Variation in phytochemical properties and expression of key genes involved in biosynthesis of hypericin using nano-capsulated and normal hormones in Hypericum perforatum L


 Hypericum perforatum is a valuable medicinal plant with anti-depressant activity. Hypericin is the major compound responsible for such activity. In the present study, the effect of four nano-capsulated hormones of 2,4-epibrassinolide, spermidine, salycilic acid and cycocel were investigated on the amount of hypericin based on HPLC analysis in two locations (Saman and Isfahan). For each hormone, the normal form was also compared with nano form. The expression patterns of the key genes (Hyp-1, pks1, pks2) for hypericin production was also evaluated using qRT-PCR. Moreover, GC-MS analysis was also performed for determination of the compounds in studied treatments. The major compounds were germacrene D (3.29–33.53%), β-caryophyllene (0-4.08) and α-longipinene (0-24.05%). In most cases, nano-hormones led to increase in these components. Significant changes were obtained in expression of key genes in hypericin synthesis as a result of nano-hormones treatments in Isfahan site. Overall, nano-hormones revealed higher increase in expression of all genes as compared with normal hormones in this site. The expression of Hyp-1, Pks1 and Pks2 was significantly increased using spermidine, 24-epibrassinolide and cycocel in Isfahan location in both nano-hormones and normal ones, while the expression of Hyp-1 was decreased in SA treatment in Isfahan location. Based on HPLC analysis, hypericin ranged from 0.21 in control to 0.51 mg 100 g− 1 DW in nano-SA in Isfahan site. Finally, the expression of the key genes were mostly elevated in colder climates and nano-form formulation.


Introduction
The secondary metabolites play an important role in human life. Many of these metabolites have been considered as key components for healing materials for pharmaceutical purposes (Bielecka et al., 2021). One of the most critical compound that is widely used as antidepressant agent is hypericin, hyperforin and their derivatives (Košutha et al., 2011). These compounds are obtained from a medicinal plant named Hpericum perforatum L. (St. John's Wort). More than 400 species have been determined in Hypericum genus (Nürk et al., 2013). Different activities has been reported for Hypericum species including antioxidant, anti-cancer, anti-depressive and anti-viral activities (Sotak et al., 2016).
The biosynthesis of secondary metabolites can be regulated by different elicitors. The chemical ones often induce by upregulating their biosynthetic genes in related pathways (Kianersi et al., 2020). Some plant hormones can act as elicitors. Jasmonic acid (JA), salicylic acid (SA), ethylene are the most relevant ones.
Nowadays, some new hormones such as epi-brasinosteroids, polyamines, have been considered as new potential chemical elicitors for changing the secondary metabolites and their related genes (Ali et al., 2008). Salicylic acid is considered as an important signal molecule in different plant species (Shahim-Ghalecheh et al., 2017).
Nowadays, the development of nanoparticulate compounds is one of the most important topics in pharmaceutical and food researches to improve their e ciency and functions (Preetz et al., 2008). The use of nanotechnology in agricultural products could play a crucial role for improving agrochemical delivery and nutrient management (Sasson et al., 2020). Among these agrochemicals, nano-hormones production is a novel subject that has not yet been fully performed. Some plant hormones are not e cient based on the treatment and experimental conditions. Furthermore, the use of nano-hormones might affect the metabolites or other health bene t compounds in plants in an e cient way. There are several reports regards the effect of normal hormones on valuable metabolites of plants including salicylic acid for thymol (Mohammadi et al., 2019), 24-epibrassinolides for lavandin in Lavandula intermedia (Aras Asci et al., 2019), cycocel in sage and peppermint (Keltawi and Croteau, 1986). The use of plant hormones and other elicitors for increasing of hypericin, hyperforin have been reported in some literatures (Shakya et al., 2019;Cirak et al., 2020). However, the use of nano-hormones have not yet reported for increasing of metabolites in plants. Besides the importance of hypericin, the essential oil components of Hypericum species were of great importance in the previous literatures (Pirbalouti et al., 2014;Seyis et al., 2020).
Nowadays, the study of the expression of key genes involved in biosynthesis of valuable compounds in medicinal plants is of great value for nding the mechanisms involved in their pathways as well as nding the way to increase such metabolites (Gharibi et al., 2019). In this regard, some valuable researches have been performed in some medicinal plants including Achillea pachycephalla (Gharibi et al., 2019), Chrysanthemum morifolium (Hodaei et al., 2018), Ocimum basilicum (Rezaei et al., 2020).
There are some key genes are responsible for producing of hypericin, hyperforin in Hypericum including hyp-1 (hypericin), pks1 (Polyketide synthase), pks2 (Polyketide synthase) (Fig. 1). Moreover, there are some reports regards the effect of plant hormones and other treatments on expression of major genes in Hypericum including gamma ray irradiation (Azeez et al., 2017), salycilic acid (Shahim-Ghalecheh et al., 2017), jasmonic acid (Amirnia et al., 2017). However, there are no reports regards the effect of nano hormones and their e ciency on hypericin production as well as the expression of the important genes (hyp-1, pks1, pks2) underlying in biosynthesis of hypericin. Moreover, besides the previous publications regards the essential oil composition of this plant, there is no report in respect to the effect of nano-hormones on essential oil components of Hypericum.
So, the goal of the present study were to assess the nano-hormones of 2,4-epibrassinolides, spermidine, salycilic acid and cycocel effects on essential oil composition based on GC-MS, hypericin amount using HPLC analysis and to evaluate the effect of these nano-hormones on expression of three major genes in hypericin production including hyp-1, pks1, pks2.

Plant materials
In this research, one genotype belonging to Hypericum perforatum L. species was cultivated in two sites in Isfahan (Goldaru company research farm, Isfahan) with the average temperature of 10°C and humidity 47% and Saman (Saedegh Abad research farm, Chaharmahal and Bakchtiari) with the average temperature of 17°C and humidity 32%. The species was determined by Dr. Hamze Shirmardi using Flora Iranica (Rechinger, 1963). The herbarium specimen number was 1966 and the sample was deposited in Charmahal and Bachtiari research center. The study was in compliance with relevant institutional, national and international guidelines and conservation policy of endangered plants. The seeds were sown into 17 ×15 cm pots on 25 February 2019. Pots contained soil with the ratio of 3:1 soil to sand. All treatments were performed in three replicates.=

Preparation of nanocapsulated hormones
Nanocapsulation was performed according to the method reported by Lagno et al. (2010) using controlled deposition of aluminum phosphate coatings. For this purpose, Xanthan (1%) were diluted in dd H 2 o and 80 mg of aluminum sulfate were dissolved in distilled water (Lagno et al., 2010). The size of nano particles was determined using Zeta sizer instrument model Malvern ZEN 3600 (UK). The protocol for nano formulation in each hormone is described as follows.

Experimental conditions and treatments
In this experiment one genotypes was evaluated in two sites of Isfahan and Saman. Four hormones were used for treatments in two forms of normal and nano-hormones including spermidine (5 mM) (S.din), 2,4-epibrassinolide (10 − 3 mM) (), salicilic acid (SA) (14mM) and cycocel (1.5g)). Spray method in 25°C and 17% moisture content was applied for treatments.

Essential oil extraction
For essential oil extraction the aerial parts were harvested at the owering stage in studied samples. The plants were dried in shade and 25°C for six days. The dried samples subjected to hydro-distillation in Clevenger apparatus for ve hours based the methods used by Tohidi et al. (2017). 2.5. GC-MS analysis GC-MS analysis was done for detection of oil components. For this purpose, the aerial part of Hypericum samples was subjected to a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with HP-5MS 5% phenyl methyl siloxane capillary column (30 m length, 0.25 mm diameter; lm thickness 0.25 µm) as well as an Agilent 5975 (Agilent Technologies, Palo Alto, CA, USA) as GC-MS/FID detection coupled with 70 eV electron impact ionization over a mass range of 39-400 m/z. Helium was applied as a carrier gas source in the column at a constant ow rate of 2 mL min − 1 in split mode 1:20 employing the following cycles: 60°C for 3 min; raising at rate of 3.0°C min − 1 to 120°C; and enhancing at rate of 15°C min − 1 to 300°C and held for 5 min. The oven temperature was initially maintained at 60°C for 4 min and subsequently, elevated to 260°C at the rate of 4°C /min. Retention indices (RI) were determined with C5-C26 alkane standards as reference. Finally, they were con rmed by a comparison of their mass spectra with those recorded in the NIST 08 (National Institute of Standards and Technology), Willey275.L (ChemStation data system), and those reported in the literature (Adams, 2007). 2.6. HPLC analysis for hypericin 2.6.1. Extract preparation The aerial parts of treated samples with nano-hormones were harvested at owering stage. The harvested parts were shade dried at 25°C, and the plant materials were ground and prepared for extraction. Methanol was extracted using 20 gr of the dried samples. The extract was shaken using 500 mL of methanol with 150 rpm at 25º C for 72h. Then, the extracts were ltered through 0.45 µm membrane (Millipore, Merck, Germany). Finally, the extracts were evaporated at room temperature (25˚C) and dried in desiccators. After ltration, the samples were kept at 4°C for HPLC analysis.

HPLC analysis
The Hypericum extracts were analyzed using HPLC system (model Agilent 1090). The HPLC elution method has been used previously by Sarfaraz et al. (2021). Hypericin (Sigma-Aldrich 56690) was used as reference compound. A 0.22 µm nylon acro-disk lter and 20 µL of the extract were used for injection.
The stationary phase had a 250 mm × 4.6 mm (5 µm) symmetry C18 column (Waters Crop., Milford, MA, USA) (10 mm × 4 mm I.D.), and the mobile one included 0.1% formic acid in acetonitrile ( ow rate of 0.8 mL min − 1 ) with the wavelength between 200-400 nm. The gradient conditions were also performed as follows: a linear step from 10-26% solvent B (v/v) for 40 min, 65% solvent B for 70 min, and nally to 100% solvent B for 75 min. The hypericin concentration was calculated based on the peak areas and their retention times. Finally, the amount were calculated based on mg /100 g of the sample dry weight.
All reagents were analytical grade (Sigma-Aldrich, USA) and the solvents used for HPLC were from Merck (Germany). 2.7. Gene expression analysis 2.7.1. Extraction of total RNA The extraction of total RNA was performed using RNA isolation kit (Dena Zist Asia Co., Iran). The quantity of RNA was measure by a Pico200 (Picodrop, UK) spectrophotometer. Putative DNA contamination was removed using DNaseI (Promega, USA), and then a cDNA synthesis kit was used to reverse transcription (Vivantis Technologies Co., Malaysia). Conserved sequences of three major genes for hypericin production viz. -hyp-1, pks1, pks2 were identi ed in the GenBank database and online ExPASy software. The primer design was performed using Oligo ver. 5 software. The primer sequences are illustrated in Table 1. The expression of key genes in hypericin production was assessed using quantitative real-time PCR in four nano-hormones and four normal ones according to those explained in treatments part. The expression was assessed by Step One Plus™ Real-Time PCR System (Applied Biosystems). A total volume of 10 µL reaction was applied including 1 µL of cDNA, 5 µL of 2× SYBR Green Master Mix (Takara Bio Co., Germany), 0.5 µL of each primer (10 pmol). The real time PCR was 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The speci city of the products was con rmed by Melting curves. The expressions amounts were measured by the simple formula viz. 2 −ΔΔCt . Finally, beta-actin as the reference gene and three technical replicates was applied.

Statistical analysis
The data were subjected to analysis of variance according to the model of completely randomized design, followed by Fisher's (protected) least signi cant difference (LSD, P ≤ 0.05) test, using SAS v9 software (SAS Institute). Correlation coe cients were calculated using the SPSS program (version 16; SPSS Inc., Chicago, IL., USA).

Gene expression
Signi cant changes were obtained in expression of key genes in hypericin synthesis as a results of nano-hormones treatments in Isfahan site (Fig. 2). As a result, Transcription of the gene Hyp-1, Pks1 and Pks2 was signi cantly increased using spermidine, Breassinosteroid and cycocel in Isfahan location in both nano-hormones and normal ones, while the expression of Hyp-1 was decreased in SA treatment in Isfahan location (Fig. 2). Among the hormones, cycocel led to the highest elevation in gene expression of all studied genes in Isfahan site. Overall, nano-hormones revealed the higher increase in expression of all genes as compared with normal hormones in this site.
Saman site has the colder climate in comparison of Isfahan and the results in this site showed a different trend for applied hormones. Despite to the Isfahan site, the highest expression of Hyp-1 was obtained when the SA treatment was applied, while the lowest was belonged to spermidine treatment. Similar trend was also obtained for two other genes. In all treatments, except for cycocel the nano-hormones showed higher expression in comparison with normal hormones. Among treatments, nano salicylic acid showed higher effect on expression of Pks2 in comparison with others.

Hypericin content
According to data analysis, amount of hypericin revealed high variation as a result of different treatments (Fig. 3). In Isfahan site, the increase in hypericin content was obtained as a result of all treatments in comparison with control. In most cases nano-hormones elevated hypericin content except for nano-SA ( Fig.). The highest and the lowest increase was observed in cycocel and SA hormones, respectively. However, some differences were attributed to the locations used in this research. In Saman location higher increase was observed in hypericin content as it can probably due to colder climate in this site in comparison with Isfahan site.
There are some reports regards the use of elicitors or plant growth regulators for increasing the hypericin content (Yamaner and Erdag, 2013;Azeez et al., 2017). Small amount of hypericin (0.21-0.51 mg 100 g − 1 DW) was obtained in this research, however the amount was in the range of some previous researches such as Azeez et al. (2017). The use of treatments in the seedlings of in vitro culture can lead to increase the hypericin content. In another similar research, the use of SA led to elevate the hypericin content from 0.25 to 0.80 mg 100 g − 1 DW in two other Hypericum species viz. H. hirsutum L. and H. maculatum (Coste et al., 2011). Higher amount of hypericin in most of nano-hormones treated samples might be due to higher absorbance e ciency of nanohormones in the leaf tissues of Hypericum plants.
Treatment with biotic or abiotic elicitors has been considered as an e cient strategy to increase the secondary metabolites (Coste et al., 2011). Among these elicitors, plant growth regulators (PGR S ) play a crucial role for enhancing the metabolites (Walker et al., 2002). The mechanism of PGR s and other elicitors is affecting the expression of secondary metabolic pathways as a defense strategy (Qian et al. 2006;Coste et al., 2011).
As illustrated in Fig. 1, PKS, Polyketide synthase; Hyp-1, phenolic oxidative coupling protein are two major key enzymes for production of Hypericin. In the present research, the related genes were applied for study of gene expression. Elicitation is considered as a major strategy for elevation of hypericin by increasing in expression of these genes. Different elicitors have been used for improvement of hpericin including jasmonic acid (Walker et al., 2002), Chitosan (Valleta et al., 2016) and Salicilic acid (Gadzovska et al., 2013), however in the present research, four nano-hormones were applied for studding the hypericin content along with the key genes expression manner. The comparison of expression patterns and hypericin content in site 1 showed similar trends in most cases. As a result, hypericin and expression of hyp-1 gene elevated by nano-hormones application except for nano-cycocel. The second location showed a different trend for some treatments (Fig. 2,3). The differences of two locations regards the expression and hypericin content might be attributed to the temperature of studied locations. Yao et al. (2019)  The effect of nano-hormones and normal ones were compared on essential oil composition of H. perforatum aerial parts. The GC-MS analysis revealed high range of compounds in two hormone types ( Table 2). As a result, the major compound was Germacrene D and ranged from 3.29% in Saman with nanobrassinostreoid (B-10 − 3 -N) treatment to 33.53% in Isfahan and S.din-5-N treatment. Most of samples revealed the elevation in this compound as compared with control. Overall, in most cases Isfahan showed higher amount of germacrene D in comparison with Saman. Moreover, in most cases Nano-hormones in most cases led to increase in germacrene D content (Table). Another major component in this research was alpha-amorphene that was ranged from 0% in some samples to 24.05% in B-10 − 3 -N of Saman. Alpha-longipinene as another major component varied from 0-10.4% in C-1.5-N. Moreover, beta-selinene ranged from 0-9.62% in B-10 − 3 -N of Isfahan. Delta-cadinene ranged from 1.4% in SA-14 of Isfahan to 4.52% in S.din-5-N of Saman. Trans-caryophyllene also ranged from 0-4.08% in B-10 − 3 of Saman. The changes in other compounds are illustrated in Table 2.           Germacrene D and β-caryophyllene have also been reported as the major component of H. perforatum in Turkish samples (Alan et al., 2010;Cirak et al., 2010), Italian (Maggi et al., 2010) and Tunesian samples (Hosni et al., 2011). Most of the reports showed similar ranges for these compounds in normal condition.
Moreover, in the present study, beta-selinene was obtained in the range of 0-9.62% that was in the range of a previous report in Turkey (5.08-19.63%) (Cirak et al., 2010). However, the discrepancy in some results can be interpreted as the harvesting phase of the samples. In the present study, for essential oil components, the samples were harvested just at the owering stage. High accumulation of sesquiterpenes such as germacrene D and β-caryophyllene in comparison with monoterpenes might probably be the result of early harvesting of the samples. Rahimmalek et al. (2009) andHodaei et al. (2017) also highlighted these form of the accumulation in the genus of Achillea and Chrysanthemum, respectively. Furthermore, they also attributed these kind of accumulation to the precursor ow in young organs with higher intensive growth than old ones and the distribution of precursors between the cytoplasm (the site of sesquiterpenes synthesis) and plastids (the site of monoterpenes synthesis).

Conclusion
The present research for the rst time assessed the effect of nano-hormones of 24-epibrassinolides, spermidine, salycilic acid and cycocel on hypericin content and related genes expression. According to the results, most of the genes were upregulated with the application of nano-encapsulated hormones. Moreover, temperature as an environmental factor was crucial in hypericin production. Among treatments, nano-cycocel showed the highest production of hypericin that can be suggested for further industrial or pharmaceutical purposes. Moreover, in the present study, the effect of these nano-hormones were compared with normal ones and interestingly the accumulation of two sesquiterpenes viz. Germacrene D and β-caryophyllene were elevated as compared with normal ones in most cases. Finally, the use of new technologies in nano related science can provide new insights and prospects for increasing of valuable metabolites in medicinal and aromatic plants. Nanophosphorus Fertilizer Stimulates Growth and Photosynthetic Activity and Improves P Status in Rice. Journal of Nanomaterials. 1-11.        Page 13/14   Figure 1 Schematic representation of biosynthetic pathway for Hypericin production. PKS, Polyketide synthase; Hyp-1, Phenolic oxidative coupling protein.