Two flavanones named (2S)-7-Hydroxy-5-methoxy-6,8-dimethyl flavanone (1), (S)-5,7-dihydroxy-6,8-dimethyl-flavanone (2), along with known chalcone, namely, (E)-2ʹ,4ʹ- dihydroxy-6ʹ-methoxy-3ʹ,5ʹ-dimethylchalcone (3) and two triterpenoids, namely, betulinic and ursolic acids (4 and 5), were isolated from the leaves of Syzygium campanulatum Korth (Myrtaceae). The structures of compounds (1 and 2) were determined on the basis of UV-visible, FTIR, NMR spectroscopies and LC-EIMS analytical techniques. Furthermore, new, simple, precise, selective, accurate, highly sensitive, efficient and reproducible RP-HPLC method was developed and validated for the quantitative analysis of the compounds (1–5) from S. campanulatum plants of five different age. RP-HPLC method was validated in terms of specificity, linearity (r2 ≤ 0.999), precision (2.0% RSD), and recoveries (94.4%–105%). The LOD and LOQ of these compounds ranged from 0.13–0.38 and 0.10–2.23 μg·mL−1, OPEN ACCESS respectively. Anti-proliferative activity of isolated flavanones (1 and 2) and standardized extract of S. campanulatum was evaluated on human colon cancer (HCT 116) cell line. Compounds (1 and 2) and extract revealed potent and dose-dependent activity with IC50 67.6, 132.9 and 93.4 μg·mL−1, respectively. To the best of our knowledge, this is the first study on isolation, characterization, X-ray crystallographic analysis of compounds (1 and 2) and simultaneous RP-HPLC determination of five major compounds (1–5) from different age of S. campanulatum plants.

Syzygium campanulatum Korth is an equatorial, evergreen, aboriginal shrub of Malaysia. Conventionally it has been used as a stomachic. However, in the currently conducted study dimethyl cardamonin or 2′,4′‐dihydroxy‐6′‐methoxy‐3′,5′‐dimethylchalcone (DMC) was isolated from S. campanulatum Korth, leaf extract. The structural characterization of DMC was carried out by making use of various techniques including UV, IR, NMR spectral followed by LC‐MS, and X‐ray crystallographic techniques. For determining the purity of compound, highly effective techniques including TLC, HPLC, and melting point were used. The cytotoxicity of DMC and three different extracts of S. campanulatum was evaluated against human colon cancer cell line (HT‐29) by three different assays. DMC and ethanolic extract revealed potent and dose‐dependent cytotoxic activity on the cancer cell line with IC₅₀ 12.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report on S. campanulatum as being a rich source (3.5%) of DMC, X‐ray crystallography, and anticancer activity on human colon cancer cells.

Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3'-hydroxy-5, 6, 7, 4'-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 μm, 3 × 150 mm) with 18 min separation time. The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products.

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 3′-hydroxy-5,6,7,4′-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 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC₅₀ 15.35 ± 4.49 µg/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.

Orthosiphon stamineus (OS) Benth is a medicinal plant and native in Southeast Asia. Pharmacological effects of OS are attributed to the presence of lipophilic flavones. However; lipophilic compounds suffer from poor aqueous solubility which limits the OS oral bioavailability and therapeutic applications. Therefore, OS was prepared in nano formulation form using liposomes from soybean phospholipids. The aim of the present study is to evaluate the in vitro genotoxicity and in vivo oral toxicity of nano liposomes of OS ethanolic extract (OS-EL). In the acute toxicity study Sprague Dawley female rats were given a single dose of the OS-EL at 5000 mg/kg/day orally and screened for two weeks after administration. In the subchronic study, three different doses of OS-EL were administered for 28 days. Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histological parameters were monitored during the study. Genotoxicity was assessed using the Ames test with the TA98 and TA100 Salmonella typhimurium strains. High-performance liquid chromatography was performed for identification and quantification of the major marker compounds in OS-EL. Heavy metal detection was performed using an atomic absorption spectrometer. The acute toxicity study showed that the LD50 of the extract was greater than 5000 mg/kg. In the repeated dose 28-day oral toxicity study, the administration of 250 mg/kg, 500 mg/kg, and 1000 mg/kg/day of OS-EL per body weight revealed no significant difference in food and water consumptions, bodyweight change, haematological and biochemical parameters, relative organ weights, gross findings or histopathology compared to the control group. The Ames test revealed that the OS-EL did not have any potential to induce gene mutations in S. Typhimurium. Analyses of these results with the information of signs, behaviour, and health monitoring could lead to the conclusion that the long-term oral administration of OS-EL for 28 days does not cause sub-chronic toxicity.

Purpose: To standardise the methanol and aqueous extracts of Ficus deltoidea leaf by developing a reverse phase high performance liquid chromatography (RP-HPLC) for determination of the ursolic acid content and to investigate their antiangiogenic activity. Methods: To prepare the water extract (FD-A), the powder of the plant was extracted with water under reflux for 24 h at 50 ˚C. The methanol extract (FD-M) was prepared using Soxhlet extractor for 24 h at 50 ˚C. The extracts were standardised for ursolic acid content by reverse phase high performance liquid chromatography (RP-HPLC), as well as by total phenolic and flavonoid contents. Antiangiogenic activity was studied using ex vivo rat aortic rings and in vivo chick chorioallantoic membrane angiogenesis models. The anti-proliferative effect of the extracts against normal human endothelial cells and two cancer cell lines was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: In rat aortic rings, methanol and water extracts inhibited the outgrowth of microvessels with IC50 values of 48.2 ± 1.1 and 62.7 ± 1.4 μg ml-1, respectively. Methanol and water extracts at doses of 100 μg disc-1 also inhibited vascularisation of chick embryo chorioallantoic membrane with inhibition values of of 62.0 ± 6.3 and 43.3 ± 4.8 %, respectively. In addition, both extracts showed potent cytotoxicity against breast and colon cancer cells while demonstrating non-cytotoxic activity against normal endothelial cells. Furthermore, the two extracts showed relatively high concentration of ursolic acid, total phenolics and flavonoids associated with potent antiangiogenic activity. Conclusion: The inhibition of angiogenesis by F. deltoidea extracts may be attributed to the relatively high ursolic acid content as well as the presence of antioxidant compounds of phenolics and flavonoids in the extracts. Keywords: Ficus deltoidea, Antiangiogenesis, Ursolic acid, Cytotoxicity, Antioxidant