While extracts of A. chinensis rhizomes have been frequently reported to have anti-inflammatory23,27, anti-tumor30–34, and anti-platelet activities35, studies on the extract of inflorescence of A. chinensis have yet to be reported. In this study, we demonstrated that GA-13-6, an ethanol extract of inflorescence parts of A. chinensis, has anti-inflammatory properties as well as antibacterial effect on oral pathogenic bacteria such as P. gingivalis, S. sanguinis, and S. mutans. To that end, we collected three different parts (aerial, underground, inflorescence) of the plant from six different regions of South Korea in two different seasons. The total 18 collected samples were initially screened for sample quality and quantity, followed by preliminary tests for the cell cytotoxicity and anti-IL-6 activity. We chose GA-13-6, an ethanolic extract of A. chinensis inflorescence, that inhibited the activation of proinflammatory cytokines most efficiently when RAW 264.7 macrophages were later induced by LPS treatment.
LPS-induced RAW 264.7 macrophages are commonly employed for studies on inflammatory reactions, where NF-κB, a pivotal transcription factor in the nucleus, is activated upon LPS binding to TLR4 on the cell membrane28,36. NF-κB plays an important role in regulating the inflammatory responses by boosting the expression of proinflammatory cytokines and inflammatory mediators, such as TNF, IL-6, iNOS, and COX237,38. TNF, a representative inflammatory cytokine, is secreted early in the immune response and is involved in the activation of inflammation and regulation of cell necrosis39. IL-6, also produced in the acute phase of inflammatory responses, contributes to host defence in both humoral and cellular immunity40. NO, an anti-inflammatory signalling molecule under normal physiological conditions, plays an important role in the pathogenesis of inflammation when over-produced upon infectious and proinflammatory stimuli41. In such abnormal situations, iNOS increases to produce more NO by converting L-arginine to L-citrulline42,43. NO is also involved in the activation of COX2 that leads to the simultaneous release of mediators, such as prostaglandin E2 (PGE2) and prostacyclin (PGI2), from the COX pathway44,45. Thus, selective inhibition of the iNOS pathway is an important strategy for controlling many chronic inflammatory diseases including, but not limited to, cognitive and cardiovascular diseases46. Especially, when the iNOS synthesis is induced by bacterial endotoxin LPS, the production of high levels of NO is delayed but prolonged47, partially explaining our Western results where the level of iNOS protein expression is inconsistent with the level of mRNA expression (Fig. 3b and 3c). The reason why the LPS induction of the macrophages should barely increase COX2 protein expression and why the expression levels of both COX2 and iNOS should increase when the macrophages were pre-treated with GA-13-6 for a longer period remain obscure, although the results suggest that GA-13-6 can counteract LPS-mediated immune responses in a dose-dependent manner (Fig. 3c).
Previously, Gil et al.26 demonstrated the anti-inflammatory effect of ACE from the rhizomes of the plant using LPS-stimulated RAW 264.7 macrophages and thioglycollate-elicited peritoneal macrophages from male C57BL/6 mice. They observed a decrease in the levels of inflammatory mediators (NO, iNOS, PGE2, and COX2) upon pre-incubation of the cells with ACE for 1 h before LPS (1.0 µg/mL) stimulation for over 24 h. In these experimental conditions, the degree of NO reduction was dose-dependent, where 25 µg/mL of ACE was enough to reduce the level of NO to half of the fully elevated level. The expression level of iNOS followed a similar pattern. The reduction levels of other proinflammatory cytokines (TNF and IL-6), however, were limited and dose-independent. In contrast, we pre-incubated the cells with GA-13-6 for 3 and 16 h to compare the effect of short- and long-term exposure before induction inflammatory responses in RAW 264.7 cells using 0.3 µg/mL of LPS while securing the cell viability. In our experimental conditions, the degrees of reduction of IL-6 and TNF were greater with short-term exposure to GA-13-6 (Fig. 2a and 2b). By contrast, the secretion of NO was greater if the cells were pre-exposed to GA-13-6 for 16 h, resulting in a more drastic decrease as the concentration of GA-13-6 increases (Fig. 2c). Likewise, the mRNA expression levels of both Cox2 and Nos2 diminished more stiffly when the cells were exposed for 16 h in a dose-dependent manner. The protein expression levels, however, were remained higher with 16 h pre-treatment of the macrophages with LPS (Fig. 3), indicating that the half-life of the mRNA is shorter than the one of the translated proteins and the longer the incubation the more the protein accumulation. Collectively, GA-13-6 exhibited not less anti-inflammatory effect than ACE from the rhizome in LPS-induced macrophages.
Astilbin is one of the major active flavonoids isolated from the rhizome of A. chinensis48–50 as well as other numerous plants and processed foods like wines, champagnes, and turtle jelly51. The anti-inflammatory activity of astilbin has been reported in T helper 17 (Th17) cells in psoriasis-like mouse model52, HaCaT cells and psoriasis-like guinea pigs model24, adjuvant-arthritis rat model48, high glucose-induced glomerular mesangial cells21, T- and B-cells in lupus mice model53, mouse J774A.1 macrophages54, human chondrocytes25, osteoarthritis mouse25, and rat models55. By contrast, astilbin showed no inhibitory effect on the production of inflammatory mediators and proinflammatory cytokines in LPS-induced RAW 264.7 macrophages (Fig. 4). Interestingly, astilbin isolated from Smilax corbularia was reported to have no inhibitory effect on NO production while blocking PGE2 release in RAW 264.7 cells induced by 1.0 µg/mL of LPS for 24 h50. Additionally, astilbin from the rhizome of Smilax glabra was reported to inhibit the production of NO and TNF but not IL-6 in RAW 264.7 cells induced by 1.0 µg/mL of LPS for 20 h49. Given that flavonoids, commonly found in photosynthesizing plants, possess anti-inflammatory activity in general51, the inconsistency of the results among the research groups may originate from the differences between cell lines, e.g., RAW 264.7 cells vs others, and the amount and duration of LPS treatment. It should be noted, however, that GA-13-6 comprises a variety of functional flavonoids17,56 that can together yield stronger anti-inflammatory activity than a single compound can50.
Periodontal disease is associated with a variety of bacteria and biofilms they form that can cause damages to the periodontal support structure, which is closely related to many systemic diseases2,57. P. gingivalis is a keystone pathogenic bacterium for the onset of periodontal disease58. Recent studies have corroborated the close relationship between the bacterium and systemic diseases including, but not limited to, cancer, cardiovascular disease59–63, diabetes64,65, rheumatoid arthritis66, and Alzheimer’s disease13–16,67−70. P. gingivalis produces several potential virulence factors, such as gingipain proteases, outer membrane vesicles (OMVs), LPS, capsule, and fimbriae15,16,69−72. Among them, gingipain proteases (Rgp and Kgp) are required for its survival while serving as primary virulence factors simultaneously69. Especially, gingipains are directly involved in the modulation of gene expression associated with dementia in the brain70. While capsule and fimbriae mediate physical interactions with host cells, LPS and OMVs trigger intracellular proinflammatory signalling pathways16,63. On the other hand, bacterial commensals or opportunistic pathogens, such as S. sanguinis, Streptococcus gordonii, and Candida albicans, may provide P. gingivalis with favourable environment for its pathogenesis when a balance of bacterial community disrupted71. Given that plant flavonoids could reduce the inflammatory responses and inhibit bacterial growth73, we assumed that GA-13-6 may contain a variety of flavonoids and could suppress the growth of oral pathogens. As expected, GA-13-6 efficiently inhibit the growth of P. gingivalis as well as S. sanguinis and S. mutans, suggesting that GA-13-6 may prevent infection and suppress ensuing inflammation if any infections occur. The difference in the inhibitory efficacy of GA-13-6 between the Gram-positive and Gram-negative bacteria as shown in Fig. 5 could be due to the difference in membrane structures, which can be disrupted by some flavonoids74. Thus, instead of using conventional antibiotics for treatment that can cause dysbiosis and antibiotic resistance, using natural extracts such as GA-13-6 may facilitate the restoration of healthy oral commensalism with minimal side effects.
In this study, we put an effort to collect three different parts of A. chinensis from a wide variety of regions in two different seasons and screen the optimal part of the plant that can effectively prevent the onset of cellular inflammation as well as the growth of oral pathogenic bacteria. By following the screening process, we for the first time found that inflorescence of A. chinensis, GA-13-6, acquired in a flowering season exhibited the best performance. We sought to employ GA-13-6 as a natural resource for the prevention and remedy of periodontal disease, a near-pandemic disease in oral cavity, and widespread dental caries as well. As expected, GA-13-6 successfully suppressed both cellular inflammation responses and oral bacterial growth. Our previous findings indicated that the aerial part of A. chinensis contains nine flavonoids including quercetin but not astilbin56. Thus, further study is needed to identify active ingredients of GA-13-6 responsible for the anti-bacterial efficacy and to define the selectivity of GA-13-6 for many other benign and harmful bacteria in oral cavity.
GA-13-6, an ethanol extract from A. chinensis inflorescence, efficiently suppressed the activation of proinflammatory cytokines and inflammatory mediators, such as TNF, IL-6, and NO, as well as the expression of COX2 and iNOS enzymes in LPS-stimulated RAW 264.7 macrophages. The anti-inflammatory efficacy of GA-13-6 is greater than the one of purified astilbin, which is one of the major effective ingredients found in A. chinensis. The antibacterial effects of the extracts were also confirmed for the first time against key oral pathogens, such as P. gingivalis, S. sanguinis, and S. mutans, indicating that GA-13-6 can inhibit bacterial infection in the oral cavity and suppress the ensuing inflammatory responses.