Study Site

This research study was performed under controlled conditions in the microbiology laboratory at the Universidad Técnica Estatal de Quevedo (UTEQ), located in the university campus “Manuel Haz Álvarez” on km 1.5 Quevedo - Santo Domingo and Finca Experimental “La María”, from the same university and located on km 7.5 or the Quevedo-El Empalme, Province Los Ríos, Ecuador.

Genetic Material

Certified seeds of cucumber var. Marketmore were obtained from the producer Agripac^® in the city of Quevedo, Ecuador.

Homeopathic Treatments

Four homeopathic medicines were assessed, each one in two different centesimal dynamizations (6CH and 12CH), of the trademark Similia^® (CDMX, MX) registered and authorized by the Health Ministry (Secretaría de Salud) in Mexico for human use. These medicines were obtained from Farmacia Homeopática Nacional (FHN^®, CDMX, MX): Silicea terra (SiT-6CH and SiT-12CH), Natrum muriaticum (NaM-6CH and NaM-12CH), Magnesia phosphorica (MaP-6CH and MaP-12CH) and Arsenicum album (ArA-6CH and ArA-12CH). The homeopathic treatments (7CH and 13CH) were prepared in the UTEQ from FHN commercial dynamizations (6CH and 12CH; Similia^®, CDMX, MX) by centesimal dilution (1:99) and dynamizations (2 min) with vortex equipment, using distilled and deionized water as dilution/agitation vehicle. These treatments were made by applying the basic homeopathic procedures (^{Mazón-Suástegui et al., 2017}, ^2018a, ^b, ^2019a, ^b; ^{Ortiz-Cornejo et al., 2017}) and the methodologies described in Farmacopea Homeopática de los Estados Unidos Mexicanos (^{SSA, 2015}).

Experimental Design

A completely randomized design with factorial arrangement (2A × 4B + 1) was applied, considering the 7^th and 13^th centesimal homeopathic dynamization as factor A (7CH and 13CH) and homeopathic medicines as factor B (SiT, NaM, MaP, and ArA). A total of eight homeopathic treatments and one control were prepared with three replicates each: T1: SiT-7CH; T2: NaM-7CH; T3: MaP7-CH; T4: ArA-7CH; T5: SiT13-CH; T6: NaM-13CH; T7: MaP-13CH; T8: ArA-13CH, and T9: Control (purified water).

During the germination stage, sterile Petri boxes were used, placing a layer of filtered paper on the base as substrate to preserve humidity; 60 seeds per treatment were used, placing 20 seeds per repetition. The seeds were submerged in each one of the treatments for 20 min and then placed in the corresponding Petri boxes. The germination assays were performed in darkness. The seeds were considered germinated when the radicle showed around 2 mm in length (^{ISTA, 2010}). During the emergence stage, the same number of seeds were used as in the previous stage, but in this case they were sown in flex-foam germination boxes with 30% of commercial substrate NOVARBO^® (Finland) and 70% of black soil, placing one seed in each one of the cavities at 1 cm in depth. The seeds were considered emerged when the seedlings arose from the substrate surface. The plant development assessment was performed in plastic pots with the substrate previously described using 45 seedlings per treatment, previously treated with the homeopathic medicines, such as indicated in the previous stage. Irrigation was performed every two days; 10 days after sowing, the treatments were sprayed both in the aerial part (stem and leaves) of the plant and substrate; spraying was performed every day in the first hours (08:00) in the morning. The tutorial and biometries were performed 21 and 55 days after sowing, respectively.

Variables Assessed

Germination rate and percentage were determined for 24 h applying the formula proposed by ^{Maguire, (1962)}. At the end of the experiments, plants were transferred to the laboratory were root, stem, and leaves were separated to assess morphology; stem (LT, for its acronym in Spanish) and root (LR, for its acronym in Spanish) length, fresh and dry biomass of the aerial part (stem and leaves) (BFPa, BSPa, for its acronym in Spanish) and root (BFR, BSR, for its acronym in Spanish) were measured. An analytical balance (Mettler Toledo^®, Model AG204, USA) was used to determine fresh and dry biomass. To obtain dry biomass, the corresponding fresh biomass in separate samples was placed in paper bags and into a drying stove (Shel-Lab^®, Model FX-5, Series-1000203, USA) at a temperature of 70 °C until its complete dehydration was obtained (approximately 72 h) Additionally, during plant development, stem diameter (DT, for its acronym in Spanish), number of leaves (nH, for its acronym in Spanish), and number of floral blooms (nF, for its acronym in Spanish) were assessed.

Statistical Analysis

The corresponding data to all morphometric variables were subjected to an analysis of variance (ANOVA) and Tukey’s test at 95% to establish statistical differences between the interactions of dilutions and homeopathic medicines. Previously, normality and homocedasticity tests were performed using the statistical software SPSS ver.22 (IBM^®, Armonk, NY 2013).

Germination

Significant differences were recorded among treatments with respect to germination rate and percentage. When the treatment was based on the 13^th centesimal dynamization (13CH) of the homeopathic medicine Arsenicum album (ArA), higher values (53.33% and 9 germinated seeds, respectively) were obtained compared with the control group with 40% and 4 germinated seeds, respectively (Figure 1).

Figure 1: Rate and percentage of germination of cucumber seeds (Cucumis sativus L.), treated with four homeopathic medicines (SiT, NaM, MaP and ArA) in two different dinamizations (7CH y 13CH). Different letters denote significant differences using the Tukey statistical test (HDS). 

The previous agrees with the results of ^{Brizzi et al (2000)} who applied different ArA dynamizations in wheat seeds, showing positive effects with this treatment. Likewise, ^{Moreno et al (2004)} indicated that the ultra-dilutions of Arsenicum album promoted germination in coffee (Coffea arabica L.) seeds, which agrees with that observed in this study and suggests that this medicine had a similar effect, accelerating the cucumber (Cucumis sativus L.) seed germination process. This result could have been a consequence of nanoparticles present in the active ingredient of the medicine, unchaining and/or accelerating the physiological processes at cellular level (these mechanisms still need to be studied further).

With respect to the morphometric variables, significant differences were also observed between the dynamization and type of interactions of the homeopathic medicines applied with a variation coefficient of 7.30%. For stem length (LT), a beneficial effect was observed in general in all homeopathic medicines used, obtaining a greater growth with MaP-13CH (6.5 cm) compared with the control group (3.7 cm). The same effect was observed in radicle length (LR) with a variation coefficient of 4.55%. In all the homeopathic treatments, similar averages were observed (10 cm) and greater than the control group (8.4 cm). This beneficial effect could have been related to the nature and origin of the MaP treatment because magnesium phosphate is obtained from the saturation of phosphoric acid transformed into magnesium salt; this mineral is of vital importance in plant development since this element is the central atom in the chlorophyll molecule. Chlorophyll is the pigment that gives the green color to plants and performs the photosynthesis process; it also intervenes in the activation of countless enzymes necessary for its development and contribution to protein synthesis. ^{Alvarado-Mendoza et al. (2017)} mentioned that the homeopathic medicine MaP reduces spore formation of the pathogen fungus Fusarium oxysporum f. sp. lycopersici in tomato.

With respect to dry and fresh weight, the variation percentage was 1.98 and 2.90% and 6.62 and 7.80% for the aerial part and radicle, respectively. It is important to note that in general, the highest values were obtained with SiT-7CH, NaM-7CH, MaP-7CH, and ArA-13CH (Table 1). To this respect, ^{Pulido et al. (2017)} demonstrated that SiT and Árnica montana homeopathic preparations increased dry biomass of broccoli seedling shoots. Additionally, Almeida (2002)² recorded an increase in basil (Ocimum basilicum L.) fresh weight with the application of homeopathic preparations of SiT-30CH. It is important to note that the 12^th centesimal Hahnemannian (12CH) homeopathic dynamization is precisely in the limits established by the Avogadro Theory for the presence of the “active principle” molecule of the initial concentrate or “Mother Tincture” (TM) to be chemically detectable, from which this homeopathic medicine was made with the corresponding serial dilution and agitation (^{Mazón-Suástegui et al., 2019a}). However, physicochemical studies on highly diluted homeopathic medicines, have undoubtedly shown that nanoparticles of the “active ingredient” exist in the MT, including in 30-CH and 200-CH centesimal dynamizations (Chikramane et al., 2012). This reference and results of this study have shown that homeopathic medicine has a biologically measurable effect beyond the simple placebo effect, which on the other hand, could not exist in plants (^{Mazón-Suástegui et al., 2018a}, ^b, ^2019a, ^b). ^{Tichavsky (2007a)} mentioned the importance of silica and justified the application of the homeopathic medicine Silicea terra (SiT) in plants because it intervenes directly in nutrition, decreasing rickets and strengthening plant tissues in general. With respect to the medicine NaM, made from marine salt, it has demonstrated to improve plant growth in basil Ocimum basilicum L., under salinity stress condition by adding NaCl (^{Mazón-Suástegui et al., 2018b}). On the other hand, NaM-12CH has been reported to increase phosphoros, iron, silica, and potassium levels in plants (Gonçalves et al., 2012).

Emergence

With respect to emergence percentage, significant differences were recorded between treatments and the control group, mainly in the emergence rate (Figure 2), and higher values (9.47 and 9.25 seeds per day, respectively) were recorded with treatment MaP, compared with the control group (8.3 seeds per day).

Figure 2: Emergence rate and percentage of cucumber seedlings (Cucumis sativus L.) treated with four homeopathic medicines (SiT, NaM, MaP and ArA) in two different centesimal dynamizations (7CH and 13 CH). Different letters denote significant differences using the Tukey statistical test (HDS). 

With respect to the morphometric variables assessed, in general and independently of the homeopathic medicine applied, a greater growth in SL was observed in plants treated with the 7^th centesimal (7CH) dynamization, with an average of 12 cm, compared with the 13^th centesimal (13CH) dynamization with an averages of 10 cm. Same as during germination, the best results in emergence were obtained with MaP-7CH (12.3 cm), which were the highest of the rest of the treatments and the control group (9.9 cm). For RL no significant differences were found. For the BFPa and BSPa, better results were observed again with SiT-13CH, with averages of 0.79 and 0.03 g, respectively, compared with the control group (0.47 and 0.02 g). As to weight gain in root (BFR and BSR), greater values were recorded with MaP-13CH (0.12 g) for fresh weight and NaM‑13CH (0.0049 g) for dry weight, statistically different to the control group that showed values of 0.04 g for BFR and 0.0017 g for BSR (Table 2). The effect of the homeopathic medicines applied could be similar to that observed during the stage previous to germination.

Plant Development

During plant development, significant differences were observed in all interactions (homeopathic medicine × centesimal dynamization) assessed. For LT, with the application of NaM-13CH plants with the greatest length (117.07 cm) were observed, followed by NaM-7CH, SiT-13CH, and ArA-13CH, compared with the rest of the treatments and the control group (78.63 cm). The homeopathic medicine Natrum muriaticum was produced from marine salt that contains almost 100% sodium chloride, and also all the different substances, compounds, and elements that seawater contains (^{Mazón-Suástegui et al., 2018b}; ^2019a). This medicine allows the treated organism (animal or plant) to assimilate, regulate and preserve the content of other salts, and intervene in water absorption by the plant and in the osmotic equilibrium of the liquids contained in its cells or tissues (^{Tichavsky, 2007}). Similarly, ^{Maute (2011)} highlighted that the medicine Arsenicum album (ArA) has been widely used in agrohomeopathy because of its beneficial action in plant development and strengthening weak plants due to hydric stress. Despite cucumber plants were the objective of this study, they were not subjected to hydric stress; the positive effect recorded in plant length could have been caused by the beneficial action of the homeopathic treatments applied, following a similar process to that described previously. For root length (LR), the highest values were obtained with SiT-7CH (49.56 cm), followed by ArA-7CH (41.27 cm) and NaM-3CH (37.21 cm) with respect to the control group (28.60 cm). ^{Pulido et al. (2017)} indicated that when the ultra-diluted dynamizations went beyond the limit established by the Avogradro Theory (1 × 10^-23) with the Silicea terra 30CH medicine, root and dry radicle length of broccoli plants cultivated in greenhouse conditions increased. With respect to fresh and dry weight (BFPa, BSPa, BFR and BSR), significant differences were obtained among the factors assessed. For BFPa plants with greater weight (30.80 g) were obtained with the application of NaM-13CH compared to the control group (17.73 g) while for the BSPa variable, greater values were obtained same with NaM-13CH (2.46 g) compared with plants treated with purified water (Control, 1.20 g). As to root weight, the plants treated with SiT-7CH showed greater fresh and dry weight (5 and 0.31 g respectively) compared with the control group with 2.6 and 0.16 g, respectively (Table 3).

With respect to the stem diameter, the plants treated with NaM-7CH) greater average diameter (5 mm) without differing statistically from plants treated with Silicea terra (SiT-7CH) with an average of 4.64 mm, compared to the control group (3.66 mm). With respect to the number of leaves per plant, the interaction NaM-3CH showed greater average value (13 leaves), statistically different than the control group (eight leaves). With respect to the average number of flowers per plant, the best result was obtained with the application of NaM-7CH since almost twice the number of flowers per plant were recorded (61) compared to the control group (33) (Table 4). Studies performed with Natrum muriaticum showed that very high dynamizations (200CH) of this homeopathic medicine have the capacity of inducing measureable biological responses in plants, promoting an increase in the amount of chlorophyll, a greater action of the RUBISCO enzyme and total protein in caupi bean Vigna unguiculata plants cultivated under salinity stress (^{Mondal et al. 2012}). These results confirm the principle of similar, which is basic in homeopathic medicine theory and practice, in the sense that the application of small doses of ultra-diluted substances and dynamized by a serial process of dilution/agitation may prevent or reverse the harmful effects of the same substance when it is applied in massive doses (^{Mazón-Suástegui et al., 2017}; ^2018a; ^2019a). On the other hand, when homeopathic medicines are applied, a primary effect of the medicine per-se should be considered and other secondary effects associated to the sphere of action of the dynamization applied. Different references are available on the effect of homeopathic medicine in weight increase of the plant species. ^{Sánchez and Meneses (2011)} demonstrated that when the medicine Calcárea fluorica 30CH was applied, the weight of the Welsh onion (Allium fitolosum) increased, but when the 30^th centesimal dynamization (30CH) of other homeopathic medicine (Calcárea carbónica) was applied, an opposite effect was recorded, reflecting a decrease in weight in the same species.