Study site and genetic material

The study was conducted under controlled laboratory conditions (Laboratorio de Fisiotecnología Vegetal, and Laboratorio de Homeopatía Acuícola-Agrícola) at Centro de Investigaciones Biológicas del Noroeste, S. C. (CIBNOR), located in the northwestern city of La Paz, B.C.S., México, at 24° 08’ 10.03” N and 110° 25’ 35.31” W, at 7 meters of altitude (^{Batista-Sánchez et al., 2017}). Certified seeds of two Capsicum annuum varieties were used. Before the experiment, a germination test was performed using the International Seed Testing Association methodology (^{ISTA, 2010}).

Experimental design and development

The experiment was conducted using a completely randomized design with a factorial arrangement of (2A × 3B × 5C), where factor A has the two varieties (Santa Fe and Jalapeño M); factor B has the three levels of NaCl (0, 50 and 100 mM); and factor C has the centesimal dynamizations (CH) of the HDBC Natrum muriaticum (NaM): NaM-7CH, NaM-13CH and Silicea terra (SiT): SiT-7CH, SiT-13CH; distilled water (DW) as control treatment, for a total of 30 treatments (Table 1) with four replicates each.

The treatments with HDBC (NaM-7CH; NaM-13CH; SiT-7CH, and SiT-13CH) were prepared in distilled water from the official medications (Natrum muriaticum 6CH, Natrum muriaticum 12 CH, Silicea terra 6CH and Silicea terra 12CH, respectively) of Similia^® brand acquired from an authorized supplier (Farmacia Homeopática Nacional^®, CDMX, México), which are registered with the Health Ministry of México and officially authorized for use in humans. During their preparation, the basic procedures established by the Mexican homeopathic pharmacopoeia (^{Secretaría de Salud, 2015}) were applied, including centesimal serial dilution (1:99) and vigorous vortex agitation (BenchMixer^®, Edison, NJ, USA), according to the technique described by ^{Mazón-Suástegui, Ojeda, García, Batista, and Abasolo (2020}a). Seeds were disinfected before sowing by immersion in a 1.5% sodium hypochlorite solution for 10 min, washed three times with deionized water to remove any disinfectant residue, placed on filter paper to dry, and then soaked for 60 min with the corresponding HDBC treatment or with distilled water (DW) in the case of the control treatment. Thirty seeds were sown per Petri dishes (150 × 15 mm) previously autoclaved, covering the bottom with a sheet of sterilized filter paper used as substrate. The dishes were moistened with 30 mL of the saline solutions (50 and 100 mM NaCl) and distilled water (0 mM NaCl), as appropriate, then subsequently incubated in a germination chamber (Lumistell^®, model IES-OS, series 1408-88-01, USA) at a temperature of 25±1 °C, 80% humidity and 12 h daily of continuous light for 14 days.

Germination percentage and rate

The germination percentage (GP) was recorded daily for 14 days, considering the seed germinated when the radicle was about 1 mm long, and the germination percentage was determined at the end of that period. The germination rate (GR) was calculated using the equation proposed by ^{Maguire (1962)}: M = n1/t1 + n2/t2 +…n30/t14; where n1, n2, … n30 are the number of seeds germinated at times t1, t2, … t14 (up to 14 days).

Morphometric variables

After 14 days, 10 seedlings were randomly selected per replicate (40 per treatment); the morphometric variable height seedling (HS) and radicle length (RL) were both determined using an image analyzer (WinRhizo^® Regent Instruments Inc. USA), whose operating principle is through direct measurements of digital images obtained by scanning different organs of the seedlings. The fresh and dry biomass of the radicle and aerial part were determined using an analytical balance (Mettler Toledo^®, model AG204, USA). The plant tissues were divided into aerial part and radicle, weighed and placed in paper bags, and introduced into a drying oven (Shel-Lab^®, model FX-5, series-1000203, USA) at a temperature of 70 ºC for 48 h until complete dehydration and weighed again to determine dry biomass.

Statistical analysis

An analysis of variance (ANOVA) was performed. When a significant difference was found between treatments, the multiple comparison test of means (Tukey’s honestly significant difference (HSD), P ≤ 0.05) was used, using the statistical program Statistica v. 10.0 for Windows (^{StatSoft, Inc, 2011}).

Germination percentage (GP)

The GP analysis showed significant differences between varieties (P = 0.0000), NaCl levels (P = 0.000010), HDBC treatments (P = 0.00001) in the interaction varieties × NaCl (P = 0.00001), varieties × HDBC treatments (P = 0.000083) and in the triple interaction varieties × NaCl × HDBC treatments (P = 0.000010). No significant difference was found in the germination rate (GR). The germination percentage decreased when NaCl increased, with the JM variety being the least affected by this stress (Figure 1A).

Figure 1: Effect of the interaction varieties × NaCl (A) and varieties × highly diluted bioactive compounds (HDBC); (B) on germination percentage of Capsicum annuum seeds. Different letters show statistical differences (Tukey’s honestly significant difference (HSD), P ≤ 0.05).  

The results found in this variable (GP) are due to the osmotic effect caused by the presence of salts in the culture medium, making it difficult for the embryo to absorb water and consequently affecting the metabolic processes at the cellular level inherent to seed germination (^{Wang, Li, and White, 2020}). This report coincides with that observed by ^{Batista-Sánchez et al. (2017)} when studying Ocimum basilicum L. germination and initial growth under saline conditions (0, 50, 100, and 150 mM NaCl), finding a decrease in GP as salt concentrations increased in the medium. Other authors, such as ^{Goykovic-Cortés, Nina, and Calle (2014)}, also reported a decrease in tomato seed germination when subjected to different types of abiotic stress. When the seeds received HDBC treatments, an increase in GP was observed (Figure 1B, Figure 2A, and Figure 2B) with respect to the control treatment (DW). For the JM variety, the greatest response was with NaM-7CH treatment and for SF with NaM-7CH and SiT-7CH treatments. This finding may be determined by stimulation at the cellular level of the biological processes that lead to germination since the applied HDBC treatments contain nanoparticles in their active ingredient capable of inducing a biological response (^{Abasolo-Pacheco et al., 2020}). Similar results were reported by ^{Mazón-Suástegui et al. (2020}b) when they applied NaM-7CH to Salicornia bigelovii (Torr) seeds and obtained a significant increase in GP with respect to the seedlings of the control treatment. The beneficial effects of HDBC treatments on the germination of Crotalaria juncea L. seeds have also been observed (Silveira, 2008¹).

Figure 2: Effect of the interaction varieties × NaCl × highly diluted bioactive compounds (HDBC) on the germination percentage of Capsicum annuum seeds. Different letters show statistical differences (Tukey’s honestly significant difference (HSD), P ≤ 0.05). 

Seedling height of Capsicum annuum

For height seedlings (HS), significant differences were observed between varieties (P = 0.001), NaCl levels (P = 0001), HDBC treatments (P = 0.0001), in the interaction varieties × NaCl (P = 0.0008), varieties × HDBC treatments (P = 0.00001) and in the triple interaction varieties × NaCl × HDBC treatments (P = 0.00001). Through the interactive analysis, a significant decrease in this variable was found when NaCl presence increased from a moderate (50 mM) to a severe (100 mM) level, with greater emphasis on the SF variety (Figure 3A). The negative effect of salt presence on this variable is determined by Cl^- and Na⁺ accumulation in the culture medium, causing a slowdown in metabolic processes, nucleic acid synthesis, enzymatic activity, and hormonal balance (^{Agüero-Fernández et al., 2019}).

Figure 3: Effect of variety × NaCl (A) and variety × highly diluted bioactive compounds (HDBC); (B) interactions on average Capsicum annuum seedling height. Different letters show statistical differences (Tukey’s honestly significant difference (HSD), P ≤ 0.05).  

When both varieties received HDBC treatments, the greatest height was observed in JM variety seedlings treated with NaM-7CH (Figure 3B), with an increase of 64% with respect to the seedlings of the control treatment (DW). The seedlings of the SF variety also responded positively to the application of the HDBC treatments NaM-7CH, NaM-13CH, and SiT-7CH. In the analysis of the triple interaction varieties × NaCl × HDBC treatments, the results also revealed an increase in HS when the seedlings were treated with greater emphasis on the NaM-7CH treatment (Table 4), even when they were subjected to saline concentrations (NaCl) from moderate (50 mM) to severe (100 mM), a favorable response was recorded for this variable. The results observed in HS when they received HDBC dynamizations can be explained by the presence of trace elements in the active ingredient; one of these is magnesium (Mg), chemically present in NaM-7CH, essential for chlorophyll molecule formation, therefore, of vital importance in photosynthesis, which is the main process of plant biomass production from nutrients and light energy (^{Mazón-Suástegui et al., 2020}b). Furthermore, Mg has a predominant role in the enzymatic activity associated with carbohydrate metabolism (^{Xiao, Hu, Chen, Yang, and Hua, 2014}).

Radicle length of Capsicum annuum seedlings

In the variable radicle length (RL), the results of the analysis showed significant differences between varieties (P = 0.00001), NaCl levels (P = 0.000001), HDBC treatments (P = 0.0001), in the interaction´s varieties × NaCl (P = 0.00001), varieties × HDBC treatments (P = 0.0002) and in the triple interaction varieties × NaCl × HDBC treatments (P = 0.00008). A decrease in this variable was observed when salt levels (NaCl) were higher for both varieties under study (Figure 4A), which has the greatest impact on the SF variety with the application of 100 mM NaCl. This result can be explained by the phytotoxicity caused by NaCl, which inhibits water absorption and hinders the development of cell division and elongation processes (^{Lamz and González, 2015}; ^{Agüero-Fernández et al., 2019}).

Figure 4: Effect of the interaction varieties × NaCl (A) and varieties × highly diluted bioactive compounds (HDBC) (B) on radicle length of Capsicum annuum seedlings. Different letters show statistical differences (Tukey’s honestly significant differences (HSD), P ≤ 0.05).  

When analyzing the data from the seedlings that received HDBC treatments, the results (RL) revealed a positive response to the NaM-7CH treatment in both varieties, with an increase of 57.80% in the JM variety and 86.30% in SF compared to the seedlings of the control treatment (DW) (Figure 4B). In the interactive analysis varieties × NaCl × HDBC treatments, an increase in RL was observed in the Capsicum annuum seedlings of the two varieties treated with NaM-7CH; for the three levels of NaCl (0, 50, 100 mM), this variable was higher than the control treatment (DW) (Table 4). This result confirms the anti-stress effect of Natrum muriaticum (NaM) previously reported by ^{Mazón-Suástegui et al. (2018)}; when the interaction of two NaM dynamizations was studied at different saline levels in the cultivation of Ocimum basilicum, an increase in tolerance of the negative effects of abiotic stress was observed in the plants that received the NaM-7CH treatment.

Fresh and dry biomass of Capsicum annuum Seedlings

No significant difference was found between varieties for fresh radicle biomass (FRB). However, a significant difference was observed between NaCl levels (P = 0.00024), HDBC treatments (P = 0.0069), in the variety × NaCl interaction (P = 00007), and in the interaction triple variety × NaCl × HDBC treatments (P = 0.007). The interactive study allowed observing the negative effect of NaCl on this variable, with an impact on both varieties when salt concentration was higher (Table 2), which may be related to salt phytotoxic characteristics when it is found in excess (^{Pan et al., 2021}). When the plants were treated with HDBC, a differential response was observed, where the JM variety recorded the highest FBR value when SiT-7CH was applied in a non-saline medium (0 mM NaCl). When the seedlings were subjected to saline conditions, the most effective treatment was NaM-7CH. For both varieties under study, a higher FRB was observed with this treatment, even when saline conditions were moderate to severe (Table 4).

In the dry radicle biomass (DRB), no significant difference between varieties was observed except for a difference between NaCl levels (P = 0.00002), HDBC treatments (P = 0.006) in the variety × NaCl interaction (P = 0.0007), and in the interaction of the triple variety × NaCl × HDBC treatments (P = 0.00768). The analysis showed a decrease in this variable when the seedlings were subjected to saline conditions (NaCl), with an impact of 8.60% in the JM variety and 44.10% in the SF variety, at a NaCl level of 100 mM (Table 2). Capsicum annuum seedlings treated with HDBC increased BSR even when subjected to moderate to severe saline stress (NaCl). A better response was noted with the treatment of SiT-7CH and NaM-7CH (Table 4). Similar results were obtained by ^{Lensi, Siqueira, and Silva (2010)}, who demonstrated the effectiveness of dynamized NaM (NaM-6CH, and NaM-30CH) in common bean plants (Phaseolus vulgaris L.) since they did not show signs of toxicity during their growth stage. On the other hand, the mechanism of action of HDBCs may involve physiological changes that lead to the formation of secondary metabolic products related to the defense mechanism of the treated plants (^{Lensi et al., 2010}; ^{Sen, Chandra, Khatun, Chaterjee, and Das, 2018}; ^{Meneses, 2024}; ^{Damiani, Givacheski, Trzimajewski, and Deboni 2024}).

The results obtained in the present study may be possible due to the direct action of the nanoparticles contained in the applied dynamizations. Silica - or the raw material from which the SiT nanomedicine is produced - has a positive influence on nutrient balance (^{Tichavsky, 2007}), increases synergies, reduces antagonisms, and also the absorption of elements that can become phytotoxic, such as excess NaCl. NaM also contains trace elements, such as magnesium (Mg), which are of great value in the synthesis of carbohydrates and important as sources of metabolic reserves and fuels (^{Mazón-Suástegui et al., 2020a}).

In the fresh biomass of aerial part (FBA P), significant differences were found between varieties (P = 0.00001), NaCl levels (P = 0.00001), HDBC treatments (P = 0.0001), in the interaction´s varieties × HDBC treatments (P = 0.00005) and varieties × NaCl × HDBC treatments (P = 0.00038), no significant difference was observed in varieties × NaCl. When analyzing the result of the interactive analysis, an increase in FBAP was observed when the seedlings were treated with nanomedicines, with greater emphasis on the NaM-7CH treatment (Table 3). In the triple interaction varieties × NaCl × HDBC treatments, the greatest response of this variable (FBAP) was observed when the JM variety seedlings were subjected to saline stress of 50 mM NaCl and received the NaM-7CH treatment, the increase concerning the control treatment (DW) at this same stress level was 63.20% (Table 4).

For the dry biomass of aerial part (DBAP), a significant difference was found between varieties (P = 0.0001), NaCl levels (P = 0.00001), HDBC treatments (P = 0.00001), in the interaction varieties × HDBC treatments (P = 0.00005) and in the triple interaction varieties × NaCl × HDBC treatments (P = 0.0003), no significant difference was found in varieties × NaCl. The results showed that the NaM-7CH treatment increased DBAP in both varieties under study (Table 3). These results coincide with those obtained by ^{Rodríguez-Álvarez et al. (2020)} when they applied NaM to C. annuum L. var. Glabriusculum plants that were subjected to abiotic stress by adding NaCl (200 mM) in a hydroponic system, observed an anti-stress effect of NaM.

When analyzing the effect of different salinity (NaCl) levels on Capsicum annuum L. seedlings and their interaction with HDBC treatments, the results revealed a differential response (Table 4). An increase in DBAP was observed when the seedlings of both varieties received the NaM-7CH treatment; even under saline conditions, the response was superior to the control treatment (DW), confirming that the highly diluted treatment used can activate defense mechanisms in the seedlings that have yet to be studied to attenuate the negative effects of saline stress (NaCl). In this same field of research, the anti-stress effectiveness of the 7CH dynamization of the NaM nanomedicine has already been proven in Ocimum basilicum and Phaseolus vulgaris (^{Mazón-Suástegui et al., 2018} and 2020a).