Phytochemical Analysis and Antimicrobial Efficacy of Macleaya cordata against Extensively Drug-Resistant Staphylococcus aureus

Abstract

The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics in livestock feed. Many nations in Europe have banned the use of antibiotics in feed, leading to higher rates of infection in livestock animals and reduced productivity for the food market. Increasingly, researchers are looking into the efficacy of phytopreparations to replace antibiotics in feed, allowing for increased animal health without the development of resistance. Macleaya cordata, or Chinese plume poppy, shows promise as a food additive. To evaluate the antimicrobial efficacy of this plant, we tested in vitro activity of M. cordata extract, as well as pure compounds sanguinarine and chelerythrine against wild-type, methicillin-resistant, and multiply-resistant strains of Staphylococcus aureus (SA1199, AH1263, and IA116, respectively). Combination tests to evaluate synergy, additivity, and antagonism within the extract were also completed for the first time. Sanguinarine and chelerythrine showed complete growth inhibition of all strains of S. aureus at concentrations ranging from 3–10 µg/mL, and were equal in activity or were more potent than the reference compound chloramphenicol. Combination studies of pure sanguinarine and chelerythrine with M. cordata extract revealed additivity or indifference of mixture components with these compounds. Because sanguinarine and chelerythrine represent the major active constituents of M. cordata, the pooled amounts of these two compounds may be useful for establishing potency for quality control purposes. This is the first report of activity of chelerythrine and sanguinarine against methicillin-resistant S. aureus AH1263 and multiply-resistant S. aureus IA116, and illustrates the promise of M. cordata extract as an alternative to antibiotics in feed additives.

Plants play a role in many medicinal practices, including Chinese traditional, Ayurvedic, and Unani medicines [1]. Medicinal plant use remains popular today, and an estimated 80% of the world’s population relies on herbal products and supplements as primary sources of medicines [2–4]. In the United States approximately 18% of adults [5] and 5% of children [6] use natural products.

Macleaya cordata (Wild.) R. Br. (Papaveraceae), or plume poppy, is a traditional Chinese herbal medicine native to China and Japan [7]. This botanical has been used to relieve muscle pain and wounds [8] and to treat insect bites and ringworm [7]. M. cordata is used in traditional Chinese medicine to treat wounds, arthritis, and Trichomonas vaginalis [8]. M. cordata extract is also antimicrobial against pathogenic bacteria [7, 9–11]. Most activities of M. cordata have been linked to quaternary benzo[c]phenanthridine- and protopine-type alkaloids [8], which are used in phytopreparations with anti-inflammatory and antimicrobial activities [7].

Antibiotic resistance is an emerging crisis worldwide [12, 13]. At least 2 million people acquire serious infections annually in the United States, leading to at least 23,000 deaths [14]. A recent report suggests that 700,000 mortalities are due to drug-resistant bacterial infections, HIV, tuberculosis, and malaria worldwide [12]. Annually, antibiotic resistance also leads to nearly $20 billion in direct healthcare costs [14]. Methicillin-resistant Staphylococcus aureus (MRSA), one of the most common antibiotic-resistant bacteria, causes over 80,000 infections and 11,000 annual deaths in the United States [14]. Numerous strains of this bacterium exist with variable resistance profiles [15].

Antibiotic additives are used to enhance feed efficiency and growth of livestock animals [16]. However, the overuse of antibiotics has increased the presence of antibiotic-resistant bacteria [16]. European Union member nations banned antibiotic food additives in 2006 to overcome this risk, increasing infections in livestock and decreasing animal production [16]. As such, alternative feed additives are being investigated, including plant extracts. Constituents within complex phytotherapies may work additively by the same or different mechanisms, they may mask activity of active components, or they may potentiate the activity of antimicrobial compounds [17, 18]. If phytotherapies are to replace antibiotics in feed, the activity of constituents and combination effects within the extract should be assessed [16].

Many safety assessment studies have been conducted on benzo[c]phenanthridine compounds sanguinarine (1, Figure 1) and chelerythrine (2, Figure 1), and sanguinarine- and chelerythrine-containing plants including M. cordata [19–27]. In pigs fed between 0.1 and 5 mg/kg body weight for 90 days, no toxic effects were witnessed, despite high levels of sanguinarine and chelerythrine in gingiva, liver, and plasma [22]. Similarly, rats fed up to 150 mg/kg sanguinarine for 14 days showed no toxic effects [20], and the LD₅₀ for rabbits treated intradermally was greater than 200 mg/kg [20]. In another study, human red blood cells were not affected by chelerythrine concentrations lower than 8.0 mM [25]. Conversely, Swiss albino mice treated at single doses ranging from 1.35 to 21.60 mg/kg sanguinarine illustrated bone marrow cells and DNA damage in a dose-dependent manner [19]. Other studies have shown that a single dose of 10 mg/kg sanguinarine caused liver damage in rats [21] and male mice [26]. These results are inconsistent with a 109-day study in which rats were fed an average of 10 mg/kg of sanguiritrin (containing high levels of both sanguinarine and chelerythrine), which found no negative effects to liver function [24]. While some studies suggest that sanguinarine and chelerythrine are safe for consumption, the recommended doses vary widely depending on cellular and animal models evaluated.

Figure 1:

Structures of sanguinarine (1), chelerythrine (2), allocryptopine (3), protopine (4), dihydrosanguinarine (5), and dihydrochelerythrine (6), abundant benzo[c]phenanthridine alkaloids found in M. cordata.

M. cordata extract and pure alkaloids contained within the extract (Figure 1) have been shown to possess antibacterial activity [7, 9–11, 15, 28]. Compounds 1 and 2 (Figure 1) are the most comprehensively studied of these compounds and have illustrated anti-MRSA efficacy [28]. Our project goals are to assess whether additional antimicrobials or combinatorial effects exist in M. cordata and to assess the efficacy of active compounds against a highly-resistant strain of MRSA (MRSA USA100 strain IA116) [29] against which activity of M. cordata has not yet been evaluated.

Bioassay-guided fractionation was completed for two stages to simplify M. cordata extract. Bioactive constituents were concentrated during the fractionation process, and multiple first-stage (MC-7 and MC-8) and second-stage fractions (MC-7–2, MC-7–3, and MC-8–4) showed higher inhibition of S. aureus growth than the M. cordata extract (Figure 2). The major compounds present in the bioactive fractions MC-7–2, MC-7–3, and MC-8–4 were tentatively identified by accurate mass as alkaloids 1–6 (Figure 3). MS-MS analysis was conducted for compounds 1 and 2 to further confirm their presence in bioactive fractions (Figures S1-S2). Fractions MC-7–2, MC-7–3, and MC-8–4 contained higher concentrations of these compounds than the extract (Table 1).

Figure 2.

Percent inhibition of S. aureus growth by M. cordata EtOAc extract (MC) and fractions resulting from chromatographic separation against Staphylococcus aureus (SA1199) [30]. The crude extract exhibited complete inhibition at high concentration, and moderate activity at low concentration. Fractionation of the crude extract yielded two fractions active at high concentration (MC-7 and MC-8). Separation of these fractions yielded three fractions active at both high and low concentrations (MC-7–2, MC-7–3, and MC-8–4) indicating that active compounds have been concentrated into these simplified fractions. * for the crude M. cordata extract, a full MIC curve was run using serial dilutions. As such, the low concentration data point represents extracts tested at 12.5 µg/mL rather than at 10 µg/mL.

Figure 3:

Chromatograms of most active second-stage fractions, illustrating that known benzo[c]phenanthridine alkaloids are most abundant in these fractions and are likely responsible for activity. Note, compounds 1 and 2 were identified by comparing with standards, and 3-6 were identified by accurate mass alone.

Table 1:

Concentrations of sanguinarine (1) and chelerythrine (2) in Macleaya cordata root extract (MC) and active second-stage fractions (MC-7–2, MC-7–3, and MC-8–4) out of a total of 100 µg/mL of sample. Active compounds 1 and 2 are increased upon fractionation, explaining the increase in activity witnessed.

Sample	Sanguinarine, 1 (µg/mL)	Chelerythrine, 2 (µg/mL)
MC	0.83 ± 0.16	1.37 ± 0.16
MC-7–2	29.6 ± 7.5	1.62 ± 0.22
MC-7–3	5.31 ± 1.2	0.26 ± 0.03
MC-8–4	0.36 ± 0.08	1.20 ± 0.16

Compounds 1 and 2 were found to have potent antimicrobial activity against growth of S. aureus SA1199 [30], with IC₅₀ values of 1.48 ± 0.10 and 1.21 ± 0.18 µg/mL, and MIC values of 3.12 and 6.25 µg/mL, respectively. Compound 3 was also evaluated and did not possess antimicrobial activity (MIC and IC₅₀ values >100 µg/mL, Figure S3). Compounds 1 and 2 were quantified in the M. cordata extract and bioactive fractions and compared to minimum inhibitory concentration (MIC) curves to determine if antimicrobial activity was explained. The IC₅₀ value of the M. cordata extract was found to be 100 µg/mL. This extract contained 1.37 ± 0.16 µg/mL of compound 2 and 0.83 ± 0.16 µg/mL of compound 1, explaining its bioactivity (Figure 4).

Figure 4:

Dose response curves of compounds 1 (A) and 2 (B) and crude M. cordata extract (C) against Staphylococcus aureus (SA1199)[30]. Concentrations of 1 and 2 have been quantified in the crude extract to assess if they are present at biologically relevant concentrations that explain the activity witnessed. * M. cordata organic extract reached 46% inhibition at 100 µg/mL. Although it did not reach 50% inhibition at this concentration, this value was chosen as the IC₅₀ value.

The activity of 1 and 2 against wild-type S. aureus is in agreement with previous research [28]. However, interactions between compounds within the extract have not been evaluated previously. It is possible that the increased activity witnessed upon fractionation was due to the separation of active constituents from inhibitors of their activity. To assess this possibility, we conducted synergy checkerboard assays [31], and combined M. cordata extract with compounds 1 and 2. Fractional inhibitory concentration (FIC) indices disentangle combinatorial effects, in which values less than 0.5 are considered to be synergistic, values between 0.5 and 4.0 are considered to be additive/indifferent, and values greater than 4.0 are considered antagonistic [32]. In combination with the crude extract, 1 and 2 had FIC values of 2.0 and 1.5, respectively, illustrating additive/indifferent effects (Table 2), further supporting our hypothesis that 1 and 2 are the major antimicrobial compounds contained within M. cordata.

Table 2:

Minimum inhibitory concentrations and IC₅₀ values for sanguinarine and chelerythrine alone and in combination with Macleaya cordata extract. FIC values were calculated using the following equation: ƩFIC = FIC_A + FIC_B = (MIC_A in combination with B/ MIC_A alone) + (MIC_B in combination with A/MIC_B alone)[31]

Treatment	MICa (µg/mL)	IC50 (µg/mL)	FIC
Sanguinarine	3.125	1.48	--
Sanguinarine + extractb	3.125	1.18	2.0
Chelerythrine	6.25	1.21	--
Chelerythrine + extractb	3.125	1.39	1.5
extract	>100	~100	--

^aefficacy against Staphylococcus aureus strain SA1199[30]

^bexpressed for sanguinarine or chelerythrine in combination with 50 µg/mL extract

A recent study suggests that sanguinarine is efficacious against S. aureus due to its ability to compromise the cytoplasmic membrane [34]. Sanguinarine-treated cells showed altered septa morphology and were predisposed to lysis due to the release of membrane-bound autolytic enzymes [34]. The presence of a methyl-substituted nitrogen ring was found to be important, as was a double bond between carbon and positively-charged nitrogen species [25]. A methylenedioxy- group present at positions 2 and 3 appears to be crucial for activity, while methyoxyl- substitutions at C-8 and C-9 allow for antimicrobial activity [25]. Because 1 and 2 both possessed activity, it appears that both methyoxyl- and methylenedioxy- substitutions at positions C-8 and C-9 allow for antimicrobial activity. Allocryptopine (3) likely did not possess activity due to its lack of a carbon-nitrogen double bond or a methylenedioxy- group at positions C-2 and C-3.

Due to the promising antimicrobial efficacy of compounds 1 and 2, we evaluated their effectiveness against two previously untested and clinically relevant strains of MRSA: MRSA USA300 LAC strain AH1263 [33] and a highly virulent and the multiply-resistant MRSA USA100 strain IA116 [29]. Compounds 1 and 2 were active against both AH1263 (1 IC₅₀=1.5 ± 0.1, MIC ≤ 6.3 µg/mL, 2 IC₅₀ = 1.2 ± 0.2, MIC ≤ 6.3) and IA116 (1 IC₅₀=2.5 ± 0.2, MIC ≤ 9.4 µg/mL, 2 IC₅₀ = 3.8 ± 0.6, MIC ≤ 9.4), and had equal or greater activity than the positive control chloramphenicol (Table 3). This is the first report of activity against AH1263 and IA116. IA1166 is highly resistant to beta-lactam antibiotics and erythromycin [29], suggesting that benzo[c]phenanthridine alkaloids 1 and 2 show promise as antimicrobial agents against highly virulent and multiply-resistant Gram-positive organisms. These compounds may also serve as markers to evaluate quality of Macleaya cordata phytopreparations used as livestock feed additives.

Table 3:

MIC and IC₅₀ data for compounds 1–2 and positive control chloramphenicol against three strains of S. aureus relative to vehicle control measured turbidimetrically by OD₆₀₀. Presented data were evaluated using four-parameter logistic curves produced using triplicate data.

Treatment	S. aureus SA1199 a		S. aureus SA1263 b		S. aureus IA116 c
	IC50	MIC	IC50	MIC	IC50	MIC
Sanguinarine (1)	1.3 ± 0.1	≤ 3.1	1.5 ± 0.1	≤ 6.3	2.5 ± 0.2	≤ 9.4
Chelerythrine (2)	1.2 ± 0.3	≤ 6.3	1.2 ± 0.2	≤ 6.3	3.8 ± 0.6	≤ 9.4
Chloramphenicol d	2.2 ± 0.5	≤ 6.3	0.8 ± 0.3	≤ 6.3	5.9 ± 2.2	≤ 19

^awild-type S. aureus with no resistance to commonly prescribed antibiotics (SA1199)[30]

^bclinically relevant methicillin-resistant strain (USA 300 LAC AH1263)[33]

^cmethicillin-resistant strain with additional resistance to erythromycin and beta-lactam antibiotics (USA 100 nasal/blood screen 116, IA116) [29]

^dchloramphenicol served as a positive control (98% purity, Sigma-Aldrich)

Experimental

General Experimental Procedures

UPLC-MS analysis was completed using a Thermo-Fisher Q-Exactive Plus Orbitrap mass spectrometer (ThermoFisher Scientific, MA, USA) connected to an Acquirity UPLC system (Waters, Milford, MA, USA) with reversed phase UPLC column (BEH C18, 1.7 μm, 2.1 × 50 mm, Waters Corporation, Milford, MA, USA). All fractions were analyzed at 0.1 mg mL⁻¹ in methanol (mass of sample per volume of solvent), with 3 μL injections. The gradient was comprised of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The gradient began with 90:10 (A:B) from 0–0.5 min, and increased to 0:100 (A:B) from 0.5–8.0 min after which it was held at 100% B for 0.5 min, before returning to starting conditions over 0.5 min and held from 9.0–10.0 min. Analysis was performed in both positive and negative modes over a m/z range of 150–1500 with the: capillary voltage at −0.7 V, capillary temperature at 310°C, S-lens RF level at 80.00, spray voltage at 3.7 kV, sheath gas flow at 50.15, and auxiliary gas flow at 15.16. The four most intense ions were fragmented with HCD of 65.0. Flash chromatography was conducted with a CombiFlash RF system (Teledyne-Isco) with a photo-diode array (PDA) detector. All chemicals were acquired through Sigma-Aldrich and were spectroscopic or microbiological grade.

Plant Material

Macleaya cordata (Willd.) R. Br. was collected on May 5, 2017, from Vance County, North Carolina (Sample # 12421, N 42°12’17.211”, W 123°19’34.60). The identity of this plant material was confirmed by Dr. Alan Feduccia, and voucher specimens were deposited at the herbarium at the University of North Carolina at Chapel Hill (NCU652633 and NCU652632).

Extraction

M. cordata stems and leaves were freeze-dried using a Labconco model Freezeone6 lyophilizer at −27 °C for 48 hours. The 121.58 g of dry mass was ground using a Wiley Mill Standard Model No. 3 (Arthur Thomas Company) and extracted in MeOH (160 g/L) at room temperature for 24 hours. This process was repeated three times. The MeOH extract was concentrated and partitioned. The extract was defatted using 10% aqueous MeOH and hexane (1:1). The dried aqueous MeOH layer was then partitioned with EtOAc/MeOH/H₂O (4:5:1), and the EtOAc layer was washed with a 1% NaCl solution. The final EtOAc extract was dried under nitrogen, yielding 5,700 mg.

Chromatographic Separation

The fractionation scheme is provided as Supplementary data (Figure S4). The EtOAc extract (5,700 mg) was separated with normal-stage flash chromatography (80 g silica gel column) with a 45.2 min hexane/CHCl₃/MeOH gradient at a 60 mL/min flow rate. Fractions MC-7 (2586 mg) and MC-8 (1501 mg) went through a second stage of normal-phase flash chromatography with a 54-minute hexane/EtOAc/MeOH gradient and were separated using a 40g silica gel column at a flow rate of 40 mL/min. Fractionation of MC-7 and MC-8 yielded 6 and 4 simplified fractions, respectively. The active fractions contained known benzo[c]phenanthridine alkaloids. Rather than pursuing isolation of known compounds, known compounds were quantified using mass spectrometry.

Quantitative analysis of known compounds and contribution to biological activity

To quantify each compound in samples, external calibration curves of standard compounds 1 and 2 (final concentrations from 0–50 µg/mL) were produced to identify the linear range using LC-MS. Each sample was re-suspended in methanol to a concentration of 0.1 mg/mL and then analyzed. Selected-ion chromatograms of each compound were plotted for each sample, and concentrations were back-calculated using the best-fit equation for the relevant calibration curve. These results were compared to biological activity data to determine which fractions possessed biologically relevant concentrations of active compounds.

Antimicrobial Assays

Antimicrobial activity was evaluated by assessing growth inhibition of a laboratory strain of Staphylococcus aureus (SA1199) [30], a clinically relevant strain of methicillin-resistant S. aureus (MRSA USA300 LAC strain AH1263) [33], and a highly virulent strain of S. aureus (MRSA USA100 strain IA116) [29] which is highly resistant to beta-lactams and erythromycin. All strains were provided by Dr. Alexander Horswill at the University of Colorado Anschutz Medical Campus. Cultures were grown from a single colony isolate in Müeller-Hinton broth (MHB) and diluted to 1.0 × 10⁵ CFU/mL based on absorbance at 600 nm (OD₆₀₀).

Samples were screened in triplicate at final concentrations of 10 and 100 μg/mL. Samples were dissolved in DMSO and diluted with MHB to prepare final concentrations containing 2% DMSO. Chloramphenicol (98% purity, Sigma-Aldrich) was used as a positive control at the same concentrations as tested extracts. Minimal inhibitory concentrations (MICs) were calculated for sanguinarine (1), chelerythrine (2), and allocryptopine (3) based on the Clinical Laboratory Standards Institute (CLSI) standard protocols [35]. Isolated compounds were added to 96-well plates in triplicate at concentrations ranging from 0–100 µg/mL in MHB. The vehicle control consisted of broth containing 2% DMSO. After an 18-hour incubation at 37 °C, OD₆₀₀ values were measured using a Synergy H1 microplate reader (Biotek). OD₆₀₀ was used to calculate growth inhibition of samples, and the MIC was defined as the lowest concentration at which no statistically significant difference between the wells that did not contain bacteria and the treated sample were found.

Checkerboard assays [31, 36] were conducted to assess the effect of EtOAc extract on activity of compounds 1 and 2. The M. cordata EtOAc extract was tested in combination with 1 or 2, with each sample ranging in concentration from 1.56–100 µg/mL. The fractional inhibitory concentration index (ƩFIC) is an algebraic equation used to define specific combination effects, where values less than 0.5 are considered to be synergistic, values between 0.5 and 4.0 are considered to be additive/indifferent, and values greater than 4.0 are considered antagonistic [32]. ƩFIC values are calculated as follows [31]:

$$\sum \text{FIC}=FI{C}_A+ FI{C}_B$$ 1

Where $FI{C}_A= \frac{MI{C}_A in presence of B}{MI{C}_A}$, and $FI{C}_B= \frac{MI{C}_B in presence of A}{MI{C}_A}$