IN VITRO STUDIES ON CALCIUM OXALATE CRYSTAL GROWTH INHIBITION OF STROBILANTHES CRISPUS EXTRACTS

 ABSTRACT Strobilanthes crispus L. (Acanthaceae) has been used locally in traditional medicine for kidney stone and related diseases. These plant extracts have the ability to inhibit the calcium oxalate crystal growth, where the ability of water extract is higher than those of the 70% acetone, methanol and acetone extracts. The ability to inhibit the calcium oxalate crystal growth of these extracts is lower than that of sodium citrate as positive control. Keywords: Strobilanthes crispus, Acanthaceae, crystal inhibition, calcium oxalat

Inhibitory effect of Labisia pumila leaf extract on angiogenesis via down-regulation of vascular endothelial growth factor

Purpose: To investigate the anti-angiogenic activity of a methanol leaf extract of Labisia pumila (ME), and its bioactive water fraction (WF), using in vitro models.Methods: The antioxidant activity and total phenolic contents of ME and WF were assessed using DPPH and Folin–Ciocalteu reagents. Antiproliferative effects of extracts towards human umbilical vein endothelial cells (HUVECs) were evaluated using MTT assay. Isolated rat aortic ring and matrigel tube formation assays were performed to assess the antiangiogenic potential of Me and its WF. Levels of VEGF protein in the cell lysates were measured using ELISA kit.Results: Among all the extracts prepared, ME and its WF showed higher total phenolic contents and exhibited moderate antioxidant effects. Significant (p < 0.001) suppression of microvessels outgrowth with half-maximal concentration (IC50) values of 20 and 26 μg/mL for ME and WF, was observed in rat aortic ring assay. ME and its WF halted proliferation and tube formation capacity of HUVECs in in vitro assays. Marked reduction in VEGF levels was observed in lysates of HUVECs treated with ME and its WF.Conclusion: Labisia pumila leaf extract and its water fraction halted angiogenesis by blocking VEGF secretion leading to inhibition of endothelial cells proliferation and differentiation which is suggested to be due to its phenolic antioxidant contents.Keywords: Labisia pumila, Anti-angiogenesis, Antioxidant, Tube formation, Rat aort

Simultaneous Determination of Two Isomers of Asarone in Piper sarmentosum Roxburgh (Piperaceae) Extracts using Different Chromatographic Columns

Purpose: To develop a rapid and reliable reverse phase high performance liquid chromatography (RPHPLC) method to quantify the two isomers of asarones in P. sarmentosum extracts using two different columns under similar analytical conditions.Methods: Two isomers, α- and β-asarone, were analyzed using two types of C 18 columns with 0.1 % orthophosphoric acid: acetonitrile: methanol (50: 40: 10) as mobile phase. The developed method was applied to determine the contents of α- and β-asarone in extracts of different parts of P. sarmentosum.Results: Column A retention times for the elution of α- and β-asarone were 11.890 ± 0.008 and 10.80 ± 0.004 min, respectively, and were significantly shorter than those of column B (15.110 ± 0.024 and 13.290 ± 0.018, respectively, p < 0.001). Column B showed better resolution (1.82 ± 0.025 of the isomers than column A (1.10 ± 0.01, p < 0.001). Both columns showed comparable sensitivity, precision and selectivity of the compounds investigated. α-Asarone level was in the range 0.36 - 5.14 % in ethanol and 50 % ethanol extracts, but absent in all water extracts. β-Asarone occurred in the range of 0.01 - 0.15 % in ethanol and 50 % ethanol extracts but was absent in all water extracts of P. sarmentosum.Conclusion: The results indicate that the developed method is a suitable quality assurance method for determining α- and β-asarone isomers in herbal extracts and food preparations.Keywords: α- Asarone, Isomers, Piper sarmentosum, Herbal extracts, Retention tim

In vitro anti-angiogenic properties of ethanolic crude extract of Vernonia amygdalina

Angiogenesis is the process of generating new blood vessels that deliver tumor cells with oxygen and essential nutrients for growth and metastasis. This study examined the in vitro antiangiogenic properties of the ethanolic crude extract from Vernonia amygdalina (VA) grown in Malaysia. The direct antiangiogenic activity of VA was evaluated on EA.hy926 cells using in vitro assessments: Cell proliferation, colony formation, migration, and cell invasion assays. VA ethanolic crude extract cytotoxic activity was evident in the antiproliferative and colony formation assays. The growth inhibition (IC50) of 50% against EA.hy926 endothelial cells was achieved after 72 h treatment at a concentration of 85.43±3.57 μg/mL. Upon 48 h treatment, colony formation was inhibited completely at 100 μg/mL while 51.94% inhibition was achieved at 50 μg/mL. Moreover, the extract showed 54.72% and 31.99% inhibitory effects against migration of cells when treated for 24 h treatment at two different concentrations, 25 μg/mL and 12.5 μg/mL, respectively. The use of 100 μg/mL VA ethanolic extract inhibited cell invasion by 35.43%, which was lower than that of 57.81% inhibition achieved by the vinblastine as a positive control. All in all, the present work clearly demonstrated the antiangiogenic properties of VA ethanolic extract that may reflect a chemotherapeutic and/or chemoprevention potential for biomedical applications

Evaluation of in vitro cytotoxicity effect of Clinacanthus nutans (Brum. f.) Lindau standardized leaf extracts

Purpose: To standardize Clinacanthus nutans (CN) leaf extracts, evaluate their contents of orientin, vitexin and isovitexin using a reversed-phase high-performance liquid chromatography (RP-HPLC) method, and also to investigate in vitro cytotoxicity of CN. Methods: CN leaf powder was macerated in distilled water, methanol, methanol (50 %), ethanol, and ethanol (50 %) over a hot water bath at 50 - 55 °C for 24 h. The extracts were standardized for total phenolic, flavonoid, proteins and polysaccharides content by ultra-violet (UV) spectrophotometry. Moreover, RP-HPLC was used to determine the contents of orientin, vitexin and isovitexin in the extracts. The anti-proliferative effect of the extracts against human colorectal carcinoma cell line (HCT116) and human colon normal cell line (CCD-18Co) was assessed using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. The most active extract was fractionated using silica gel flash column chromatography to produce 20 fractions. All the fractions were subjected to the MTT test. Results: The extracts showed modest cytotoxicity against HCT-116 and non-cytotoxicity against CCD18Co cell lines. Of all the extracts tested, the methanol extract (CN-M) showed the highest activity of all the extracts and had the highest content of flavonoid and phenolic compounds. Twenty fractions were obtained from this extract. Fraction nos. F3, F4, F14 and F16 showed significant (p < 0.05) cytotoxicity against HCT-116, with F14 having the highest activity. Conclusion: Fraction F14 has the potential to be developed to anti-colon cancer agent. However, further studies including chemical profiling, mechanism of action and safety profile of this fraction are required

Angiogenesis: Managing the Culprits behind Tumorigenesis and Metastasis

Deregulated angiogenesis has been identified as a key contributor in a number of pathological conditions including cancer. It is a complex process, which involves highly regulated interaction of multiple signalling molecules. The pro-angiogenic signalling molecule, vascular endothelial growth factor (VEGF) and its cognate receptor 2 (VEGFR-2), which is often highly expressed in majority of human cancers, plays a central role in tumour angiogenesis. Owing to the importance of tumour vasculature in carcinogenesis, tumour blood vessels have emerged as an excellent therapeutic target. The anti-angiogenic therapies have been shown to arrest growth of solid tumours through multiple mechanisms, halting the expansion of tumour vasculature and transient normalization of tumour vasculature which help in the improvement of blood flow resulting in more uniform delivery of cytotoxic agents to the core of tumour mass. This also helps in reduction of hypoxia and interstitial pressure leading to reduced chemotherapy resistance and more uniform delivery of cytotoxic agents at the targeted site. Thus, complimentary combination of different agents that target multiple molecules in the angiogenic cascade may optimize inhibition of angiogenesis and improve clinical benefit in the cancer patients. This review provides an update on the current trend in exploitation of angiogenesis pathways as a strategy in the treatment of cancer.Ashwaq H. S. Yehya is funded by TWAS (The Academy of Sciences for the Developing World, Italy). Chern Ein Oon is supported by L’Oréal-UNESCO for Women in Science National Fellowship (304/CIPPM/650806/L117) and MAKNA Cancer Research Award (304/CIPPM/650859/M122)

Antiangiogenic properties of Koetjapic acid, a natural triterpene isolated from Sandoricum koetjaoe Merr

Background: Angiogenesis, the formation of new blood vessels, has become an important target in cancer therapy. Angiogenesis plays an important role in tumor growth and metastasis. Koetjapic acid (KA) is a seco-A-ring oleanene triterpene isolated from S. koetjape. The solvent extract of this plant species was shown previously to have strong antiangiogenic activity; however the active ingredient(s) that conferred the biological activity and the mode of action was not established. Given the high concentration of KA in S. koetjape, an attempt has been made in this study to investigate the antiangiogenic properties of KA.Results: Treatment with 10-50 μg/ml KA resulted in dose dependent inhibition of new blood vessels growth in ex vivo rat aortic ring assay. KA was found to be non-cytotoxic against HUVECs with IC40.97 ± 0.37 μg/ml. KA inhibited major angiogenesis process steps, endothelial cell migration and differentiation as well as VEGF expression.Conclusions: The non-cytotoxic compound, KA, may be a potent antiangiogenic agent; its activity may be attributed to inhibition of endothelial cells migration and differentiation as well VEGF suppression

Flavonoids-Rich Orthosiphon stamineus Extract as New Candidate for Angiotensin I-Converting Enzyme Inhibition: A Molecular Docking Study

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 30-hydroxy-5,6,7,40-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure–activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX: BioSolveIT’s LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC50 value (45.77 � 1.17 �g/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC50 15.35 � 4.49 �g/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% � 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE

Ethyl-p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions

OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and angiogenesis-related diseases