudmani for their collaboration in the plant material collection campaigns. This was carried out in accordance with the Nagoya Protocol. We extend our sincere gratitude to the community of the Calchaquí Valleys (Tucumán, Argentina) for their invaluable cooperation and support. The language of the manuscript was reviewed by Luisa Montivero.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Photograph of Larrea cuneifolia in Monte region from Argentina. (A) summer (B) spring (C) winter (D) autumn.
Figure 1. Photograph of Larrea cuneifolia in Monte region from Argentina. (A) summer (B) spring (C) winter (D) autumn.
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Figure 2. Flowchart of procedure to obtain and characterize L. cuneifolia extracts.
Figure 2. Flowchart of procedure to obtain and characterize L. cuneifolia extracts.
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Figure 3. Principal Component Analysis (PCAs) of the phytochemical composition of aerial parts of L. cuneifolia collected at Ampimpa site 1 y2, Tío Punco site 1 y 2, Fuerte Quemado site 1 y 2, and Los Poleos site 1 y 2. A1, Ampimpa site 1; A2, Ampimpa site 2; TP1, Tío Punco site 1; TP2, Tío Punco site 2; FQ1, Fuerte Quemado site 1; FQ2, Fuerte Quemado site 2; LP1, Los Poleos site 1; LPS2, Los Poleos site 2; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; FT, total flavonoids; RS, reducing sugars; P, soluble proteins.
Figure 3. Principal Component Analysis (PCAs) of the phytochemical composition of aerial parts of L. cuneifolia collected at Ampimpa site 1 y2, Tío Punco site 1 y 2, Fuerte Quemado site 1 y 2, and Los Poleos site 1 y 2. A1, Ampimpa site 1; A2, Ampimpa site 2; TP1, Tío Punco site 1; TP2, Tío Punco site 2; FQ1, Fuerte Quemado site 1; FQ2, Fuerte Quemado site 2; LP1, Los Poleos site 1; LPS2, Los Poleos site 2; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; FT, total flavonoids; RS, reducing sugars; P, soluble proteins.
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Figure 4. Heatmap depicting the phytochemical composition of aerial parts of L. cuneifolia, collected in A1, Ampimpa site 1; TP1, Tío Punco site 1; LP1, Los Poleos site 1; LPS2, Los Poleos site 2; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; TF, total flavonoids.
Figure 4. Heatmap depicting the phytochemical composition of aerial parts of L. cuneifolia, collected in A1, Ampimpa site 1; TP1, Tío Punco site 1; LP1, Los Poleos site 1; LPS2, Los Poleos site 2; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; TF, total flavonoids.
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Figure 5. Heat map depicting the phytochemical composition of aerial parts of L. cuneifolia extracts, collected at Ampimpa site 1 in different seasons and times of day. A1, Ampimpa site 1; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; TF, total flavonoids.
Figure 5. Heat map depicting the phytochemical composition of aerial parts of L. cuneifolia extracts, collected at Ampimpa site 1 in different seasons and times of day. A1, Ampimpa site 1; I, winter; O, autumn; P, spring; V, summer; M, morning; MM, midday; T, afternoon; TPC, total phenolic compounds; TF, total flavonoids.
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Figure 6. (I) HPLC profile of L. cuneifolia extracts, collected at Ampimpa S1 in (A) autumn/morning; (B) spring/morning; (C) spring/midday; (D) spring/afternoon; (E) winter/morning. Peak 1: 3,3′,4′-trihydroxy-4 methoxy 7,7′-epoxylignan; peak 2: 3,4,3′,4′-tetrahydroxy-6,7′ cyclolignan; peak 3: Nordihydroguaiaretic acid; peak 4: 3,3′-dihydroxy-4-methoxy 7,7′- epoxylignan; peak 5: 4-[4-(4-hydroxy-phenyl)-2,3 dimethyl-butyl]-benzene-1,2-diol; peak 6: Methyl nordihydroguaiaretic acid; peak 7: Methyl nordihydroguaiaretic acid isomer. (II) UV absorption spectra (196–375 nm) of peak 2 (A) and peak 3 (B).
Figure 6. (I) HPLC profile of L. cuneifolia extracts, collected at Ampimpa S1 in (A) autumn/morning; (B) spring/morning; (C) spring/midday; (D) spring/afternoon; (E) winter/morning. Peak 1: 3,3′,4′-trihydroxy-4 methoxy 7,7′-epoxylignan; peak 2: 3,4,3′,4′-tetrahydroxy-6,7′ cyclolignan; peak 3: Nordihydroguaiaretic acid; peak 4: 3,3′-dihydroxy-4-methoxy 7,7′- epoxylignan; peak 5: 4-[4-(4-hydroxy-phenyl)-2,3 dimethyl-butyl]-benzene-1,2-diol; peak 6: Methyl nordihydroguaiaretic acid; peak 7: Methyl nordihydroguaiaretic acid isomer. (II) UV absorption spectra (196–375 nm) of peak 2 (A) and peak 3 (B).
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Figure 7. Locations of L. cuneifolia harvesting in Valles Calchaquies, Tucumán, Argentina.
Figure 7. Locations of L. cuneifolia harvesting in Valles Calchaquies, Tucumán, Argentina.
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Table 1. Total lignan content in L. cuneifolia samples collected at Ampimpa site 1 at different times of the year (summer, autumn, winter and spring) and hours of the day (morning, midday and afternoon) by using a spectrophotometric method.
Table 1. Total lignan content in L. cuneifolia samples collected at Ampimpa site 1 at different times of the year (summer, autumn, winter and spring) and hours of the day (morning, midday and afternoon) by using a spectrophotometric method.
Total Lignans (µg NDGAE/g DW)
SummerAutumnWinterSpring
Morning518.5 ± 5.78 Ba496.60 ± 3.93 Ab511.24 ± 3.53 Bb517.84 ± 7.06 Ba
Midday558.7 ± 6.12 Cb533.24 ± 2.30 Bc519.73 ± 4.63 Ab564.36 ± 6.11 Cb
Afternoon517.42 ± 2.61 Ca282.59 ± 5.79 Aa384.89 ± 4.81 Ba581.84 ± 3.12 Db
NDGAE, nordihydroguaiaretic acid equivalent; DW, dry weight of extract. Data were analyzed by one-way ANOVA for each factor (season and time of day) independently, followed by Tukey’s multiple comparison test (p ≤ 0.05). Different capital letters in the same line indicate significant differences between seasons, and lowercase letters indicate significant differences between times of day.
Table 2. Quantification of the different compounds identified in the extracts of L. cuneifolia from samples collected in autumn, spring and winter at different times of the day using HPLC-DAD.
Table 2. Quantification of the different compounds identified in the extracts of L. cuneifolia from samples collected in autumn, spring and winter at different times of the day using HPLC-DAD.
Compounds AutumnSpringWinter
MorningMorningMiddayAfternoonMorning
RT. (Minutes)ABCDE
µg of Lignans in NDGA Equivalent/mg of Soluble Principle (Dry Weight of Extract)
13,3′,4′-trihydroxy-4 methoxy 7,7′-epoxylignan31.510.2 ± 4.710.1 ± 4.89.9 ± 1.611.8 ± 7.98.5 ± 5.5
23,4,3′,4′-tetrahydroxy-6,7′ cyclolignan34.221.2 ± 3.673.0 ± 0.220.8 ± 0.024.6 ± 2.019.5 ± 5.2
3Nordihydroguaiaretic acid41.19182.1 ± 2.9273.4 ± 2.5291.8 ± 2.8253.6 ± 3.4187.6 ± 3.1
43,3′-dihydroxy-4-methoxy 7,7′- epoxylignan47.74.2 ± 0.38.6 ± 0.3 9.3 ± 2.58.8 ± 4.86.5 ± 0.5
54-[4-(4-hydroxy-phenyl)-2,3 dimethyl-butyl]-benzene-1,2-diol53.710.1 ± 1.440.6 ± 4.214.6 ± 1.713.6 ± 1.38.3 ± 5.2
6Methyl nordihydroguaiaretic acid55.812.8 ± 3.614.8 ± 3.918.2 ± 5.117.5 ± 1.110.8 ± 0.5
7Methyl nordihydroguaiaretic acid isomer59.620.5 ± 0.528.4 ± 5.233.4 ± 1.330.9 ± 3.021.3 ± 3.7
RT, retention time; A: autumn/morning extract; B: spring/morning extract; C: spring/midday extract; D: spring/afternoon extract; E: winter/morning extract; NDGA, nordihydroguaiaretic acid.
Table 3. Antioxidant activity of extracts obtained from plant material from the Ampimpa Site, at different times of the year (summer, autumn, winter and spring) and hours of the day (morning, midday and afternoon).
Table 3. Antioxidant activity of extracts obtained from plant material from the Ampimpa Site, at different times of the year (summer, autumn, winter and spring) and hours of the day (morning, midday and afternoon).
SC50 (µg GAE/mL)
SummerAutumnWinterSpring
Morning2.323 ± 0.012 Ba3.838 ± 0.122 Db3.227 ± 0.011 Cb1.560 ± 0.021 Aa
Midday2.250 ± 0.009 Ba3.434 ± 0.009 Da3.071 ± 0.023 Ca1.647 ± 0.155 Aa
Afternoon2.299 ± 0.092 Aa4.977 ± 0.043 Cc3.644 ± 0.072 Bc2.116 ± 0.185 Ab
GAE, gallic acid equivalent; Data were analyzed by one-way ANOVA for each factor (season and time of day) independently, followed by Tukey’s multiple comparison test (p ≤ 0.05). Different capital letters in the same line indicate significant differences between seasons, and lowercase letters indicate significant differences between times of day.
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Barrera, M.C.; Rosa, M.D.; Zampini, I.C.; Isla, M.I. Impact of Daily and Seasonal Variation on the Phytochemical Profile of Larrea cuneifolia in Northwestern Argentina. Plants 2025, 14, 3332. https://doi.org/10.3390/plants14213332

AMA Style

Barrera MC, Rosa MD, Zampini IC, Isla MI. Impact of Daily and Seasonal Variation on the Phytochemical Profile of Larrea cuneifolia in Northwestern Argentina. Plants. 2025; 14(21):3332. https://doi.org/10.3390/plants14213332

Chicago/Turabian Style

Barrera, María Celeste, Mariana Daniela Rosa, Iris Catiana Zampini, and María Inés Isla. 2025. "Impact of Daily and Seasonal Variation on the Phytochemical Profile of Larrea cuneifolia in Northwestern Argentina" Plants 14, no. 21: 3332. https://doi.org/10.3390/plants14213332

APA Style

Barrera, M. C., Rosa, M. D., Zampini, I. C., & Isla, M. I. (2025). Impact of Daily and Seasonal Variation on the Phytochemical Profile of Larrea cuneifolia in Northwestern Argentina. Plants, 14(21), 3332. https://doi.org/10.3390/plants14213332

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