Comparison of Different Extraction Procedures for Isolating Bacteriostatic and Anti-pseudorabies Virus Components from Rodgersia Sambucifolia (Hemsl)

Medicinal plants are considered a repository of bioactive ingredients. Rodgersia sambucifolia (Hemsl), a herbaceous perennial originating from East Asia, has demonstrated bacteriostatic and antiviral properties. The aim of this study was to identify an effective extraction procedure for isolating potential bacteriostatic and antiviral compounds from the plant. The crude extracts were obtained using ethanol, methanol or acetone, and stepwise extracted with ethyl acetate, n-butanol or water. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and diameters of the inhibition zone were evaluated for bacteriostatic efficacy. These tests were carried out with Enterococcus faecalis , Enterococcus faecium, Shigella dysenteriae , Streptococcus lactis , Staphylococcus aureus , and Streptococcus lactis . The antiviral activities of prevention, treatment, and neutralization, utilizing pseudorabies virus, were evaluated using swine testicular cell viability. The active ingredients from the best bioactivity extraction procedure were analyzed using liquid chromatography-mass spectrometry (LC-MS). When ranked highest to lowest, the bacteriostatic and antiviral effects were obtained in the order of the crude extracts prepared from ethanol, methanol, and acetone. Bacteriostatic and antiviral effects were highest in the stepwise extraction of ethyl acetate, followed by the extraction of n-butanol and water. The main compounds identified by LC-MS in the ethyl acetate phase in descending order of content were: bergenin, palmitic acid, baicalein, linoleic acid, chrysin, γ -linolenic acid, catechin gallate, catechin, ursolic acid, and baicalin. This study provides a research basis for the development of bacteriostatic and antiviral drugs from traditional Chinese herbal medicines.

The excessive use of antimicrobials in the clinic has rendered antimicrobial resistance one of the most severe global public health problems. Additionally, viral pathogens are highly harmful and have proven difficult to impossible to treat with therapeutic medicines available at the current time. The pseudorabies virus (PRV) is one of the most common viral pathogens threatening the global swine industry. 1 It has been detected in at least 44 countries since its discovery in the early 20 th century. The virus causes reduced fertility in sows, respiratory disease in adult pigs, nervous system disorders and high piglet mortality. 2 According to reports, the estimated losses due to PRV infection in the United States swine industry ranged from $21 million to nearly $33 million per year. 3 Medicinal plants are considered a repository of bioactive ingredients possessing varied therapeutic features. A unique approach to both the overuse of antibiotics and identification of effective antiviral agents is investigation of Chinese herbal medicines (CHM) that have been associated with antibacterial and antiviral effects. 4 For example, glycyrrhizin, a bioactive compound of licorice root, has been used as a traditional CHM for a variety of beneficial clinical effects including anti-inflammatory properties. Diammonium glycyrrhizate, derived from glycyrrhizin, is more stable, soluble and has significant antiviral activity. In particular, it inhibits cellular infection by PRV and reduces apoptosis during PRV infection. 5 Rodgersia sambucifolia (Hemsl) (RSHE) is a herbaceous perennial originating from East Asia (i.e. Rodgers flower, rhizome of featherleaf). In China, known as Hong Jiang or Jin Yue Tuo, it has been used as a traditional Chinese medicine, exhibiting suitable pharmacological activities and little toxicity or side effects. 6,7 The plant is known for its active component, bergenin, which has antinociceptive and anti-inflammatory properties; however, other active components are less explored. In particular, the components associated with antibacterial and antiviral properties are especially compelling. 8 Different extraction procedures can influence bacteriostatic and antiviral effects of natural products. 9 Ethanol (ET) and ethyl acetate extracts of Rodgersia aesculifolia (Batal) exhibited the best antiproliferative effects on Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae and Bordetella bronchiseptica. 10,11 The antiviral activity of the n-butanol and ethyl acetate extracts was better than that of the acetone extract against Coxsackie B group I-VI viruses. The bioactivities of these extracts, however, were the same for the Herpes simplex type I virus. 12 The aim of this study was to compare different extraction methods in order to identify an efficient extraction procedure for isolating bacteriostatic and antiviral components from Rodgersia sambucifolia (Hemsl). The effect of the extraction methods on potential bacteriostatic and antiviral activity was evaluated on six bacteria and PRV under in vitro conditions. Additionally, liquid chromatography-mass spectrometry (LC-MS) was used to investigate and identify active ingredients in the R. sambucifolia extracts. The objectives of this study, therefore, were to identify an effective extraction procedure that optimized bacteriostatic and antiviral efficacy and to identify the main ingredients in the extracts that might contribute to the plant extract's biological activity. Study findings provide a basis for the development and use of a natural antibacterial and antiviral drug.

Preparation of Rodgersia sambucifolia (Hemsl) extracts (RSHE)
Rodgersia sambucifolia (Hemsl) was purchased from a R. sambucifolia plantation in China a . Its crude extracts were obtained as follows: the plant was dried and granulated with a 60 mesh sieve and mixed with ET, methanol (MT) or acetone b (AT). The powder was further dispersed by ultrasonication for 15 minutes and overnight magnetic rotation at room temperature. The yield rate of the crude extracts was calculated after filtration, rotary steaming, concentration and freeze-drying.
The extraction phase of R. sambucifolia was obtained as follows: the crude extracts of ET, MT and AT were concentrated and extracted 3 times with ethyl acetate, n-butanol or distilled water. The extraction liquids from these 3 steps were combined, and yield rate of crude extract was calculated after rotary steaming, concentration and freeze-drying.

In vitro bacteriostatic test
The bacteriostatic efficacy test was performed by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and bacterial growth inhibition zones. These tests were conducted against Enterococcus faecalis, Enterococcus faecium, Streptococcus lactis, Shigella dysenteriae, Klebsiella pneumonia, and Staphylococcus aureus c .
The broth dilution assay determined the MIC and MBC values of RSHE extracts using two-fold serial dilutions (100 to 0.195 mg/ml). The bacterial solution (100 µL of 10 6 CFU/ml) was incubated for 18 hours. Iodonitrotetrazolium chloride blue was added as an indicator of microbial growth. The MIC value was visually determined when the solution turned colorless. The minimum bactericidal concentration without any growth was considered the MBC. The inhibition zone was determined by the Oxford cup assay. 13 Three concentrations of RSHE (100 µL each of 100 mg/ml, 50 mg/ml, and 25 mg/ml) were added into Oxford cups in triplicates. The blank control contained distilled water. The evaluation criteria for the bacteriostatic zone followed a previous study. 14

In vitro anti-pseudorabies virus (anti-PRV) test
Swine testicular (ST) cells were obtained from a standard supplier d . Following a previous report, the cells were revived and cultured. 15 The procedure was initiated by thawing cells in a 37°C water bath and collected after centrifugation at 1000 rpm for 4 minutes. 16 Culture medium (2 ml) was added and the cells were incubated (37°C, 5% CO 2 ) and grown to a 90% confluence. The culture medium was collected, twice washed with 1ml of phosphate buffered saline and digested by 0.25% trypsin. The collected cells were gently blown into suspension and divided into 2-3 culture flasks which were placed in a cell incubator (37°C, 5% CO 2 ). Cell concentration and survival rate were calculated.
The ST cells were then mixed with lyophilization solution and divided into sterile lyophilization tubes which were frozen overnight at -80°C with gradient cooling. The tubes were transferred to a liquid nitrogen tank for storage.
To assess a safe RSHE concentration, the ST cells were incubated for 24 hours with 10 RSHE doses (200, 100, 50, 25, 12.5, ..., 0.78 µg/ml; 6 replicates of each dose). The ST cells (10 4 cells) were cultured on a 96-well culture plate and infected with 10 -1 to 10 -8 doses of the PRV solution into eight replicates. The PRV was isolated at the Preventive Veterinary Laboratory of the College of Veterinary Medicine, Yunnan Agricultural University (virus titer = 10 5.6 TCID 50 /ml). 17 The TCID 50 was calculated from the cytopathic effect.
The antiviral effect of RSHE was evaluated according to cell viability using the CCK-8 kit for prevention, treatment and neutralization of PRV e . The prevention efficacy was evaluated as follows: a safe concentration of RSHE was first added to 10 4 ST cells, and then the cells were infected with 100 µL of PRV (100 TCID 50 ). The treatment efficacy was assessed as follows: ST cells (10 4 ) were first infected with 100 µL of PRV (100 TCID 50 ); the safe concentration of RSHE was then added. The neutralization efficacy of PRV was evaluated by mixed liquid incubation 100 µL of PRV (100 TCID 50 ) + safe concentration RSHE. The PRV control group was infected for 4 hours and then incubated until 80% of the cells showed pathology (around 2 hours).

Screening of the potential active components from RSHE
The best biological extraction phase (with maximum antibacterial and antiviral properties) was selected for active ingredients screening. The potential active components in RSHE were analyzed by LC-MS. The parameter settings were: column temperature 40°C, flow rate 400 µL/min, sample loading 2 µL. For positive ion mode: Phase A was 0.1% formic acid in water; Phase B was 0.1% formic acid in acetonitrile. For anion mode: Phase A was water (with 2 mM ammonium acetate); Phase B was acetonitrile. The flow elution gradient was: 0-1.5 min, 95% A, 5% B; 2.5 min, 90% A, 10% B; 14 min, 60% A, 40% B; 22-25 min, 5% A, 95% B; and 26-30 min, 95% A, 5% B. For electrospray ionization mass spectrometry: the ion source was the atomizing pressure of 60 PSI (pounds/square inch); auxiliary pressure was 60 PSI; air curtain pressure was 35 PSI; temperature was 650°C; spray voltage was 5000 V (positive ion mode) or -4000 V (negative ion mode).

Statistical analysis
The extraction rate of RSHE = (W/50)×100% with "W" as the weight of the extract, that is, the extraction rate of RSHE is the ratio of the weight of the extract to 50 g RSHE. The data from the bacteriostatic and anti-PRV tests were statistically analyzed by commercial software f using a one-way ANOVA test. Statistical significance was p < 0.05.

Figure 1:
The highest to the lowest extraction rate was obtained for the ET, MT, AT solvents, and the n-butanol phase (NBA), ethyl acetate phase (AC), and aqueous phase (AQ).

In vitro bacteriostatic tests (MIC, MBC, Oxford cup with bacteriostatic zone tests)
The ET-AC extract achieved the most effective inhibition based on MIC results against Enterococcus faecalis and Streptococcus lactis (MICs = 0.39 mg/ml). The ET-AC extract against Shigella dysenteriae, the MT-NBA extract against Enterococcus faecalis and Streptococcus lactis and the AT-AC against Staphylococcus aureus and Streptococcus lactis had an MIC value of 0.78 mg/ml. The MBC results (0.78 mg/ml) for the best inhibition were consistent with the MIC results. Study findings, therefore, demonstrated RSHE inhibited the proliferation of six bacteria and the highest inhibition was observed in the ET-AC extract ( Table 1). The zone of inhibition of bacterial growth resulted in 27 combinations of bacteria tested being sensitive to RSHE, 21 of which were associated with ethyl acetate ( Table 2). The RSHE, therefore, inhibited bacterial proliferation, and the AC extract displayed a notable antibacterial effect. than the control group. Cell viabilities of 200~100 µg/ml of MT, MT-AC, MT-NBC and MT-AQ were substantially lower than those of the control group. The cell viability of 200~100 µg/ml of AT, 200~50 µg/ml of AT-AC, 20~50 µg/ml of AT-NBC, and 200 µg/ml of the AT-AQ extracts were significantly lower than that of the control group. These specific concentrations of RSHE, therefore, were toxic to ST cells, while the other concentrations were safe. According to the cell viability of these 3 types of RSHE, the safe concentration of RSHE ranged from 0.78 to 12.5 µg/ml (Figure 2).

In vitro anti-pseudorabies virus (anti-PRV) test
The cell viability of the PRV groups was significantly lower than the control groups (p<0.05), indicating the successful infection of the ST cells by PRV. The viability of ST cells in the groups treated with 12.5 µg/ml of ethanol extract of n-butanol phase, 12.5 µg/ml of MT-AC, and 3.13 µg/ml of AT-AC was significantly higher than that of the PRV groups. The viability of these cells increased by 30.54% (p<0.001), 35.66% (p<0.001), and 22.52% (p<0.01), respectively. These results revealed the prevention efficacy of RSHE (Figure 3).
The ST cell viability of the groups treated with 1.56 µg/ml of ET, MT, and AT was significantly higher than that of the PRV groups. The cell viability increased by 12.26% (p<0.01), 12.34% (p<0.01) and 22.48% (p<0.01), respectively. These results revealed the treatment efficacy of RSHE (Figure 4).

DISCUSSION
In this study, different extraction procedures were used to evaluate the bacteriostatic and antiviral efficacies of the Chinese herbal medicine, Rodgersia sambucifolia (Hemsl). The order of stepwise extraction for the 3 solvents was AC, NBA and AQ, which was consistent with previous reports describing higher bacteriostatic effect in the AC extract than other organic phases. 12,18 Study findings then demonstrated that the ethyl acetate extraction of AC yielded the most active bacteriostatic/antiviral ingredients, which were further analyzed by LC-MS. The main compounds identified in this extract included: bergenin, palmitic acid, baicalein, linoleic acid, chrysin, γ-linolenic acid, catechin gallate, catechin, ursolic acid, and baicalin. Findings from this study provide a basis for the use and development of natural antibacterial and antiviral drugs from Rodgersia sambucifolia (Hemsl). Study findings demonstrated RSHE had a notable antibacterial effect. The ET-AC extract achieved the most effective inhibition based on MIC results against Enterococcus faecalis and Streptococcus lactis and the AT-AC against Staphylococcus aureus and Streptococcus lactis. The RSHE demonstrated inhibition of 6 bacteria (ET-AC greatest effect) and the zone of inhibition of bacterial growth resulted in 27 combinations of bacteria demonstrating sensitivity to RSHE (21 of these with ethyl acetate).
Specific antiviral assessment demonstrated 1.56 µg/ml concentration of the ET, MT, and AT-AC extracts of R. sambucifolia exhibited positive treatment efficacy in PRV-infected ST cells. The prevention efficacies of the MT and ET-AC groups were better than those of other extracts. The MT-NBA extract had the most effective PRV neutralization action. These results indicated that the dose might impact antiviral activities. The bacteriostatic and antiviral mechanisms of RSHE could be related to direct killing, inhibition of virus replication or adsorption into cells.
Similar to the flavonoid containing extracts of R. sambucifolia, studies have shown other flavonoids such as myricetin, and flavonoid containing herbs such as Panax notoginseng, and Radix isatidis have an antiviral effect on PRV. Myricetin can effectively inhibit PRV infection and prevent PRV from entering cells early in infection. It can regulate NF-κB and mitogen-activated protein kinase pathways to inhibit viral replication and control the inflammatory response and apoptosis caused by viral infection. 19 Panax notoginseng polysaccharides inhibit PRV infection by interfering with PRV adsorption and replication in vitro. 20 Radix isatidis polysaccharide can inhibit PRV replication, prevent infection, and kill the virus. 21 LC-MS results of the RSHE in the present study indicate the presence of a variety of flavonoids with antibacterial, antitumor, antiviral and immune functionenhancing properties. Components such as baicalein, gallic acid, catechin, and quercetin (flavonoid components) have been associated with bacteriostatic and antiviral properties. [22][23][24][25][26] The flavonoid components baicalein, baicalin and catechin are used for their antibacterial, anti-PRV, anti-influenza effects and the treatment of viral pneumonia. 27,28  The present study sought to define an optimal procedure for extraction of antibacterial and antiviral biocompounds from Rodgersia sambucifolia Hemsl. The antibacterial and antiviral effects of ethanolic, methanolic, acetonic extracts and their isolates after sequential extraction with ethyl acetate, n-butanol and water were determined. The ethyl acetate phase displayed the best bacteriostatic and anti-PRV properties, therefore, was considered the best extraction procedure and was subjected to LC-MS analysis. The main active ingredients determined by LC-MS included the flavonoid components of baicalin, baicalein, chrysin, quercetin, and polyphenol components: bergenin, gallic acid, catechin. These isolates may be related to R. sambucifolia's bacteriostatic and anti-PRV effects but await further mechanistic study for proof of this hypothesis. In conclusion, Rodgersia sambucifolia (Hemsl) with its variety of active ingredients has broad application potential and this study provides a basis for developing bacteriostatic and antiviral agents from this traditional Chinese medicine.