GRAPHIC SUMMARY

Source: authors

1. INTRODUCTION

Hypericum perforatum, known as St. John’s wort, has been used as a medicinal plant to treat different human diseases, mainly mild and moderate depression (Ng et al., 2017). The biological activity of H. perforatum is attributed to more than ten classes of secondary metabolites, including anthraquinones/naphthodianthrones, phloroglucinol, flavonoids, xanthones, volatile oils, vitamin C, tannins, proteins, carotenoids, and coumarins. However, hypericin and hyperforin have been the main compounds studied in this medicinal plant because of their well-known antidepressant effects (Mullaicharam and Halligudi, 2018).

Hypericin, an anthraquinone derivative, is naturally found in the yellow flowers of H. perforatum. It has antidepressant activity, resulting from an inhibitory effect on the neuronal uptake of norepinephrine, dopamine, γ-amino butyric acid and L-glutamate. It is accumulated in specialized morphological secretory structures know as dark glands (Gaid et al., 2016). Previous studies have reported that hypericin concentrations will depend on different factors, such as planting and harvesting time, the phenological stage of plants at harvest and use of appropriate treatments for phytosanitary maintenance (Southwell and Bourke, 2001).

Native from Europe, Asia, and North Africa, the species H. perforatum can abundantly grow up on pastures, roadsides, and environments modified by human activity (Crompton et al. 1998). However, the adaptation and cultivation of H. perforatum in Brazil is still ineffective, because the plant does not reach a proper size (~ 60 cm) and does not reach an adequate stage to flower. Considering the high therapeutic potential of H. perforatum, it needs to be grown in ecological systems, so that there are no negative changes in the content of medicinal compounds that provide efficiency against diseases, such as depression (Faron et al., 2004).

Regulated by Normative Instruction No. 17/2014 by the Ministry of Agriculture, Cattle and Supplying for organic production (Ministério da Saúde, 2014b), homeopathic preparations have proven to be an effective and residue-free technology for use in agriculture (Teixeira and Carneiro, 2017; Sen et al., 2018). The application of homeopathic preparations can help the cultivation of medicinal plants on a more sustainable basis, eventually improving plant growth, and secondary metabolites biosynthesis and accumulation (Pereira et al., 2019).

In addition to the choice of residual-free treatments in medicinal plants (Ministério da Saúde, 2014a), the period of cultivation of the species needs to be determined, as this factor will also be determinant for the production of biomass and the biosynthesis of bioactive compounds. According to Soni et al. (2015), the growing season influences the availability and the amounts of bioactive compounds in medicinal plants, determining their phytotherapeutical potential. Planting and/or harvesting at the wrong time may impair the yield of secondary metabolites pharmacologically relevant, so it is very important to identify the best seasons for cultivation. In this sense, this study aimed to evaluate the use of high dynamized dilutions and the influence of seasons on the vegetative growth and content of phenolic compounds in Hypericum perforatum plants.

2. MATERIALS AND METHODS

Cultivation of Hypericum perforatum: The experiments were carried out in a culture room with controlled temperature and light at the Laboratory of Plant Health and Homeopathy and also in a greenhouse at the Epagri Experimental Station, located in the city of Lages (50º 19’46.93” W, 27º 48’28.746” S), Santa Catarina state, southern Brazil.

Two experiments were performed as follows: The Spring/Summer experiment, from October 2017 to January 2018 and the Summer/Autumn experiment, from February 2018 to May 2018. H. perforatum seeds were acquired from Feltrin Seeds®, showing a 64% germination rate, according to the manufacturer.

For production of the seedlings, the seeds were sown in a sowing tray filled with vermiculite and black earth, in a 2:1 ratio. The experiment used a randomized block design and the sowing trays were separated into blocks and transferred to a growth room at 25 ºC and 16h/8h photoperiod, under luminous intensity set up at 2.338 LUX provided by LED lamps. The sowing trays were placed on a plastic tray containing 200 mL until the seeds’ emergence, 15 days. Five treatments were used, consisting of homeopathic preparations in the 12CH (twelfth order of the Hahnemannian centesimal dilution) of Kali carbonicum, Natrum muriaticum, Phosphorus, and Silicea terra and distilled water as a control. The matrices of the homeopathic preparations were acquired in a compounding pharmacy at 6CH. The preparations at 12CH were made according to the Brazilian Homeopathic Pharmacopeia (Ministério da Saúde, 2011).

The selection of homeopathic preparations was carried out using repertory language and in consultation with medical sources. The main characteristics of the species H. perforatum were analyzed, such as: sensitivity to cold, need for constant water and light, photosensitivity, and fragility. Using the materials listed, and according to the Homeopro® software, it was determined which homeopathic best approached the level of similarity.

All experiments were performed in a double-blind analysis, namely, the operator was unaware of the treatment to be used. H. perforatum seedlings were treated twice a week, dispensing 1 mL of homeopathic preparations per sowing cell directly in the soil, totaling eight applications in a 30-day experimental period.

Six weeks after sowing, the treated seedlings, with approximately 6 cm in height, were transplanted into pots (four plants per pot) and taken to a greenhouse. The experimental plot consisted of 12 plants and 4 repetitions, totaling 48 plants per treatment. 8.7liters pots containing vermiculite, black soil, and sheep manure were used (1:1:1, v/v/v). 195 g natural phosphate per 360 liters of compost were used. In the greenhouse, homeopathic treatments were applied twice a week again and extended for one month until the end of the experiment, at 60 days after sowing.

After seed germination (15 days), two assessments per week were performed to measure the height of the main branch. The measurements were done from the base of the stem up to the highest leaf, and 17 evaluations of the main branch were carried out. At the end of the experiment (75 days), plant height, total number of branches and shoot dry weight were evaluated.

Sample collection and dark gland count: All the samples of the Spring/Summer experiment were collected in February 2019; whereas, for the Summer/Autumn experiment samples were harvested in June 2019. Dark glands were counted from a destructive sample of five leaves from each plant, totaling 240 leaves per treatment. The counting of dark glands was performed using a stereoscopic microscope (25x) on the adaxial face of the leaves. The remaining materials (leaves and roots) were placed in a force-air drying oven for 48 h at 50 ºC.

Preparation of hydroalcoholic extracts: After comparing the data obtained in the general average of the dark gland count, the samples treated with the homeopathic preparation Silicea terra 12CH and the control ones were selected for further chromatographic analysis. The extracts were obtained by maceration using commercial ethyl alcohol 92% (v/v) and 36g and 29g dry shoot samples of the control plants and Silicea terra­-treated plants, respectively, from the Spring/Summer experiment. Similarly, for the Summer/Autumn experiment, 1.05 g and 1.39g for plants treated with water and Silicea terra, respectively. Grinding was performed with 48 plants that were divided into four repetitions. The material was kept under maceration at room temperature and protected from light for seven days. After filtration, the solvent was removed by rotary evaporation at 45 ºC to obtain the crude hydroalcoholic extract. The extracts obtained for each maceration step were combined, frozen, and lyophilized, yielding four crude extracts per treatment.

Ultra-Performance Liquid Chromatography (UPLC): The hydroalcoholic preparations of H. perforatum extracts were submitted to UPLC analysis for hypericin characterization. The samples were prepared by removing the nonpolar components by solid-liquid extraction using n-hexane as an extractor solvent. 0.5 g hydroalcoholic extract were added in 2 mL of n-hexane, and the procedure was repeated twice. All samples were analyzed in triplicate. The analytical standard of the hypericin compound (Sigma-Aldrich 95%) was used for identification of the target compound, according to its retention time and UV-vis spectroscopic profile.

The experiments were conducted using an Ultimate 3000 RS UPLC System (Thermo-Fisher Scientific, USA) as proposed by Brolis et al. (1998). Chromatographic separation was performed by using a reverse-phase C18 column (FR-Thermo Scientific 250×4.6 mm, 5 μm) coupled with a C18 guard column (Phenomenex®), thermostatized at 33°C, and a diode array detector (DAD). The mobile phase was eluted at flow rate at 1mL/min using the following linear gradient program (Brolis et al., 1998): A) water acidified with phosphoric acid 85% (99.7: 0.3 v/v); B) acetonitrile; C) methanol. Injection volume was 10 µl, and hypericin detection was achieved at wavelengths 240 nm, 270 nm, 320 nm, and 400 nm.

Statistical analysis: For the study variables (height of the largest branch, number of branches and shoot dry weight) one way analysis of variance (ANOVA - F test) was used for each season, and the assumptions of the model were checked using the Bartlett test (homoscedasticity) and the Shapiro-Wilk test (normality). In cases in which the assumptions of the model were not satisfied, the transformation proposed by Box-Cox was used, applying the optimal lambda for transformation. In cases where there was a significant effect of the treatments, the means were compared by Tukey’s test.

To describe the behavior of the plant height variable over time, the logistic model was used, given by:

[Ecuación 1]

where: ${\mathrm{\beta }}_{1i}$ represents the parameter associated with the asymptote for the ith treatment, ${\mathrm{\beta }}_{2i}$ is the parameter representing the numerical value associated with time at the curve’s inflection point for the ith treatment. At this point the height value will be$\frac{{\mathrm{\beta }}_{1i}}{2}$ . ${\mathrm{\beta }}_{3i}$ is the scale parameter associated with the ith treatment, tj is the time in days associated with jth observation and is the error associated with the jth observation of the ith treatment.

The treatments were compared using the confidence intervals of the model parameters. All analyses were performed with the aid of scripts written in the R language, considering a 5% significance level.

Retention time (min) and mean of the peak area (%) are presented for the chromatographic analyses performed by UHPLC.

3. RESULTS AND DISCUSSION

Cultivation: The vegetative growth of H. perforatum in the Spring/Summer experiment was higher (p < 0.05) in plants treated with the homeopathic preparation with Silicea terra compared to the ones treated with Phosphorus, but it was similar to that of the other treatments. In the Summer/Autumn experiment, the treatments with Natrum muriaticum and Kali carbonicum provided the highest vegetative growth, and were statistically different from the control (Figure 1).

Figure 1.
Vegetative growth of the main branch of Hypericum perforatum plants with high dilution treatments. A) Spring/Summer experiment; B) Summer/Autumn experiment.
Source: authors.

The plants treated with Silicea terra were higher in comparison to those treated with Phosphorus, but their height was similar to that of plants treated with Kali carbonicum, Natrum muriaticum, and to the height of the control treatment in the Spring/Summer experiment. In the Summer/Autumn experiment, Natrum muriaticum-treated plants differed from the Silicea terra-treated ones, but not from homeopathic preparations with Kali carbonicum and Phosphorus.

As far as growth period is concerned, the Spring/Summer plants treated with Silicea terra took 4 and a half days longer, on average, to reach 50% of the estimated height than plants treated with Phosphorus. For the Summer/Autumn experiment, Silicea terra was shown to act on plant height differently when compared to the Spring/Summer experiment, with a shorter time to reach 50% of the estimated height in comparison to other treatments.

Table 1.
Vegetative growth of Hypericum perforatum over time in two growing seasons and treated with homeopathic preparations.

Source: Authors. *Means followed by the same letter in the column within each variable do not differ significantly from one another (5% significance). Kali-c = Kali carbonicum; Nat-m = Natrum muriaticum; Phos = Phosphorus; Sil = Silicea terra.

**Estimated time for plant height to reach 50% of total growth. The parameter ${\mathrm{\beta }}_1$ corresponds to the height (cm) of the plant over time and the parameter ${\mathrm{\beta }}_2$ represents the number of days that the plant reaches 50% of the height estimated by the asymptote.

At the end of the experiment, the height of the longest branch in Spring/Summer plants was lower when the plants were treated with the homeopathic preparations Kali carbonicum and Natrum muriaticum. Phosphorus, Silicea terra, and the control treatments presented the highest averages. For the Summer/Autumn experiment, no statistical differences were detected for the treatments (Table 2).

Table 2.
Average height of the longest branch and number of branches (± standard error) of Hypericum perforatum plants treated with homeopathic preparations in two growing seasons.

Source: Authors. *Means followed by the same letter in the column do not differ significantly from one another (5% significance). ns= non-significant. Kali-c = Kali carbonicum; Nat-m = Natrum muriaticum; Phos = Phosphorus; Sil = Silicea terra.

The average number of branches of H. perforatum plants in the Spring/Summer experiment was higher with the Silicea terra treatment, unlike the Natrum muriaticum-treated plants, but similar to control and the other treatments. In the Summer/Autumn experiment, there were no statistical differences among the treatments for height and number of branches.

Regarding the influence of homeopathy on agriculture, Bonato and Silva (2003) state that homeopathic preparations behave like energy and, when dynamized, the wave frequency remains fixed, with variation only on its amplitude, thus being able to alter the responses in a negative or positive way. Homeopathic medical materials act in the form of vibration, acting on the amplitude of the wave and consequently reflecting on biological organisms (Kolisko and Kolisko, 1978; Silva et al., 2005). Andrade and Casali (2011) state that medical materials react on the electromagnetic field differently, depending on the vitality of the plant. Thus, the performance in the organisms will depend on the chosen homeopathic preparation, the Hahnemannian centesimal used, and even the application method.

Dark glands: The number of dark glands present in the leaves of H. perforatum did not differ according to the homeopathic preparations and the control (Table 3).

Table 3.
Number of dark glands in the leaves of Hypericum perforatum over time in two growing seasons and treated with homeopathic preparations.

Source: Authors. *Means followed by the same latter in the column do not differ significantly from one another (5% significance). ns = non-significant.

Treatments	Dark glands (nº)
	Spring/Summer experiment	Summer/Autumn experiment
Kali carbonicum 12CH	14.76 ± 0.44 ns	12.75 ± 0.54 ns
Natrum muriaticum 12CH	15.39 ± 0.41	13.37 ± 0.37
Phosphorus 12CH	14.64 ± 0.65	12.20 ± 0.43
Silicea terra 12CH	16.41 ± 0.56	13.28 ± 0.34
Control	16.24 ± 0.51	12.54 ± 0.38
Mean and error	15.51 ± 0.24	12.83 ± 0.19

The number of dark glands may vary according to the growth stage of H. perforatum plants and, consequently, it affects the production of hypericin. Kladar et al. (2015) reported that phenological stages, such as flowering (pre-bloom, full-bloom, and post-bloom), influence the secondary metabolite pathways of plants.

Chromatographic analysis: The hypericin substance was targeted, but there was no peak in the crude extract chromatograms that corresponded to its retention time. Other peaks with absorptions at wavelengths 240 nm, 270 nm, 320 nm were detected, but they could not be identified. In the Spring/Summer experiment, the hydroalcoholic extracts of the control and Silicea terra-treated plants presented 11 peaks in the chromatograms, while in the Summer/Autumn experiment, 12 peaks were detected in both treatments. The detected peaks mostly showed more nonpolar compounds that appeared in the final retention times.

Rizzo et al. (2019) reported that the biosynthesis of hypericin and even other metabolites are related to the development of dark glands. The authors explained that leaves are the parts used in therapeutic treatment, but flowers are the ones that have more dark glands and, consequently, more hypericin. At the time of cultivation performed in this study, there was no time to flowering, which presumably determined the non-detection of the compound. In this study, as H. perforatum plants were grown only for 75 days, one could speculate that plants did not reach the growth and developmental stages required for the biosynthesis and accumulation of hypericin, regardless of growing seasons.

4. CONCLUSIONS

Homeopathic remedies affected plants of H. perforatum differently during the growing seasons. Higher averages were observed in plant cultivation when planting started in Spring compared to Summer. The compound of interest, hypericin, was not detected in any sample of leaves of H. perforatum. This suggests the need for a longer cultivation time so that biosynthesis and accumulation of such secondary metabolite can occur in the dark glands.

Acknowledgments

The FAPESC Project 2015TR1067 and the Support Fund for the Maintenance and Development of Higher Education (FUMDES, in Portuguese), through the Santa Catarina University Scholarship Program UNIEDU.

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Additional information

HOW TO CITE: Nunes, A., Coelho, D., Soldi, C., Werner, S., Maraschin, M., Boff, P., and Boff, M. (2021). Vegetative growth of Hypericum perforatum L. plants treated with high dynamized dilutions over different growing seasons. Revista de Investigación Agraria y Ambiental, 12(2), 39 – 49. DOI: https://doi.org/10.22490/21456453.4237

AUTHOR'S CONTRIBUTION: First author: Methodology, research, data analysis, conceptualization, writing, original outline. Second author: Methodology, data analysis, review. Third author: Methodology, data analysis, review. Fourth author: Methodology, data analysis, review. Fifth author: Methodology, data analysis, review. Sixth author: Resource acquisition, supervision, review. Seventh author: Resource acquisition, supervision, review.

CONFLICTO DE INTERESES: Los autores declaran no tener ningún conflicto de intereses.

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