Phenanthrenes from Arundina graminifolia and in vitro evaluation of their antibacterial and anti-haemolytic properties

Abstract Chemical investigation and activity test of Arundina graminifolia led to the isolation of six phenanthrenes: blestriarene A (1), shancidin (2), densiflorol B (3), ephemeranthoquinone (4), coelonin (5) and lusianthridin (6). The isolated compounds demonstrated antibacterial and anti-haemolytic activities. It was found that compounds 1 and 2 had medium antibacterial activity against Staphylococcus aureus, Bacillus subtilis and Escherichia coli, with MICs of 20–40 μg/mL and MBCs of 40–320 μg/mL. Bactericidal mechanisms were explored. Rupture of cell wall and membrane and leakage of nuclear mass were observed by transmission electron microscopy (TEM). Moreover, compounds 1–3 attenuated the erythrocyte damage. Compounds 1 and 2 showed significant anti-haemolytic activity with inhibition rate about 50% at 16 μg/mL.


Introduction
Arundina graminifolia D. Don (Orchidaceae) has been traditionally used against snake bites, food poisoning, jaundice, arthritis and lung infection in Dai nationality (Traditional Chinese Medicine dictionary 1986). Stilbenoids such as bibenzyls (Liu et al. 2004;Hu et al. 2013 Du et al. 2014), phenanthrenes (Liu et al. 2005), benzofurans ) and benzopyrans (Gao 2013) were found in A. graminifolia and reported activities such as cytotoxicity (Liu et al. 2012), antiviral (Gao et al. 2012) and antioxidant activity (Liu et al. 2011). Phenanthrene and stilbenoid with similar structure skeletons (Wang et al. 2014;Zhao et al. 2016) also existed in other plants of the same orchidaceae family. This study aimed to investigate phytochemicals of A. graminifolia as well as antibacterial and anti-haemolytic activities of isolated phenanthrenes. Moreover, antibacterial mechanisms were observed by TEM.
Two-fold dilution method in 96-well plates was applied to evaluate antibacterial activity of the isolated phenanthrenes against four gram-positive and gram-negative bacteria consisting of Staphylococcus aureus ATCC 29213, Bacillus subtilis CMCC 63501, Escherichia coli CMCC 44825 and Salmonella choleraesuis ATCC 13312. Compounds 1-4 exerted antibacterial activity against S. aureus, B. subtilis and E. coli (Table S1). Compounds 1 and 2 showed notably antibacterial activity with MICs from 20 μg/mL (S. aureus and E. coli) to 40 μg/mL (B. subtilis).
In particular, MBCs of compounds 1 and 2 against S. aureus were 40 and 80 μg/mL, respectively. Besides, compound 3 possessed better antibacterial activities against S. aureus and B. subtilis (gram-positive bacteria strains) with MICs and MBCs in a range of 20-80 μg/mL and 160-320 μg/mL. To the best of our knowledge, compounds 1-4 were reported for their antibacterial activity for the first time.
Two sensitive bacterial strains, namely S. aureus and E. coli, were selected to explore the antibacterial mechanisms of compound 1. TEM photographs demonstrated that normal S. aureus and E. coli were intact and had discernible cell wall and membrane (Figure 2(a) and 2(d)). After treatment with compound 1 at MIC, cells with broken cell wall and membrane were captured (Figure 2(b) and 2(e)). Massive and vital intracellular contents drained out from cells (Figure 2(c) and 2(f )). Taken together, compound 1 might increase bacterial membrane permeability and directly result in structure destruction to kill bacteria. Haemolysin secreted by S. aureus plays an important role in the pathogenesis of S. aureus infection. For example, the absence of haemolytic gene in S. aureus reduced the incidence of peritoneal, breast and pulmonary infection (Qiu 2012). Therefore, compounds 1-3 exhibiting considerable antibacterial activity against S. aureus were selected to investigate anti-haemolytic effect. The result exhibited that compounds 1 and 2 possessed 50% inhibition rate at 16 μg/mL and compound 3 showed 50% inhibition rate at 128 μg/mL. In addition, the morphological changes of erythrocytes were observed. Compared with round, uniform and integral profiles of normal erythrocytes (Figure 3(a)), erythrocytes cultured with S. aureus haemolysin appeared lysed, shrunk and abnormal (Figure 3(b)). However, erythrocytes treated with both haemolysin and compound (compounds 1 and 2) maintained their shape with neat edges and surfaces (Figure 3(c) and 3(d)). These results indicated compounds 1-2 from A. graminifolia alleviated the haemolysin cytotoxicity towards erythrocytes.

Conclusion
Phenanthrenes isolated from A. graminifolia were investigated for antibacterial and anti-haemolytic activities. Compounds 1 and 2 inhibited the growth of S. aureus, B. subtilis  and E. coli by breaking the integrity of the cell walls and membranes. Meanwhile, compounds 1-3 showed anti-haemolytic activity on erythrocytes haemolysis caused by bacterial, which suggested these phenanthrenes had promising value in treatment of infectious diseases and toxicity caused by exotoxin. Our work provided basic data for utilisation of phenanthrenes as new antibacterial and detoxifying agents.

Disclosure statement
No potential conflict of interest was reported by the authors.