Anti-angiogenic potential of ardisia crispa roots ethanolic extract and its quinone-rich fraction in mice

Angiogenesis is the process of blood vessel formation which plays a crucial role in normal physiology, and also in the progression of various chronic diseases such as cancer, arthritis and such. As targeting angiogenesis has become an important strategy in the search of treatments of various debilitating ailments, it is a need-based study to identify a natural source of anti-angiogenic agent that may halt the progression of angiogenesis event. Ardisia crispa, locally known as “mata itik” (Family: Myrsinaceae) has been used in traditional Malay medicine to treat various ailments related to inflammation. Ardisia crispa roots have been shown to treat various inflammation-related diseases in several previous studies. As angiogenesis is strongly correlated with inflammation, the aim of the present study was to evaluate anti-angiogenic potential of the hexane partition of Ardisia crispa roots ethanolic extract (ACRH) and its quinone- rich fraction (QRF) on several experimental models, namely Miles vascular permeability test, murine air pouch granuloma and mouse sponge implantation test. Preliminary cyclooxygenase and soy lipoxygenase inhibitory study were also conducted to elucidate the possible pathways involved. Preliminary phytochemical screening of ACRH indicated the abundant presence of flavonoid, triterpene and tannin. Quinone- rich fraction (QRF) was separated from ACRH (38.33% w/w) and further isolated to yield a compound, namely fAC-2, indicated by a single TLC spot at Rf: 0.76. The compound was later found to be impure, when later analysed with GC-MS. Nevertheless, fAC-2 was elucidated to possess a major constituent of a benzoquinonoid compound (2-methoxy-6-undecyl-1, 4-benzoquinone), when compared with the standard data. Both ACRH and QRF were also quantified using high performance liquid chromatography (HPLC). For toxicity study, the LD50 value of ACRH was found to be 617.02 mg/kg. In Miles vascular permeability assay, the lowest dose of both ACRH and QRF (10 mg/kg) produced significant reduction in VEGF-induced hyperpermeability compared to vehicle control. In murine air pouch granuloma, ACRH and QRF displayed significant and dose-dependent angiogenic and inflammatory inhibition, in which significant reduction of vascular index and granuloma tissue weight was observed at high dose (100 mg/kg). ACRH and QRF were also shown to possess selective COX-2 inhibitory properties which were dose-dependent, though COX-1 inhibition was also observed in a lower percentage. On the other hand, ACRH and QRF did not exhibit LOX inhibitory activity. Interestingly, fAC-2 showed its selectivity towards the inhibition of COX-2, instead of COX-1, and showed to be a moderate LOX inhibitor. Thus, it can be concluded that Ardisia crispa roots showed potential anti-angiogenic properties by partly mediating COX-2 activity, as shown in the in vitro screening, and it is postulated that fAC-2 (2-methoxy-6-undercyl-1, 4-benzoquinone) displays dual COX-2 and LOX once it is purely isolated in a large scale and tested in vivo

Angiogenesis inhibitors from natural sources

A multi-target strategies targeting on various biochemical and physiological pathways implicated in tumour pathogenesis should be developed with the ultimate aim to manage patients with cancer and reduce the normal-tissue toxicity. Tumor angiogenesis has been recently discovered as an important strategy in treating cancer as most tumors rely on angiogenesis to survive, develop, invade and metastasize. Targeting angiogenesis to inhibit the progression of tumorigenesis has recently been a focus in developing novel anti-cancer development. This is mainly due to the specificity that anti-angiogenic possesses: it targets on newly-formed blood vessels and spares the existing ones. With that being said, inhibiting angiogenesis is now considered a promising strategy in the development and selection of new anti-cancer drug candidates. To date, there are cytotoxic drugs which also exhibit antiangiogenic activity but not angiogenesis inhibitors in whole. In this chapter, we will be discussing selected natural sources including marine products which have been investigated for their antiangiogenic activities. Various methods in validating the effects as well as their possible multiple pathways will also be contended in this chapter

Ardisia crispa roots inhibit cyclooxygenase and suppress angiogenesis

Background: In our previous studies conducted on Ardisia crispa roots, it was shown that Ardisia crispa root inhibited inflammation-induced angiogenesis in vivo. The present study was conducted to identify whether the anti-angiogenic properties of Ardisia crispa roots was partly due to either cyclooxygenase (COX) or/and lipoxygenase (LOX) activity inhibition in separate in vitro studies. Methods: Benzoquinonoid fraction (BQ) was isolated from hexane extract by column chromatography, and later analyzed by using gas chromatography–mass spectrometry (GC-MS). Anti-angiogenic effect was studied on mouse sponge implantation assay. Ardisia crispa ethanolic rich fraction (ACRH), quinone-rich fraction (QRF) and BQ were screened for COX assay to evaluate their selectivity towards two isoforms (COX-1 and COX-2), The experiment on soy lipoxygenase (LOX) inhibitory assay was also performed to determine the inhibitory effect of ACRH, QRF and BQ on soy LOX. Results: BQ was confirmed to consist of 2-methoxy-6-undecyl-1,4-benzoquinone, when compared with previous data. Antiangiogenesis study exhibited a reduction of mean vascular density (MVD) in both ACRH and QRF, compared to control. In vitro study showed that both ACRH and QRF inhibited both COX-1 and COX-2, despite COX-2 inhibition being slightly higher than COX-1 in BQ. On the other hand, both ACRH and QRF were shown to have poor LOX inhibitory activity, but not BQ. Conclusions: In conclusion, ACRH and QRF might possibly exhibit its anti-angiogenic effect by inhibiting cyclooxygenase. However, both of them were shown to possess poor LOX inhibitory activity. On the other hand, BQ displayed selectivity to COX-2 inhibitory property as well as LOX inhibitory effect

The toxic effects of p-Cresyl Sulfate on bone metabolism

Background p-Cresyl Sulfate (pCS) is a uremic toxin that has been implicated in kidney disease, cardiovascular risks, endothelial dysfunction and neuropathies. In chronic kidney disease (CKD), pCS progressively accumulates in the body as the dysfunctional kidneys have a reduced ability to excrete toxins normally. pCS accumulated as a consequence of CKD cannot be removed from th body through dialysis, hence leading to further accumulation. This ultimately leads to bone loss which correlates with the worsening of CKD. As such, pCS could play a part in the development of bone loss with CKD. The objective of this review is to further understand the comprehensive effects pCS has on the bone

Synergistic action of compounds isolated from the hexane extract of Ardisia crispa root against tumour-promoting effect, in vitro

An isomeric mixture of α,β-amyrin (triterpene) and 2-methoxy-6-undecyl-1,4-benzoquinone (quinone) isolated from the Ardisia crispa root hexane (ACRH) extract was reported to possess anti-inflammatory properties in vivo. Considering the close association between inflammation and cancer, on top of the lack of antitumour study on those compounds, this study aimed to determine the potential of both compounds against tumour promotion in vitro, either as single agent or in combination. Triterpene and quinone compounds, as well as triterpene–quinone fraction (TQF) and ACRH were subjected to inhibition of Epstein–Barr virus-early antigen (EBV-EA) activation assay for that purpose. Compared with curcumin (positive control), inhibition against EBV-EA activation occurred in the order: ACRH>TQF ≥ curcumin>α,β-amyrin ≥ 2-methoxy-6-undecyl-1,4-benzoquinone. These findings reported, for the first time, the antitumor-promoting effect of α,β-amyrin and 2-methoxy-6-undecyl-1,4-benzoquinone from the roots of A. crispa, which was enhanced when both compounds act in synergy

The hexane fraction of Ardisia crispa Thunb. A. DC. roots inhibits inflammation-induced angiogenesis

Background: Ardisia crispa (Myrsinaceae) is used in traditional Malay medicine to treat various ailments associated with inflammation, including rheumatism. The plant's hexane fraction was previously shown to inhibit several diseases associated with inflammation. As there is a strong correlation between inflammation and angiogenesis, we conducted the present study to investigate the anti-angiogenic effects of the plant's roots in animal models of inflammation-induced angiogenesis.Methods: We first performed phytochemical screening and high-performance liquid chromatography (HPLC) fingerprinting of the hexane fraction of Ardisia crispa roots ethanolic extract (ACRH) and its quinone-rich fraction (QRF). The anti-inflammatory properties of ACRH and QRF were tested using the Miles vascular permeability assay and the murine air pouch granuloma model following oral administration at various doses.Results: Preliminary phytochemical screening of ACRH revealed the presence of flavonoids, triterpenes, and tannins. The QRF was separated from ACRH (38.38% w/w) by column chromatography, and was isolated to yield a benzoquinonoid compound. The ACRH and QRF were quantified by HPLC. The LD50 value of ACRH was 617.02 mg/kg. In the Miles vascular permeability assay, the lowest dose of ACRH (10 mg/kg) and all doses of QRF significantly reduced vascular endothelial growth factor (VEGF)-induced hyperpermeability, when compared with the vehicle control. In the murine air pouch granuloma model, ACRH and QRF both displayed significant and dose-dependent anti-inflammatory effects, without granuloma weight. ACRH and QRF significantly reduced the vascular index, but not granuloma tissue weight.Conclusions: In conclusion, both ACRH and QRF showed potential anti-inflammatory properties in a model of inflammation-induced angiogenesis model, demonstrating their potential anti-angiogenic propertie

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin−/VCAM-1− (–/–), E-selectin+/VCAM-1− (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin−/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a “quiescent” subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction

The hexane fraction of Ardisia crispa

BACKGROUND: Ardisia crispa (Myrsinaceae) is used in traditional Malay medicine to treat various ailments associated with inflammation, including rheumatism. The plant’s hexane fraction was previously shown to inhibit several diseases associated with inflammation. As there is a strong correlation between inflammation and angiogenesis, we conducted the present study to investigate the anti-angiogenic effects of the plant’s roots in animal models of inflammation-induced angiogenesis. METHODS: We first performed phytochemical screening and high-performance liquid chromatography (HPLC) fingerprinting of the hexane fraction of Ardisia crispa roots ethanolic extract (ACRH) and its quinone-rich fraction (QRF). The anti-inflammatory properties of ACRH and QRF were tested using the Miles vascular permeability assay and the murine air pouch granuloma model following oral administration at various doses. RESULTS: Preliminary phytochemical screening of ACRH revealed the presence of flavonoids, triterpenes, and tannins. The QRF was separated from ACRH (38.38% w/w) by column chromatography, and was isolated to yield a benzoquinonoid compound. The ACRH and QRF were quantified by HPLC. The LD(50) value of ACRH was 617.02 mg/kg. In the Miles vascular permeability assay, the lowest dose of ACRH (10 mg/kg) and all doses of QRF significantly reduced vascular endothelial growth factor (VEGF)-induced hyperpermeability, when compared with the vehicle control. In the murine air pouch granuloma model, ACRH and QRF both displayed significant and dose-dependent anti-inflammatory effects, without granuloma weight. ACRH and QRF significantly reduced the vascular index, but not granuloma tissue weight. CONCLUSIONS: In conclusion, both ACRH and QRF showed potential anti-inflammatory properties in a model of inflammation-induced angiogenesis model, demonstrating their potential anti-angiogenic properties

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro) vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ-andmicrovascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin(-)/VCAM-1(-) (-/-), E-selectin(+)/VCAM-1(-) (E-sel+), E-selectin(+)/VCAM-1(+) (+/+), and E-selectin(-)/VCAM-1(+) (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-kappa B and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a "quiescent" subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction

Pharmacological inhibition of focal adhesion kinase 1 (FAK1) and anaplastic lymphoma kinase (ALK) identified via kinome profile analysis attenuates lipopolysaccharide-induced endothelial inflammatory activation

Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies