Plant material collection

The plant tissues were collected in plantations in Huizotlaco, Coacuilco and Contepec in Hidalgo, México (Figure 1, Table 1). Collecting of the leaves, stems and flowers was done during the flowering stage in May 2014. The fruits of the plants from each site were collected 32 weeks after pollination and cured with the traditional process (Beneficio Primero de Mayo, Papantla, Veracruz) by the curing master Veremundo Rodríguez.

Figure 1 Geographic location of the collection sites of vanilla structures in the Huasteca Hidalguense, México. S1: Huizotlaco, S2: Coacuilco, S3: Contepec. 

Quantitative analysis by thin-layer chromatography (TLC)

Preparation of extracts. From crushed fresh leaves, stems and flowers, and hexane and methanol in 1:5 proportion (tissue:solvent), extracts with different polarity were obtained (^{Recio-Iglesias, 1999}); the extract from the cured pod was obtained only with methanol because of the low availability of the sample (^{Jadhav et al., 2009}). The samples were placed in a sonicator (Auto Science, model A5515OB) for 30 min, at a frequency of 5.5, they were kept in maceration at room temperature for 24 h, filtered and stored in glass vials at -20 °C until the time of their analysis.

Identification of groups of compounds. 15 to 20 μL of the extracts were applied to silica gel plates 60, F₂₅₄ (Sigma-Aldrich). The eluents and chromogenic agents were specific for the detection of each group of metabolite (^{Wagner and Bladl, 1996}); in the case of flavonoids the plates were visualized with ultraviolet light (UVLMS-38 El series 3UV™ Lamp) at 365 nm.

Quantitative analysis

Leaves and stems were obtained from the superior and inferior ends and the middle region of a meter of cutting from the plant, and small pieces were sectioned, extracted with methanol and kept in the same manner as described before. The humidity content was determined for each tissue to express the concentration of the secondary metabolites based on dry matter (DM).

Total phenolic compounds. These compounds were quantified with the method proposed by ^{Singleton et al. (1999)}, at 725 nm in a UV-Vis spectrophotometer (Evolution 300, Thermo Scientific). The standard curve (y=1.6571x-0.016, R²=0.9932) was prepared with gallic acid (Sigma). The results were expressed in mg gallic acid equivalent per 100 g of dry matter.

Total tannins. These compounds were quantified with the method described by ^{Makkar et al. (1993)}, at 725 nm in a UV/ VIS spectrophotometer (Evolution 300 Thermo Scientific). The results were expressed in mg of tannic acid (Sigma) per 100 g of dry matter, based on the standard curve equation (y=1.6571x-0.016, R²=0.9932).

Condensed tannins. These compounds were quantified with the method proposed by ^{Makkar et al. (1993)}, at 550 nm in a UV/VIS spectrophotometer (Evolution 300 Thermo Scientific). The results were expressed in mg of tannic acid (Sigma) per 100 g of dry matter, according to the following equation:

where A is absorbance, 78.26 is the correction factor, % DM is the percentage of dry matter.

Flavonoids. These compounds were quantified according to the method suggested by ^{Chang et al. (2002)} at 415 nm in a UV/ VIS spectrophotometer (Evolution 300, Thermo Scientific). The results were expressed in equivalent mg of quercetin (Sigma) per 100 g of dry matter, according to the standard curve equation (y=6.0143x-0.0084, R²=0.9984).

The curing process of fruit was carried out following the description by ^{Xochipa-Morante et al. (2016)}.

Statistical analysis

Contingency tables were obtained to analyze the results of the qualitative tests by thin-layer chromatography. ANOVA was performed for the concentration of total phenolic compounds, total tannins, condensed tannins and flavonoids, from nine repetitions of leaf and stem, and four of flower and cured pod, from each collection site. The difference between means per collection and between tissues was evaluated with the Tukey test (α=0.05) with the SAS statistical package version 9.0 (^{SAS Institute Inc., 2002}).

Qualitative analysis by thin-layer chromatography

The most abundant phytochemical groups, identified by TLC, varied between the tissues (Table 2): 1) terpenoids (17 to 20 bands) are produced commonly in vegetative tissues, flowers and occasionally roots (^{Dudareva et al., 2004}); their presence in vanilla could be because it is the most abundant and diverse group of plant secondary metabolites in chemical structure, and their role is important in plant-insect, plant-pathogen, and plant-plant interactions (^{Paschold et al. 2006}); 2) saponins (16 to 18 bands) are a type of terpenoid; they usually participate actively in the regulation processes of plant growth and the variations in their distribution, composition and concentration in plants is attributed to the species’ reactions to the environment (^{Moses et al., 2014}), and 3) flavonoids (14-18 bands); their synthesis in plants is recognized as a reaction to infection from microorganisms (^{Dixon et al., 1983}), and they help combat oxidative stress and act as growth regulators (^{Kumar and Pandey, 2013}). In general, the leaves presented a higher diversity of metabolites (19 to 22 bands), perhaps because of their higher metabolic activity, since in addition to photosynthesis, exposure to light modulates the synthesis and presence of phytochemicals that protect plants against pathogens or herbivores; these compounds may even be deposited outside the leaves as waxes and cutin (^{Vivanco et al., 2005}).

The presence of metabolites by tissue depended on the collection site. The flavonoids showed more bands (6) in leaves and stems from Coacuilco; the leaves and stems from Contepec stood out for the higher number of saponins (7 bands), terpenoids in leaf (7), and total phenolic compounds in cured pod (3). In the tissues from the three collection sites there were less bands (1-2) of tannins and alkaloids (Table 2). ^{Shanmugavalli et al. (2009)} documented similar results, since they detected traces of these two groups in leaves and stems of V. planifolia under natural conditions, in India.

Quantitative analysis

The principal groups of phytochemicals in tissues showed coefficients of variation (CV) of between 7 and 15 %, the highest corresponded to condensed tannins. In all the variables, highly significant differences were observed (p<0.0001) (Table 3).

The concentration of phytochemical components varied widely between the collection sites. The plant material from Contepec had the highest concentration of total phenolic compounds, and total tannins. The material from Coacuilco had the highest concentration of flavonoids and condensed tannins, and from Huizotlaco it presented the lowest concentration of all the components evaluated (Table 4).

The cured pod presented the highest concentration of total phenolic compounds (Table 4), apparently because they contribute widely (1 000 to 3 000 mg 100 g^-1 DM) to the aroma of the cured pods (Shina et al., 2008) and favor the antioxidant activity of cured pod extracts (^{Rojas-López and Cañizares-Macías, 2013}).

The leaf was the tissue with highest concentration of flavonoids, but its content was considerably less than in tea (Camelia sinensis) (611 mg 100 g^-1 DM), which has high antioxidant capacity (^{Pereira et al., 2014}) (Table 4). The cured pod showed the highest concentration of total tannins, but corresponded to less than half in some of fresh fruits, such as cranberry (233 mg 100 g^-1) (^{Vázquez-Flores et al., 2012}), or even fruits that are cured like roasted coffee (270 mg 100 g^-1) (^{Savolainen, 1992}). The leaves showed the highest concentration of condensed tannins and the stems followed; these compounds can contribute to the protection of the tissues, which like others are exposed to pathogens, herbivores and UV radiation (^{Brillouet et al., 2013}).

The highest concentration of total phenolic compounds was observed in flowers and cured pod from Contepec, compared to the other collection sites and tissues. This agreed with the results of the aroma components (mainly phenolic compounds) in cured pods from the same collection site, which showed high concentration of vanillic acid and medium concentration of vanillin (^{Delgado-Alvarado et al., 2016}). The leaves, stems and flowers from Coacuilco and the cured pod from Contepec showed a higher concentration of flavonoids and total tannins (Table 5). The highest concentration of condensed tannins was shown by stems from Coacuilco, leaves from Huizotlaco, and flowers and cured pod from Contepec (Table 5). These differences between collection sites can be attributed to the conditions of each site; Coacuilco is at a higher altitude compared to the other sites (Table 1), so the temperature differs and can generate stress in the plants and favor the production of these compounds.

Cola parchycarpa and Cola lepidota grow in different geographic and environmental conditions and the collection site determines the presence and concentration of phytochemicals in the vegetative structures (^{Ene-Obong et al., 2016}). Also, in V. planifolia the differences between tissues can also be associated to the development stage and growth conditions of the plant, since the younger tissues tend to concentrate a higher amount of phytochemicals (^{Palama et al., 2010}). These metabolites are usually transported from the root to the stems and the leaves, via xylem or phloem, and stored in reproductive structures such as flowers and pods (^{Wink and Schimmer, 2010}).

Based on this, the collection region of greatest interest could be Coacuilco, because the leaves and stems collected there presented the highest concentration of total flavonoids and total tannins, and Contepec because the pods and flowers presented the highest concentration of total phenolic compounds and condensed tannins. In contrast, all the plant tissues in Huizotlaco showed the lowest concentration of secondary metabolites. These results allow suggesting the convenience of researching the effect of other factors that favor the synthesis of metabolites in the vanilla plant.