Reagents

All reagents and solvents used in the study were of analytical grade. Distilled H₂O, NaNO₂ (Sigma-Aldrich, 237213), HCl (HYCEL, 51349-2N19), CH₃COOH (J.T. Baker, 10433191), NaOH (Karal, 6080) and L-DOPA standard (Sigma-Aldrich, PHR1271, with a purity of 99.7 %) were used.

Figure 1

Faba bean-producing localities in the central-western part of the state of Puebla.

Plant material

During November - December 2022 twenty-four local faba bean varieties were collected in seven localities in the municipalities of Tlahuapan and Chiatzingo in Puebla (Figure 1 ,Table 1). Municipalities from the central-western part of the state of Puebla that had high production of green faba bean + grain faba bean consecutively during the period 2015 - 2019 (SIAP & Servicio de Información Agroalimentaria y Pesquera, 2023).

Environmental Variables of the Localities

The values of the environmental variables of the study localities—mean annual temperature (MAT), mean annual precipitation (MAP), and mean annual solar radiation (MASR)—were obtained using metadata from the 2023 Geoinformation Portal of the National Commission for the Knowledge and Use of Biodiversity (CONABIO & , 2023). Subsequently, the necessary information for each locality was extracted using ArcMap software version 10.8. Altitude data were obtained from records provided by the National Institute of Statistics and Geography (INEGI, 2012) (Table 1).

Morphological Characterization of Faba Bean Seeds

To measure seed dimensions, the methodology of Salamanca-Bautista, Delgado-Alvarado, Herrera-Cabrera, Mendo-Za-Castillo, and Conde-Martínez (2018) was used with modifications. From each local faba bean variety, 10 seeds were randomly selected, and their length and width were recorded using a digital caliper. This procedure was repeated three times for each set of 10 seeds. To determine the weight of 100 seeds (W100S), 100 seeds were randomly selected and weighed using a precision balance (OHAUS/H-5852), with the measurement performed in triplicate.

Sowing

Seeds from the 24 local varieties were sown on February 16, 2023, in the experimental field of the Center for Technological Innovation in Protected Agriculture (CITAP) (18° 55' 52” N, -98° 23' 59” W). The soil was prepared (plowing, harrowing and furrowing) and moistened three days before sowing. The sowing was conducted using a randomized complete block design, following the farmer’s traditional tillage practices. Seeds were planted at a depth of 5 cm, with two seeds per hill and a spacing of 20 cm between hills. Each plot (120 x 60 cm) represented one replicate (28 seeds), and three replicates per local variety were established. During plant development, the field was irrigated by drip with potable water for 2 hours in the morning, three times per week (Figure 2). The species authenticity of the plant material used was based on the morphological descriptors of the International Board for Plant Genetic Resources (IBPGR, 1985) and Duc (1997).

Figure 2

Sowing and development of the V. faba plant.

Agro-Environmental Variables

Temperature (°C), relative humidity (%RH), and solar radiation (W/m²) (Figure 3) were recorded every hour during the cultivation of the faba bean plants using a weather station (ADCON TELEMETRY, addVANTAGE Pro 6.5) installed at the CITAP experimental field.

Plant Age Identification, Collection, and Processing of Plant Material

At the emergence stage, when the shoot breaks through the soil surface (stage 09) (Meier, 2018), the shoots were marked with colored raffia to facilitate the collection of plants 20 days after emergence (DAE ± 1). The plants were harvested approximately one month after sowing. Five plants per plot were harvested, with roots removed by a transverse cut. The plants were then weighed using a precision balance (OHAUS/H-5852) and dried in the shade at room temperature (a process lasting between 20 and 30 days, depending on plant size). Once dried, the plants were weighed again, and the moisture content (%H) was measured using a thermobalance (Adam, PMB 53, Milton Keynes, U.K). This process allowed for the determination of biomass per plant in terms of dry matter, with results reported as the mean value of five plants per plot. The dried plants were subsequently ground using a coffee grinder (Hamilton Beach, Model: 80393, U.S.A.), and the resulting powder was sieved with a No. 20 mesh (841 μm) and stored in food-grade polypropylene bags under refrigeration (4 ± 1 ºC) for further analysis.

Figure 3

Data of agro-environmental variables during the cultivation of V. faba plants 20 days after emergence. Each box represents 24 data per day. The first and third quartiles represent the largest distribution of values, while the second quartile indicates the median of values. The arms represent the minimum and maximum values and the outer points represent the outliers.

L-DOPA Content

Statistical Analysis

Twenty-four treatments with three replications each were evaluated. The morphological variables of V. faba seeds, along with the results of biomass, L-DOPA content, and yield in V. faba plants, were analyzed using one-way ANOVA (PROC ANOVA) with the SAS statistical package (SAS version 9.4, SAS Institute, Cary, NC, USA). Significant differences between treatments were determined using Tukey's Honestly Significant Difference (HSD) test (p ≤ 0.05). Additionally, multivariate principal component analysis (PCA) and cluster analysis (applying Ward's method and squared Euclidean distance) were performed using IBM SPSS Statistics for Windows, version 27 (IBM Corp., Armonk, NY, USA) and JMP (Pro version 16), utilizing the mean values of environmental variables (Table 1), morphological variables, faba bean plant biomass, L-DOPA content, and yields. Subsequently, the most common characteristics in each group were identified through means testing and bidirectional clustering.

Characteristics of Faba Bean Seeds

The morphological variables—width, length, and hundred-seed weight—showed significant differences (p ≤ 0.01) among the local varieties. Local variety T1 exhibited the highest values for width (19.76 mm), length (30 mm), and hundred-seed weight (W100S) (295.82 g). Conversely, local variety C4 had the lowest values for width (13.89 mm) and length (20.92 mm), while local variety T12 had the lowest W100S (122.37 g) (Table 2). In terms of seed dimensions and hundred-seed weight, and considering seed size characteristics from other studies on Mexican bean cultivars (Herrera-Cabrera, Miranda-Trejo, & Delgado-Alvarado, 2010; Salamanca-Bautista et al., 2018), the seeds were classified into small, medium, and large categories (Table 2).

Biomass, L-DOPA Content, and Yield

Plant biomass exhibited significant differences (p ≤ 0.05) among local varieties, while L-DOPA content and yield showed highly significant differences (p ≤ 0.01) among local varieties. Plant biomass ranged widely from 1.56 to 3.76 g DM, with local variety T1 having the highest value, though it was statistically different only from local variety C5 (Table 3). The L-DOPA content in the local varieties ranged from 127.91 to 179.52 mg g⁻¹ DM, with a mean of 154.51 mg g⁻¹ DM (Figure S1, Appendix A). Local variety T7 had the highest L-DOPA content, but it was not significantly different (p ≥ 0.05) from eighteen other local varieties (T1, T2, T3, T4, T6, T9, T10, T11, T12, T14, T15, T16, T17, T18, C1, C4, C5, C6).

Initial trials of L-DOPA for PD in the 1960s revealed transient improvements in rigidity and tremor but were limited by side effects such as nausea and vomiting. The addition of a DOPA decarboxylase inhibitor (carbidopa or benserazide) in 1967 was a breakthrough, reducing peripheral metabolism of L-DOPA to dopamine and minimizing side effects, thereby enhancing its ability to cross the blood-brain barrier (Riederer & Horowski, 2023). It remains uncertain whether L-DOPA from plants would require co-administration with carbidopa or if it could be effective on its own, given that plant tissue also contains other bioactive compounds that might provide additional benefits for PD (Fuentes-Herrera et al., 2022). It is important to consider that each patient requires specific doses, so it is necessary to know the contents of this bioactive in the plant, given that, patients on existing medication might experience dopaminergic overstimulation if consuming natural L-DOPA sources (Ramírez-Moreno, I, O, Y, & , J. M., Salguero B. I., ., y . 2015. . , 30 (6), 375 - 391. DOI: 10.1016/j.nrleng.2013.08.006, 2015).

The L-DOPA content in the faba bean plants, with some local varieties showing up to twice the content reported by Fuentes-Herrera et al. (2023) (67.30 - 88.04 mg g^-1 DM). This discrepancy may be attributed to differences in the varieties used and the drying methods employed. In this study, plants were dried in the shade at room temperature (22 - 25°C), whereas Fuentes-Herrera et al. (2023) used a drying temperature of 38°C. L-DOPA is known to be thermolabile and can degrade with increased temperature. For example,Kai-Bin, Xiao-Yu, Shan-Lian, and Ai-Ping (2016) found that drying at temperatures of 40 - 100°C reduced L-DOPA content by 60% in V. faba flowers, likely due to oxidative decomposition or accelerated degradation of the metabolite.Zhou, Alany, Tuan, and Wen (2012) also demonstrated that L-DOPA undergoes oxidation through thermal action (37 - 80°C), following first-order kinetics.

Regarding L-DOPA yield, a wide range was observed among the local varieties (223.27 - 584.21 mg plant⁻¹), with local variety T1 showing the highest yield per plant and local variety C5 the lowest (Table 3).

Both metabolite content and plant biomass are essential parameters for evaluating the yields of bioactive compounds (Fuentes-Herrera et al., 2023). In this study, plant biomass was found to be a determinant of L-DOPA yield. For instance, local variety T1, despite not having the highest L-DOPA content (155.80 mg g^-1 DM), exhibited the highest L-DOPA yield (584.21 mg plant^-1) due to its superior biomass (3.76 g DM). Conversely, local variety T7, which had the highest L-DOPA content (179.52 mg g^-1 DM), had a lower biomass (2.09 g DM), resulting in a lower L-DOPA yield (363.86 mg plant^-1) (Table 3). This underscores the critical role of biomass in determining L-DOPA yield. Similarly,Etemadi, Hashemi, Randhir, Zandvakili, and Ebadi (2018) reported that while drought stress increased L-DOPA concentrations in faba bean seedlings, it compromised metabolite yield due to reduced biomass production. Thus, higher biomass correlates with higher L-DOPA yields (Table S1, Appendix A).

Although the study identified local varieties with high L-DOPA content (e.g., T7) and yield (e.g., T1), no statistically significant differences were found among several local varieties (Table 3). Consequently, local faba bean varieties from these production localities could potentially be cultivated to produce L-DOPA for human consumption or to develop supplements or nutraceuticals for PD treatment or other ailments, since it has also been documented that L-DOPA has also been shown to improve male sexual behavior and reproductive functions (Tangsrisakda et al., 2022). Given the complexity and expense of synthetic L-DOPA production, deriving L-DOPA from natural sources such as plant tissue is more feasible, providing an economical and enantiometrically pure product (Patil, Apine, Surwase, & Jadhav, 2013; Tesoro et al., 2022). Cultivating faba bean plants to produce biomass for L-DOPA could also enhance economic opportunities for bean producers.

Principal Component Analysis and Cluster Analysis

The dispersion of the 24 local V. faba varieties represented in the space by the first three principal components explained 87.25% of the cumulative variation of the 11 variables analyzed (Table 4). The first principal component (PC1) explained 62.47% of the total variation and was primarily associated with seed morphological variables, environmental variables, and L-DOPA yield. The second principal component (PC2) accounted for 13.88% of the explained variation and was associated with L-DOPA content. The third principal component (PC3) explained 10.90% of the total variation and was mainly associated with plant biomass. For each component, this association was considered by taking as a selection threshold the eigenvectors, positive or negative, greater than 0.570 (Table 4).

Based on the configuration obtained from the first three principal components, five groups were differentiated (Figure 4). Group one (G I) consisted of local variety T1. Group two (G II) was composed of local varieties T2, T4, T7, T10, and T16. Group three (G III) comprised local varieties T3, T6, T8, T9, T11, T13, T14, T15, and T17. Group four (G IV) included local varieties T5, T12, T18, and C1, while group five (G V) consisted of local varieties C2, C3, C4, and C5 (Figure 4).

Figure 4

Dispersion of twenty-four local V. faba varieties defined by the first three principal components (PC) from seven bean-producing localities in the state of Puebla, Mexico.

The groups located on the positive axis of PC1 are characterized by having large-sized local varieties with high L-DOPA yields (G I and G II). In contrast, groups on the negative axis consist of local varieties from environments with a mean annual temperature higher than 13°C, mean annual precipitation around 900 mm, and mean annual solar radiation (MASR) of 22 MJ/m² (G V). The groups located on the positive axis of PC2 include local varieties that produce a high amount of L-DOPA (G II), whereas those on the negative side produce less (G I). In PC3, the groups located on the positive axis consist of cultivars that develop a high amount of plant biomass (G I and G III) (Figure 4).

Cluster analysis shows that, with a squared Euclidean distance of 3.98 in the dendrogram, four clusters were observed, in which the local varieties of Groups G I and G II from the principal component analysis were united into a single group. At a squared Euclidean distance of 3.60, the five clusters previously defined by the principal component analysis were identified, corroborating the results obtained (Figure 5).

Figure 5

Bidirectional hierarchical clustering constructed using Ward's method of twenty-four local V. faba varieties from seven producing localities in the state of Puebla, Mexico.

Using the test of means (Table S2, Appendix A) and bidirectional clustering of the dendrogram (Figure 5), the specific characteristics of each group were identified. Group G I was primarily characterized by local varieties from higher altitude areas (2,776 masl) and higher mean annual rainfall (900 mm). This group had large seeds, developed plants with high biomass, and presented high L-DOPA yields. This group is represented by local variety T1 from the locality of Santa Cruz Moxolahuac.

Group G II also consisted of seeds from higher altitude zones (2,665 - 2,807 masl) with similar environmental characteristics to those of G I. Seeds in this group were medium to large, with high L-DOPA content, and medium to high plant biomass content and yield of this bioactive compound. Local varieties in this group are from the localities of Santa Cruz Moxolahuac (T2), San Francisco de la Unión (T4), San Juan Cuauhtémoc (T7), and Ignacio Manuel Altamirano (T10, T16).

Group G III was characterized by seeds primarily from areas of medium to higher altitude (2,572 - 2,807 masl), with a mean annual temperature (MAT) of 13°C, mean annual precipitation (MAP) of 700 mm, and mean annual solar radiation (MASR) of 17.5 - 18.6 MJ/m². This group had medium-sized seeds and average values for the other variables analyzed. It includes local varieties from the localities of San Francisco de la Unión (T3, T17), Guadalupito las Dalias (T6), San Juan Cuauhtémoc (T8), and Ignacio Manuel Altamirano (T9, T11, T13, T14).

Group G IV was identified by local varieties from areas with an altitude range of 2,572 - 2,782 masl, MAT of 13°C, MAP of 700 - 900 mm, and MASR of 17.5 - 22.16 MJ/m² (Table 1). This group was characterized by small seeds, and low to medium values for plant biomass and L-DOPA content and yields. Local varieties in this group are from the localities of both municipalities, Tlahuapan (T5, T12, T18) and Chiautzingo (C1, C6) (Table 1).

Group G V was identified by local varieties from environments with MAT values of 14°C, MAP of 900 mm, and high MASR. These local varieties had small seeds, developed small plants, and exhibited low L-DOPA content and yields (Figure 5). This group consists solely of local varieties from the municipality of Chiautzingo in the locality of San Nicolás Zecalacoayan (C2, C3, C4, C5).

Principal component analysis illustrated the relationships between environmental, morphological, biomass, and L-DOPA content and yields variables with respect to the variability found in the local faba bean varieties. The PCA results, which identified five groupings consistent with those from cluster analysis, indicated that L-DOPA content present in the plant is not defined by the place of origin of the seed, since the groupings obtained in the study revealed that some groups presented local varieties from different localities (e.g., G II and G III), even from different municipalities (e.g., G IV), suggested that L-DOPA content variability possibly is attributed to genetic differences among local faba bean varieties rather than the place from which the seed was collected. In addition, bidirectional hierarchical clustering clearly showed that seed size can be an indicator of the amount and yield that can be obtained from L-DOPA, regardless of where the seed comes from, since large seeds were clustered together found to provide higher L-DOPA content and yield (e.g., G I and G II) than small seeds (e.g., G IV and G V) (Figure 5).Kai-Bin et al. (2016) also demonstrated this behavior, but in flowers of faba bean plants, where it was observed that L-DOPA content in flowers was strongly associated with seed size. Moreover, also in this study it was found a positive correlation between seed size and biomass content (p ≤ 0.05) (Table S1, Appendix A), indicating that larger seeds generally produce higher biomass, which in turn influences L-DOPA yield, regardless of seed origin.

The study identified significant variability in the morphological characteristics of faba bean seeds across different localities. In the PCA and cluster analysis the clustered shown that the higher altitude localities (2,665 - 2,807 masl), seeds were predominantly medium to large in size with substantial 100-seed weights (249.08 - 295.82 g), as observed in the G I and G II. Conversely, smaller seeds with lower 100-seed weights (122.37 - 169.01 g) were more common in lower altitude areas (2,460 - 2,665 masl), such as in G IV with local varieties C1 and C6 from San Antonio Tlatenco and GV with local varieties C2, C3, C4 and C5 from San Nicolás Zecalacoayan (Figure 5; Figure 4). This finding aligns with Herrera-Cabrera et al. (2010), who also reported larger seeds in higher altitudes (2,700 - 3,400 masl) and smaller seeds in lower altitudes (2,200 - 2,500 masl), regardless of local variety.

The influence of mean annual solar radiation (MASR) on seed size was also noted; local varieties in areas with higher MASR had smaller seeds (e.g., G V), while those in areas with lower MASR had larger seeds (e.g., G I and G II) (Figure 5). However, this study did not find conclusive evidence of the effects of temperature and precipitation on seed characteristics. This may be due to the proximity of the localities, which could limit the range of agro-environmental variation necessary to discern their impact on faba bean seed morphology. Previous research has indicated that temperature can affect seed characteristics in other species. For instance,Razzaque, Heckman, and Juenger (2023) demonstrated that increasing mean annual temperature negatively impacted seed mass in Panicum hallii.

According to the (WHO, 2023), levodopa with carbidopa remains the most effective PD treatment, but accessibility, affordability, and availability issues persist, particularly in low- and middle-income countries. V. faba plant tissue could offer a biologically and economically viable source of L-DOPA. Nonetheless, studies on microbiological safety and potential toxicity, as well as comprehensive in vivo assays, are needed to confirm the safety and efficacy of L-DOPA derived from faba bean plants.

Current PD treatments alleviate symptoms but do not halt disease progression (Valadez-Barba et al., 2021). Recent findings suggest that faba bean plants at 20 DAE contain L-DOPA along with other compounds such as flavonoids, rutin, and isoorientins. These compounds, in conjunction with L-DOPA, might not only alleviate PD symptoms but also offer potential neuroprotective effects (Fuentes-Herrera et al., 2023).

The utilization of V. faba for L-DOPA production exemplifies a bioeconomic approach, leveraging plant biomass to produce vital bioactive compounds for PD. The bioeconomy emphasizes the sustainable use of biological resources, integrating knowledge, science, and technology to deliver innovative and sustainable solutions. It fosters synergies across agriculture, medical research, and food science (IACGB & International Advisory Council on Global Bioeconomy, 2020), promoting new value chains and rural development through the circular economy approach (IICA, 2019). The present research is the first to assess L-DOPA content and yield in plants of local faba bean varieties from Puebla, Mexico, highlighting their potential to obtain L-DOPA. The use of the species for this proposal could help increase the V. faba production and consumption in México, benefit farming communities, and, in turn, contribute to regional rural development.