Rosmarinic Acid Content and Antioxidant Activity in Ehretia asperula Zollinger et Moritzi Cell Suspension Cultures

In the 1840s, Zollinger and Moritzi reported the first description of E. asperula Zollinger et Moritzi, a medicinal plant belonging to the Boraginaceae family (Riedl, 1997). This plant is native to China, Myanmar, Thailand and Vietnam. It is mainly found in Vietnam’s northern highland regions, particularly in the province of Hoa Binh (Tram et al., 2021). Traditional medicine has made extensive use of its aerial parts to treat a variety of diseases, especially to prevent liver disease, diabetes, hypertension, jaundice and acne (The Asia Foundation, 2012). Studies indicated that E. asperula Zollinger et Moritzi extracts had cytotoxic activity against HepG2, MCF-7, MDA-MB-231 and Hela cells (Nguyet et al., 2018; Tuyen et al., 2022) and could resist retinal cell death (R28) (Le et al., 2021).
       
Phenolic compounds are significant antioxidants with chemopreventive activities such as antioxidant, anticancer, antimutagenic and anti-inflammatory properties (Kumar et al., 2023). Their antioxidant capacity depends on the amount and location of their hydroxyl groups, which can quickly transfer H⁺ to reactive oxygen species (Wafa, 2024). One of the main phenolic compounds in E. asperula Zollinger et Moritzi is rosmarinic acid (RA) (Le et al., 2021).
       
Species in the Lamiaceae and Boraginaceae family have significant concentrations of RA. Recently, RA has been attracted because of its beneficial biological properties, including antioxidant, anti-inflammatory, anti-tumor, anti-allergic, antimicrobial, antiviral and cardioprotective properties (Kim et al., 2015; Nadeem et al., 2019). In addition to being obtained from wild plants, RA has been biosynthesized in vitro, such as in callus of Zataria multiflora Boiss (Françoise et al., 2007), hairy roots of Agastache foeniculum (Pursh) Kuntze (Nourozi et al., 2016), Lepechinia caulescens (Vergara-Martínez et al., 2021) and Agastache rugosa (Yeo et al., 2023), cell suspension cultures of Satureja khuzistanica (Sahraroo et al., 2018), shoots of Melissa officinalis L. (Vanda et al., 2019),  roots of Ocimum basilicum L. (Biswas, 2020) and Origanum dictamnus L. (Sarropoulou et al., 2023).
       
This study describes the establishment of E. asperula  Zollinger et Moritzi cell suspension cultures to obtain RA from biomass. It compared RA content in cell biomass, callus, field-grown and in vitro plants. The antioxidant activity of these materials was tested in vitro using the DPPH test.

Plant material
 
The dark green leaves of field-grown E. asperula Zollinger et Moritzi plants (6-year-olds) were used to extract RA (Fig 1A). The leaves in vitro were used for callus induction and RA extraction (Fig 1B).
 

 
Cultures of callus and cell suspension
 
The leaves in vitro were cultivated on Gamborg (B5) medium supplied with 0.4 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.1 mg/L BA (benzyl adenine) and 30 g/L glucose to create callus. Every five weeks, friable callus was subcultured.
       
To establish a suspension of cells, 1 g fresh weight (FW) of friable callus from the second subculture was transferred into liquid B5 medium (20 mL) added with 30 g/L glucose, 0.4 mg/L NAA (naphthalene acetic acid) and 0.1 mg/L BA. on a shaker fixed at 90 rpm.
       
After three weeks, cell suspensions were transferred to a B5 liquid medium supplemented with 45 g/L glucose, 0.4 mg/L NAA and 0.1 mg/L BA. After four weeks, 50 mg/L of chitosan was added to the medium to stimulate RA production in 48 hours.
       
The cultures were conducted in dark condition, at a temperature of 25±2°C and humidity of 70±2%.
       
The study was carried out from October 2021 to December 2022 in the laboratory of Thu Dau Mot University, Vietnam.
 
Analysis of RA content
 
After the treatment with chitosan, the biomass of cell suspension cultures was harvested and dried at 50°C and RA content was determined.
 
Extraction of RA
 
One gram of dry powders, including field-grown plant leaves, in vitro leaves, callus and biomass from cell suspension cultures (Fig 2), were mixed with 12 mL of 50% ethanol and extracted for three hours at 70°C and 200 rpm using a magnetic stirrer (Tram et al., 2022). The extracts were filtered and the solutions were retained to analyze RA.
 

 
Determination of RA
 
Spectrophotometry analysis of RA
 
200 μL extract solutions were mixed with 4.6 mL ethanol and 200 μL of a 0.5 M zirconium oxide chloride solution. After five minutes, a spectrophotometer was used to measure the reaction mixture’s absorbance at 362 nm. Equation y = 0.0224x - 0.0332 (R² = 0.9992) was used to calculate the concentration of RA, using RA (Sigma-Aldrich) at a concentration range of 2-40 μM as a standard (Öztürk et al., 2010).
 
HPLC analysis of RA
 
Before injection into the HPLC system for RA analysis, extract solutions were passed via a 0.45 ìm membrane filter. The injected volume was 20 μL.
       
The HPLC column was C18 (4,6 × 250 mm, 5 µm, Shim-pack GIST, Japan). The mobile phase flow rate for RA was maintained at 1 mL/min at 30°C and its detection wavelength was 280 nm. The values for retention time (RT) were 10 minutes. The isocratic conditions were 80% A and 20% B. Mobile phase A consisted of methanol and mobile phase B consisted of water containing 0.1% acetic acid (Adham, 2015).
       
The calibration curve was prepared by plotting concentration versus an area with the equation y = 4377.6x - 4932.9 (R² = 0.9993).
 
Analysis of antioxidant capacity (DPPH assay for scavenging free radicals)
 
After filtering through filter papers, ethanolic extracts were evaporated at 50°C to dryness. The extracts were subjected to a DPPH free radical scavenging test, utilizing the methodology described in prior research (Marinova and Batchvarov, 2011). The extracts were dissolved in absolute alcohol in a series of concentrations: 2000, 1000, 500, 250, 125 and 62.5 µg/mL. Ascorbic acid was the positive control and absolute alcohol was the negative control. The test of the DPPH free radical scavenging assay was presented in Table 1. After 30 minutes of dark, room-temperature incubation, the mixture’s absorbance at 517 nm was determined. The following formula was used to determine the percentage of inhibition of DPPH free radicals:
   

 
Analytical statistics
 
Using Statgraphics Centurion XV software, the data were statistically processed with a 5% significance level. The mean and standard deviation of each triple experiment are displayed in all experimental data.

E. asperula Zollinger et moritzi cell suspension cultures
 
Cell suspension cultures were started using the friable callus of E. asperula Zollinger et Moritzi (Fig 3A). Single cells and small clumps were present in the dark yellow cell suspensions after four weeks of culture (Fig 3B).
 

 
RA in E. asperula Zollinger et moritzi cell suspension cultures
 
This study used spectrophotometry and HPLC methods to determine the RA content in extract samples from E. asperula Zollinger et Moritzi.
       
In Table 2, RA is a phenolic compound with significant content in the tested extracts. In vitro leaves had the highest concentration, followed by callus and field-grown leaves and the lowest concentration was in cell suspension cultures. RA content in the extracts measured by the HPLC method was higher than that measured by the spectrophotometry method.
 

 
DPPH test for scavenging free radicals in the extracts
 
The results showed that the extracts created yellowish solutions on a purple background, demonstrating antioxidant activity in the DPPH experiment (Fig 4).
 

       
A comparative analysis of the antioxidant activity of the extracts from leaves of field-grown and in vitro E. asperula Zollinger et Moritzi plants, callus and biomass of cell suspension cultures was presented in Fig 5.
 

       
The inhibition percentage of DPPH free radical scavenging by leaves in vitro and callus extracts was provided as 90.3% and 90.99% at the concentration of 2000 ìg/mL, which have more potent antioxidant activity than the extracts of field-grown leaves (82.24% at the concentration of 2000 μg/mL) or biomass of cell suspension cultures (65.53% at the concentration of 2000 μg/mL) (Fig 5).
       
From the standard curve showing the linear correlation between the concentration of the sample test and the value of % inhibition of DPPH free radicals (Fig 5), the DPPH inhibition IC₅₀ value of the experimental extracts was interpolated (Table 3). The leaves in vitro extracts had the strongest anti-radical activity (IC₅₀ = 871.54 μg/mL) in the extracts. However, ascorbic acid’s IC₅₀ value (27.62 μg/mL) was higher than the E. asperula Zollinger et Moritzi extracts.


       
In Table 2, RA contents in E. asperula Zollinger et Moritzi extracts were high. The lowest RA concentration in the biomass of cell cultures was due to a portion of RA dissolved in the liquid medium (Tram et al., 2022). In particular, the RA contents recorded by the HPLC method were higher than those measured by spectrophotometry. The HPLC method is susceptible, has good quantitative ability and a higher level of accuracy than the spectrophotometry method. The RA content of the extract from field-grown leaves reached 8.94%, equivalent to the RA content from E. asperula Zollinger et Moritz leaves extracted with 50% ethanol (9.95%), as reported by Ly (2016). The RA content in E. asperula Zollinger et Moritzi cell suspension cultures (74.64 mg/g DW) was equivalent to that in Thymus lotocephalus shoot cultures (78.57 mg/g DW) (Gonçalves et al., 2019), higher in Dracocephalum moldavica L. cell suspension cultures (27.2 mg/g DW) (Weremczuk-Jeżyna et al., 2017).
       
About the assessment of antioxidant activity, the tested extract samples had antioxidant capacity equivalent to the extracts from in vitro E. asperula Zollinger et Moritzi plants grown under a fluorescent lamp (IC₅₀ = 1101.10 µg/mL) in the experiment of Tram et al., (2018). In particular, the extract from the biomass of cell suspension cultures had a lower antioxidant capacity than others, possibly due to some biologically active substances (especially RA) being secreted from the cells during growth and dissolved in the culture medium (Tram et al., 2022).
       
The experimental extract samples of E. asperula Zollinger et Moritzi also had an antioxidant capacity equivalent to or higher than the antioxidant capacity of some previously studied herbs, such as Allamanda neriifolia root, stem and leaf extracts with IC₅₀ values of 713.44, 1397.24 and 936.86 μg/mL, respectively (Hung et al., 2014), extracts from Sauropus androgynus, Polyscias fruticosa, Portulaca oleracea L. with IC₅₀ values of 993.85, respectively; 2110.08 and 2835.33 µg/mL (Mai et al., 2017).
       
A strong negative correlation was found between the RA contents in the experimental extracts and the IC₅₀ values (r = -0.934 for the spectrophotometric method, r = -0.756 for the HPLC method). RA plays an essential role in the bioactivity of E. asperula Zollinger et Moritzi extracts. The antioxidant activity of RA is well known due to its four phenolic hydrogens and two catechol (1,2-dihydroxy benzene) rings (Bhatt et al., 2013). A significant correlation between total phenolic contents (especially RA contents) from the cultures in vitro and antioxidant activity has been reported in many previous studies (Shiga et al., 2009; Samarakoon et al., 2016; Weremczuk-Jeżyna et al., 2017; Gonçalves et al., 2019).

These results suggested obtaining a large RA concentration is possible when using E. asperula Zollinger et Moritzi cell suspensions, callus, plantlet in vitro or field-grown leaves. Therefore, E. asperula Zollinger et Moritzi cell suspensions is a good candidate for in vitro RA biosynthesis. In addition, the DPPH assay of the E. asperula Zollinger et Moritzi extracts has significant in vitro radical scavenging potential, suggesting that it can protect cells from oxidative stress.

There is no conflict of interest.