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Background: Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila has been used traditionally for the treatment of several ailments such as gonorrhoea, dysmenorrhoeaand as tonics for females females.AimsAims: To investigate the standardization procedure of Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila extract (LPE) and evaluation of its nanoliposomes.Methods:Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila Labisia pumila extract was standardized by high performance liquid chromatography (HPLC), and gallic acid, caffeicacid, rutin and 2,4 2,4-Di -tert tert-butylphenol were quantified in the extract. Then, the standardized extract was prepared as nanoliposomeusing soy bean phospholipid by the film method and after that was characterized by zetasizer, zeta potential, UV -Visspectrophotometer and FTIR techniques techniques.Results:For the standardization, the mean percentage recovery values of the concentration studied were 98.49 98.49±1.43, 97.01 97.01±2.04,97.7097.70±1.55 and 99.43 99.43±3.04 % for gallic acid, caffeic acid, rutin and 2,42,4-Di -tert tert-butylphenol, respectively. The accuracy values werebetween 95.06 and 104.86% for the marker compounds, while the corresponding precision values were betwee n 0.09 and 5.18% forwithinwithin-day and between between-day analysis, respectively. The average particle size for LLP was 174.20±4.58 nm with zeta potential ofparticles surface charge from −43.40 to − 44.40 mV. The polydispersity index was 0.19±0.02 and the morpholo gy and presence ofliposomes were further confirmed by transmission electron microscopy which revealed the presence of spherical liposomes of < 200nm.Conclusion:HPLC method for the simultaneous determination of selected marker compounds has been develop ed; the method wasreliable, repeatable and reproducible. The method was successfully applied in standardization of LPE. LPE was successfully pr eparedas nanoliposome using soybean phospholipid.

Syzygium myrtifolium Walp. (Syzygium campanulatum Korth), Myrtaceae, is locally known as “Kelat paya” in Malaysia. Traditionally, it is used as a remedy for stomach pain. The goal of the present study was to investigate the toxicological potential as well as the antidiarrhoeal and antispasmodic activities of betulinic acid, dimethyl cardamonin and standardized non-formulated and nano-formulated ethanol and supercritical fluid extracts prepared from leaves of S. myrtifolium. The standardized extracts did not produce in vivo toxicity. Both the compounds and standardized extracts showed dose-dependent antidiarrhoeal activity by examining changes in the percentage of liquid stools and percentage of defecation frequency. Dimethyl cardamonin and standardized extracts showed antispasmodic activity on the isolated ileum of guinea pigs. Compared with hyoscine-N-butylbromide, dimethyl cardamonin and standardized extracts produced significant, potent antagonizing activity in ileum contractions induced with acetylcholine. Furthermore, the antagonistic potential of S. myrtifolium active markers against muscarinic type M2 and M3 receptors was investigated by molecular docking.

Background Uterine fibroids are a common type of solid tumor presenting in women of reproductive age. There are very few alternative treatment available from conventional treatment involving surgeries. Labisia pumila var. alata or locally known as 'Kacip Fatimah' was widely used as traditional medicine in Malaysia. This plant has been used to maintain a healthy female reproductive system. The present study aimed to evaluate anti fibroid potential of L. pumila extracts through in vitro apoptosis activity against uterine leiomyoma cells (SK-UT-1) and in uterine leiomyoma xenograft model. Evaluation of bioactive markers content were also carried out. Methods Apoptotic induction of the extracts was determined by morphological examination of AO/PI dual staining assay by flourescent microscopy and flow cytometry analysis on Annexin V-FITC/PI stained cells. In vivo study was done in immune-compromised mouse xenograft model. HPLC analysis was employed to quantify marker compounds. Results Morphological analysis showed L. pumila induced apoptosis in a dose dependent manner against SK-UT-1 cells. In vivo study indicated that L. pumila significantly suppressed the growth of uterine fibroid tumor. All tested extracts contain bioactive marker of gallic acid and cafeic acid. Conclusion This work provide significant data of the potential of L. pumila in management of uterine fibroids. .

Orthosiphon aristatus (Blume) Miq., Lamiaceae, is a medicinal plant from Southeast Asia. Pharmacological effects of O. aristatus are attributed to the presence of lipophilic flavones. This study aimed to carry out accelerated stability studies on O. aristatus ethanolic extract and its nano liposomes. The extracts were exposed to four different temperatures at 30, 40, 50 and 60 °C for 6 months. The samples were analyzed at 0, 1, 2, 3, 4, 5 and 6 months by high performance liquid chromatography using rosmarinic acid, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin and eupatorin as markers. Different chemical kinetic parameters of the markers were evaluated by Arrhenius equation to predict shelf life (t90) at different storage conditions and at room temperature. Moreover, the stability of O. aristatus ethanolic extract and O. aristatus nano liposomes were analyzes by chemical fingerprinting using FTIR spectroscopy, principal component analysis and hierarchical clustering analysis. The degradation of markers in both O. aristatus ethanolic extract and O. aristatus nano liposomes followed the first order degradation reaction (dependening on their initial concentration). The loss of marker compounds in O. aristatus ethanolic extract, stored at 30, 40, 50 and 60 °C for six months were up to 25, 52, 72 and 89% for all compounds, respectively. However, in O. aristatus nano liposomes 16, 71, 85 and 100% of compounds were lost during 6 months of storage at 30, 40, 50 and 60 °C, respectively. Therefore, the markers in O. aristatus nano liposomes seems to be more stable at a temperature below 30 °C compared to O. aristatus ethanolic extract. However, markers present in O. aristatus ethanolic extract are more stable at a higher temperature (above 30 °C). principal component analysis or hierarchical clustering analysis analyses were applied to the FTIR results in order to demonstrate the discrimination between extracts based on the storage conditions. The results show that the functional group of the components in the extracts and their chemistry relationship is influenced by the temperature setup indicating the extracts are not stable during the storage conditions. Keywords: Stability, Nano liposomes, HPLC, FTIR, PCA

This study was conducted to develop an ethanolic solid dispersion of Orthosiphon stamineus (ESD) using polymers as carriers, namely polyvinylpyrrolidone (PVP), poloxamer 188 (P188), and poloxamer 407 (P407) via the solvent evaporation method. The purpose of preparing the formulation is to improve the solubility of the lipophilic flavones, namely sinensetin (SIN), eupatorine (EUP), and 3′-hydroxy-5, 6, 7, 4′-tetramethoxyflavone (TMF) and caffeic acid derivatives, namely rosmarinic acid (RA). The optimized ESD was characterized using High-Performance Liquid Chromatography (HPLC), Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) fingerprints, and physicochemical methods (particle size, zeta potential, TEM, and SEM). The effect of pH on stability and solubility in buffer and water, in-vitro release, and antioxidant properties (DPPH assay) indicated that the nano-formulation ESD using polymers (PVP/P407) with a ratio of extract to polymers (1.0:1.1:0.3 w/w) enhanced the lipophilic flavones (TMF=3.56 ± 0.01% w/w, SIN=2.46 ± 0.01% w/w and EUP=7.87 ± 0.01% w/w) and RA (20.66 ± 0.01% w/w) compared to the same compounds in ethanolic extract (P< 0.0001) with particles size less than 200 nm. In conclusion, the successful development of ESD using water-soluble copolymers (PVP/P407) has enhanced the solubility of lipophilic flavones and other compounds (RA), thereby further improving its pharmacological properties.

Sinensetin was identified in the leaves of Orthosiphon stamineus by thin-layer chromatography, GC coupled with mass spectrometry (MS), UV, IR and 1 H-NMR spectroscopy. Enrichment of sinensetin by using different mixture solvent system and the quantification analysis by thin-layer chromatography-imaging densitometric method using as external standard method showed the highest concentration of 0.32% of sinensetin in the mixture solvent of acetone–water (70:30) when compared to methanol–water (1:1) of only 0.15% in the leaves of Orthosiphon stamineus. The TLC densitometer, although yields slightly higher values than the other analytical methods, is preferred due to its simplicity, ease and low cost.

Syzygium campanulatum Korth is a plant, which is a rich source of secondary metabolites (especially flavanones, chalcone, and triterpenoids). In our present study, three conventional solvent extraction (CSE) techniques and supercritical fluid extraction (SFE) techniques were performed to achieve a maximum recovery of two flavanones, chalcone, and two triterpenoids from S. campanulatum leaves. Furthermore, a Box-Behnken design was constructed for the SFE technique using pressure, temperature, and particle size as independent variables, and yields of crude extract, individual and total secondary metabolites as the dependent variables. In the CSE procedure, twenty extracts were produced using ten different solvents and three techniques (maceration, soxhletion, and reflux). An enriched extract of five secondary metabolites was collected using n-hexane:methanol (1:1) soxhletion. Using food-grade ethanol as a modifier, the SFE methods produced a higher recovery (25.5%‒84.9%) of selected secondary metabolites as compared to the CSE techniques (0.92%‒66.00%).

The objective of the present study is to develop accelerated stability of Orthosiphon stamineus standardised ethanolic extract (SEE) and its solid dispersion (ESD). The stability study of SEE and ESD has been performed using high-performance liquid chromatography (HPLC) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analyses. The spectroscopic datasets of ESD were applied to the principal component analysis (PCA) to extract the maximum information of the ATR-FTIR spectra. SEE and ESD were stored at three different temperatures with two different humidity conditions (30 °C/75% RH, 40 °C/75% RH and 60 °C/85% RH) for six months. Overall, the degradation of marker compounds; rosmarinic acid (RA), 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) at high temperature (60 °C/85% RH) was higher compared to low temperature (30 °C/75% RH) for both samples. Moreover, the degradation of RA, TMF, SIN and EUP in ESD was slower compared to SEE. The deterioration of marker compounds for both samples followed the first-order reaction kinetics. The shelf life of SEE and ESD is based on the estimated shelf life RA, TMF, SIN, and EUP present in the samples. The shelf life of RA, TMF, SIN, and EUP in ESD were significantly enhanced (p < 0.001) compared to the same markers in SEE with EUP was showing the highest shelf life (15 months), while RA showed the lowest shelf life (7 months) when stored at the temperature below 30 °C. The shelf life of all marker compounds in SEE was less than two months when stored at the same temperature (below 30 °C). Based on ATR-FTIR fingerprinting datasets analysed with PCA, ESD kept at 30 °C/75% RH were still preserved of its chemical properties, which indicates that low temperature is better to keep the formulation.

Objectives: The effects of water and 50% ethanolic-water extracts of Orthosiphon stamineus Benth (OS) on cell proliferation and apoptotic activity against uterine leiomyosarcoma (SK-UT-1) cells were investigated. Methods: Anti-proliferation effect was evaluated through cell cycle analysis whereas apoptotic activity was determined via screening and quantifying using fluorescence microscopy and flow cytometric analysis, respectively. The effect of extracts on molecular mechanism was studied using real-time reverse transcription polymerase chain reaction and Western blotting. Results: Cell cycle flow cytometric analysis showed the induction of cell cycle arrests were behaves in a p53-independent manner. The examination using fluorescence microscopy and Annexin V flow cytometry revealed the presence of morphological features of apoptotic bodies. Downregulation of anti-apoptotic gene (Bcl-2) supports the apoptotic activity of OS extracts although poorly induce PARP-1 cleavage in Western blot analysis. The extracts also inhibit the SK-UT-1 growth by suppressing VEGF-A, TGF-β1 and PCNA genes, which involved in angiogenesis and cell proliferation. Conclusion: This study demonstrates that O. stamineus extracts are able to inhibit proliferation and induced apoptosis of uterine fibroid cells and is worth further investigation.

Composite films that utilize seaweed as a matrix and oil palm shell (OPS) nanoparticles as a reinforcing material were developed. The effects of loading OPS nanoparticle (0%, 1%, 5%, 10%, 20%, and 30%) into seaweed films were determined by analyzing the physical, mechanical, and morphological properties of the films. The seaweed-based film incorporated with OPS nanoparticles at a high concentration (20% w/w) achieved the highest tensile strength (44.8 MPa) and Young’s Modulus (3.13 GPa). However, the film’s hydrophobicity (contact angle = 47.3o) and percentage of elongation at break (2.10%) were reduced. Moreover, it was observed that excessive loading of nanofillers (> 20%) reduced the tensile strength and hydrophilicity of the film. This phenomenon was attributed to the agglomeration of OPS nanoparticles and the formation of large voids on the film surface. Thus, the relative effectiveness of the various tested nanofiller contents in enhancing the mechanical strength of the composite film were found to be ranked in the following order: 20%, 10%, 5%, 30%, and 1%.

Syzygium campanulatumKorth 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 fromS. campanulatumKorth, 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 ofS. campanulatumwas 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 IC5012.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report onS. campanulatumas being a rich source (3.5%) of DMC, X-ray crystallography, and anticancer activity on human colon cancer cells.

Nigella sativa, commonly referred as black cumin, is a popular spice that has been used since the ancient Egyptians. It has traditionally been used for treatment of various human ailments ranging from fever to intestinal disturbances to cancer. This study investigated the apoptotic, antimetastatic, and anticancer activities of supercritical carbon dioxide (SC-CO2) extracts of the seeds of N. sativa Linn. against estrogen-dependent human breast cancer cells (MCF-7). Twelve extracts were prepared from N. sativa seeds using the SC-CO2 extraction method by varying pressure and temperature. Extracts were analyzed using FTIR and UV-Vis spectrometry. Cytotoxicity of the extracts was evaluated on various human cancer and normal cell lines. Of the 12 extracts, 1 extract (A3) that was prepared at 60°C and 2500 psi (~17.24 MPa) showed selective antiproliferative activity against MCF-7 cells with an IC50 of 53.34±2.15 μg/mL. Induction of apoptosis was confirmed by evaluating caspases activities and observing the cells under a scanning electron microscope. In vitro antimetastatic properties of A3 were investigated by colony formation, cell migration, and cell invasion assays. The elevated levels of caspases in A3 treated MCF-7 cells suggest that A3 is proapoptotic. Further nuclear condensation and fragmentation studies confirmed that A3 induces cytotoxicity through the apoptosis pathway. A3 also demonstrated remarkable inhibition in migration and invasion assays of MCF-7 cells at subcytotoxic concentrations. Thus, this study highlights the therapeutic potentials of SC-CO2 extract of N. sativa in targeting breast cancer.

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.

Background: Orthosiphon stamineus (OS) Benth is a medicinal plant and native in Southeast Asia. Previous studies have shown that OS leaves possess antioxidant, cytotoxic, diuretic, antihypertensive, and uricosuric effects. These beneficial effects have been attributed to the presence of primary and secondary metabolites such as polyphenols, amino acids, and flavonoids. Objective: To develop and validate an high-performance liquid chromatography (HPLC)-diode array detector (DAD) method combined with solid-phase extraction that involves precolumn derivatization with O-phthaladehyde for simultaneous analysis of free amino acids in OS leaves extracts. Materials and Methods: OS leaves were extracted with water (OS-W), ethanol (OS-E), methanol (OS-M), 50% ethanol (OS-EW), and 50% methanol (OS-MW). The extracts were treated by C18 cartridge before derivatization, resulting in great improvement of separation by Zorbox Eclipse XDB-C18 column. Results: The HPLC–DAD method was successfully developed and validated for analyzing the contents of free amino acids in OS extracts. The results showed that l-aspartic acid with 0.93 ± 0.01 nmol/mg was the major free amino acid in OS-W extract. However, in OS-E, OS-M, OS-EW, and OS-MW, l-glutamic acid with 3.53 ± 0.16, 2.17 ± 0.10, 4.01 ± 0.12, and 2.49 ± 0.12 nmol/mg, respectively, was the major free amino acid. Subsequently, l-serine, which was detected in OS-W, OS-E, and OS-M, was the minor free amino acid with 0.33 ± 0.02, 0.12 ± 0.01, and 0.06 ± 0.01 nmol/mg, respectively. However, l-threonine with 0.26 ± 0.02 and 0.19 ± 0.08 nmol/mL in OS-EW and OS-MW, respectively, had the lowest concentration compared with other amino acid components. Conclusion: All validation parameters of the developed method indicate that the method is reliable and efficient to simultaneously determine the free amino acids content for routine analysis of OS extracts. SUMMARY The HPLC-DAD method combined with solid phase extraction was successfully developed and validated for simultaneous determination and quantification of 17 free amino acids in Orthosiphon stamineus (OS) Benth extractsOS extracts were found to be rich in free amino acid contentL-aspartic acid was the major free amino acid in OS water extract while, in OS ethanol, methanol, 50% ethanol and 50% methanol extracts, L-glutamic acid was the major free amino acidL-serine was the minor free amino acid in OS water, ethanol and methanol extracts while, in OS 50% ethanol and 50% methanol extracts, L-threonine had the lowest concentration compared to other amino acid components. Abbreviations used: HPLC-DAD: High-Performance Liquid Chromatography with Diode-Array Detection, OS: Orthosiphon stamineus, OS-W: Orthosiphon stamineus water extract, OS-E: Orthosiphon stamineus ethanol extract, OS-M: Orthosiphon stamineus methanol extract, OS-EW: Orthosiphon stamineus 50% ethanol extract, OS-MW: Orthosiphon stamineus 50% methanol extract, OPA: O-phthaladehyde, SPE: Solid Phase Extraction, UV: Ultraviolet, LOD: Limit of Detection, LOQ: Limit of Quantification, RSD: Relative Standard Deviation.

Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.

Objective The aim of the present study is to develop liquid chromatography (LC)/Time-of-flight mass spectrometry (TOF/MS) profile for methanol and water extracts of Orthosiphon stamineus leaf using SEN and RA as flavonoid and non-flavonoid polyphenolic markers in the extracts. The study also evaluates in vitro nitric oxide radical scavenging effect of the extracts. Method Orthogonal Time of Flight Mass Spectrometer equipped with HPLC separation module was used in the analyses of the extract. The in vitro nitric oxide scavenging activity of the extracts was measured according to the method described by Rao. Results The qualitative analysis of the extracts performed with HPLC–TOF/MS confirmed the presence sinensitin (SEN) and rosmarinic acid (RA) in the extracts. The extracts showed in vitro nitric oxide scavenging activities. Conclusions The HPLC–TOF/MS method could be employed for quality determination of herbal medicinal products and formulations containing O. stamineus . The extracts may play a significant role in prevention of degenerate disease due to its ability to scavenge nitric oxide radical.

This study aimed to investigate the antitumorigenicity of xanthones-rich extract from Garcinia mangostana L., Clusiaceae, fruit rinds which was obtained by a simple recrystallization of 75% ethanolic extract. α-Mangostin content of the extract was determined qualitatively by TLC and quantitatively by HPLC, and total xanthones content was quantified by UV spectrophotometry. The extract was evaluated for cytotoxicity, apoptosis and antitumorigenicity on HCT 116 human colorectal carcinoma cells. α-Mangostin was found to be the main constituent of the extract which was 71.2±0.1%, and the total xanthones content was 95±4.8% (wt/wt). The extract showed potent dose dependent cytotoxicity with IC50 value 9.2 μg/mL. Apoptosis studies revealed activation of caspases 3 and 7, DNA fragmentation, chromatin condensation and loss of mitochondrial membrane potential. Studies on cell migration and colony formation indicate reduced cell migration ability and clonogenicity of treated HCT 116 cells at sub-inhibitory concentrations. Taken together, the cytotoxic effect of the xanthones extract is mediated through the mitochondrial pathway of apoptosis. The reduced cell migration and clonogenicity of HCT 116 cells might prevent both primary and metastatic tumor growth in vivo which will be the topic of our future work using the metastatic orthotopic colon cancer model.

Syzygium aromaticum (L.) Merr. & L.M. Perry, Myrtaceae, is an evergreen tree with anticarcinogenic, antimutagenic, aphrodisiac, antimicrobial, antioxidant and antiinflammatory properties. This study aims to investigate the anti-breast cancer effect of extracts from leaves, stem and bark of S. aromaticum and to develop a method for preparation of betulinic acid fraction from the leaves. Betulinic acid, ursolic acid and oleanolic acid contents of the extracts were determined by HPLC. A betulinic acid fraction was prepared by simple crystallization of leaves extract and was characterized by HPLC and mass analysis. Anti-breast cancer effects were studied on MCF-7 and MDA-MB-231 cells. The extracts were found to contain high levels of betulinic acid particularly the leaves extract which contained 17% wt/wt. The betulinic acid fraction contains 75% betulinic acid. Cytotoxicity testing reveals high and selective cytotoxic effect of the stem extract on MCF-7 cells with IC50 33±1.6 µg/mL. Cytotoxic effect of the stem extract was due to activation of apoptotic machinery of cell death. Combination studies of stem extract with tamoxifen reveals antagonistic effect at high concentration of tamoxifen and enhancement effect at low concentration. The selective cytotoxicity of the stem extract of S. aromaticum on MCF-7 is not due to betulinic acid but due to other constituents yet to be discovered.

FTIR spectroscopy was used together with multivariate analysis to distinguish six different species ofPhyllanthus. Among these speciesP. niruri,P. debilisandP. urinariaare morphologically similar whereasP. acidus,P. emblicaandP. myrtifoliusare different. The FTIR spectrometer was used to obtain the mid-infrared spectra of the dried powdered leaves in the region of 400–4000 cm−1. The region of 400–2000 cm−1was analyzed with four different pattern recognition methods. Initially, principal component analysis (PCA) was used to reduce the spectra to six principal components and these variables were used for linear discriminant analysis (LDA). The second technique used LDA on most discriminating wavenumber variables as searched by genetic algorithm using canonical variate approach for either 30 or 60 generations. SIMCA, which consisted of constructing an enclosure for each species using separate principal component models, was the third technique. Finally, multi-layer neural network with batch mode of backpropagation learning was used to classify the samples. The best results were obtained with GA of 60 gens. When LDA was run with the six wavenumbers chosen (1151, 1578, 1134, 609, 876 and 1227), 100% of the calibration spectra and 96.3% of the validation spectra were correctly assigned.

Purpose: To investigate the toxicity of Labisia pumila standardized extract (LPE) and its liposomal extract (LLP).Methods: For acute toxicity study, LPE or LLP was orally administered (2000 mg/kg) in single doses to Sprague Dawley rats and the routine activity of the rats was continuously monitored for a total of 14 days. After 14 days of treatment, all rats were sacrificed and their vital organs were excised, weighed and macroscopically examined, while for a repeated dose toxicity study, the rats were orally administered with LPE or LLP at the selected doses (250, 500 and 1000 mg/kg) for a period of 28 days. The animals were sacrificed (anaesthetized by sodium pentobarbitone and blood was collected by cardiac puncture), followed by examination of their body organs and blood serum.Results: LPE and LLP at 2000 mg/kg did not produce mortality or significant changes in the general behaviour, body weight and organ gross appearance of the rats. In repeated dose toxicity study no significant changes in, growth, organ weights, haematological parameters, biochemical values and histological features of vital organs of the treated groups, compared to the control group.Conclusion: The no-adverse-effect-level for LPE and LLP is (1000 mg/kg/day) when administered orally for 28 days.Keywords: Labisia pumila, Acute oral toxicity, Repeated dose toxicity

Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05) at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.

The acute toxicity of standardized extract of Orthosiphon stamineus was studied in Sprague Dawley rats. The rats were administered a single dose of 5000 mg/kg body weight (BW) orally on Day 0 and observed for 14 days. There were no deaths recorded and the animals did not show signs of toxicity during the experimental period. The effect of the extract on general behavior, BW, food and water intake, relative organ weight per 100 g BW, hematology and clinical biochemistry were measured. All the parameters measured were unaffected as compared to the control. The acute toxicity LD50 was estimated to be >5000 mg/kg BW.

Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r2 > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 μg/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.

Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r2 > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 μg/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.

Hibiscus sabdariffa tea is a widely used medicinal beverage and a treatment for high blood pressure and high blood cholesterol in many parts of the world. Many studies on H. sabdariffa have been conducted including extraction and identification of main biocompounds. However, information on the effects of processing the plant is scarce. This is important as sample processing procedure influence the composition of the end product. Hence, the main objective of this present study was to examine the effect of sample processing (non-extracted, ethanol extract and water extract) on H. sabdariffa composition. Fourier Transform Infrared (FTIR) was used for the process of identification. The powdered sample of H. sabdariffa (FT34) was obtained from a local company in Peninsula Malaysia. A fresh sample obtained from the same company was processed in the Phytochemistry Laboratory, Institute for Medical Research and labelled as FT35. Sample and potassium bromide (KBr) were mixed (1:250) to form a 1-2 mm transparent disk under 9.80 psi in vacuum. The FTIR Spectra were recorded with 32 scans and 0.2 cms-1 OPD speed. Spectra of FT34 and FT35 raw samples indicated obvious differences in the range of 1500-1135 cm-1. The FT34 ethanol extract using trifluoroacetic acid (TFA) showed that the peak at 1629 cm-1 was the highest in the range of 1800-1500 cm-1, whereas for FT35, the highest peak was 1739 cm-1. The peak at 1071 cm-1 of FT35 was the only one compatible to standard dephinidin-3-O-sambubioside and cyanidin-3-Osambubioside which are used for qualification of sample content. In fact, both standards showed up as different chromatographs in thin layer chromatography. Water extract of FT35 showed a peak at 1676 cm-1 which was not detected in water extract spectrum of FT34, while the pattern of spectrum varied within the range of 1300-400 cm-1. Second derivative spectra enhanced the comparable base peaks of both sample and the target standards. There were five matched ethanol extract base peaks, indicating the macrofingerprint of H. sabdariffa. Two dimensional correlation spectrum of FT34 raw powder showed different correlation spot especially in the cluster of 1425 cm-1 to 1743 cm-1 compared with FT35. The three-stage infrared spectroscopy comprehensively analysed the holographic spectra and hierarchically characterized the integrated constituents involved.

Background 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). Methods 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. Results 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. Conclusions 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.

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.

Background Studies on selected metabolites profiling of Orthosiphon stamineus extracts using chromatographic and spectroscopic techniques combined with chemometric tools have not been fully elucidated. Thus present study was performed to profile selected metabolites in O. stamineus leaves extracts using HPLC and FTIR combined with chemometric tools and correlated with biological activities. Methods Five different extracts were prepared using three methods; maceration, soxhlet and reflux. The extracts were analyzed using UV-Vis, HPLC and FTIR techniques. Analysis of selected primary and secondary metabolites was also evaluated. The antioxidant and cytotoxic activities of the extracts were evaluated. Chemometric tools were employed to classify the extracts based on HPLC analysis and FTIR fingerprints. Results The ethanolic extract using maceration characterized high content of phenolics and flavonoids, (rosmarinic acid and eupatorin) with high antioxidant activity. Ethanolic (50 %) and methanolic extracts using soxhlet showed high proteins and glycosaponins. Water extracts using reflux and maceration showed high polysaccharides. Methanolic extract (50 %) using soxhlet and methanolic extract using maceration showed strong cytotoxic effect against MCF7 and HCT116 cell lines, respectively. Antioxidant and cytotoxic activities showed significant correlation with selected primary and secondary metabolites. HPLC fingerprints combined with chemometrics showed the extracts have been clustered based on selected major peaks profile. FTIR fingerprints combined with chemometrics showed that the extracts have been clustered based on protein and polysaccharide contents. Conclusion Ten different extracts of O. stamineus have showed significant differences in the content of selected primary and secondary metabolites as well as the biological activities. Chemometric tools were able to classify and discriminate the distinctive features of extracts thus can be correlated with the biological activities.

Objective: To investigate the effect of methanolic extract of Melastoma malabathricum (M. malabathricum) in thrombocyte counts in mice. Methods: Methanolic extract of M. malabathricum corresponding to 1.5 to 2 mg/10 g body weight in saline was administered to mices via oral route. Control group was given normal saline. Twenty five microlitres of blood were drawn at 0 h and thereafter at 1, 2, 4, 8, 12, 20 h after dosing via tail bleeding technique. The thrombocytes were counted in the triple laminated middle 25 squares of the haemocytometer using light microscope. Results: The control group showed a moderate rise after the administration of saline whereas the M. malabathricum treated group shows significant rise within 2nd h in thrombocyte counts with an increment of 51.64% compared to baseline count. Conclusions: Based on the results, it can be concluded that M. malabathricum could be a potential remedy in treating thrombocytopenic condition.

This study evaluates the primary and secondary metabolite profiles of Eurycoma longifolia Jack (EL) stems and leaves to determine whether it can be utilized for therapeutic purposes as the roots. A total of six types of extracts were tested. The extracts showed high content of glycosaponins, polysaccharides, proteins and phenolics. The presence of flavonoids and phospholipids was also detected. High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography (HPLC) analysis showed the presence of bioactive marker of EL root, eurycomanone and 14,15AŸ-dihydroxyklaineanone in stem and leaf extracts. Primary and secondary metabolites identified were reported to associate with the enhancement of ergogenic and aphrodisiac activities in animal and human subjects. The result shows that stem and leaves of E. longifolia has the potential for therapeutic purposes.

Xanthones are a group of oxygenated heterocyclic compounds with anticancer properties, but poor aqueous solubility and low oral bioavailability hinder their therapeutic application. This study sought to prepare a xanthones extract (81% α-mangostin and 16% γ-mangostin) in polymeric nanoparticles and to investigate its intracellular delivery and cytotoxicity toward colon cancer cells. The nanoparticles were prepared in Eudragit RL100 and Eudragit RS100 by the nanoprecipitation method at drug loading and entrapment efficiency of 20% and >95%, respectively. Freeze-drying of bulk nanoparticle solutions, using glucose or sucrose as cryoprotectants, allowed the collection of nanoparticles at >95% yield. Solubility of the xanthones extract was improved from 0.1 µg/mL to 1250 µg/mL. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) of the freeze-dried final formulation showed the presence of cationic round nanoparticles, with particle size in the range of 32–130 nm. Scanning electron microscopy (SEM) showed the presence of nanospheres, and Fourier transform infrared (FTIR) spectroscopy indicated intermolecular interaction of xanthones with Eudragit polymers. Cellular uptake of nanoparticles was mediated via endocytosis and indicated intracellular delivery of xanthones associated with potent cytotoxicity (median inhibitory concentration26.3±0.22 µg/mL). Presented results suggest that cationic nanoparticles of xanthones may provide a novel oral drug delivery system for chemoprevention or treatment of intestinal and colon tumors.

Obesity is one of the major public health problems worldwide and it is generally associated with many diseases. Although synthetic drugs are available for the treatment of obesity, herbal remedies may provide safe, natural, and cost-effective alternative to synthetic drugs. One example of such drugs is Melastoma malabathricum var Alba Linn (MM). Although several studies have been reported for the pharmacological activities of MM, there is no report on the anti-obesity effect of MM. The aim of the present study is to evaluate the anti-obesity potential of methanolic extract of MM. The anti-obesity effect of MM on rats fed with a high-fat diet was investigated through determination of the changes in body weight, fat weight, organ weights, and blood biochemicals. The animals in this study were divided into three groups: a normal group with a standard diet (N), a control group fed with high-fat diet (C), and a MM treatment group fed with high-fat (HFD + MM) diet for 8 weeks. There was no significant difference in the amount of food intake between control and HFD + MM treatments. These results also suggest that MM does not induce a dislike for the diet due to its smell or taste. The study shows that MM significantly prevented increases in body weight, cholesterol, LDL, HDL, and total lipids that resulted from the high-fat diet. MM also decreased the epididymal fat (E-fat) and retroperitoneal fat (R-fat) weights and phospholipid concentrations induced by the high-fat diet. On the basis of these findings, it was concluded that MM had anti-obesity effects by suppressing body weight gain and abdominal fat formation.

Tephrosia apollinea is a perennial shrublet widely distributed in Africa and is known to have medicinal properties. The current study describes the bio-assay (cytotoxicity) guided isolation of (-)-pseudosemiglabrin from the aerial parts of T. apollinea. The structural and stereochemical features have been described using spectral and x-ray crystallographic techniques. The cytotoxicity of isolated compound was evaluated against nine cancer cell lines. In addition, human fibroblast was used as a model cell line for normal cells. The results showed that (-)-pseudosemiglabrin exhibited dose-dependent antiproliferative effect on most of the tested cancer cell lines. Selectively, the compound showed significant inhibitory effect on the proliferation of leukemia, prostate and breast cancer cell lines. Further studies revealed that, the compound exhibited proapoptotic phenomenon of cytotoxicity. Interestingly, the compound did not display toxicity against the normal human fibroblast. It can be concluded that (-)-pseudosemiglabrin is worthy for further investigation as a potential chemotherapeutic agent.