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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.

Cosmos caudatus is a traditional medicinal plant from the family Compositae. The fresh leaves are consumed as appetizer and for health benefits such as antioxidant activity, to improve blood circulation and to promote the formation of healthy bones. Our study aims to develop a reverse phase high performance liquid chromatography method for quantification of rutin, quercitrin and quercetin in C. caudatus extracts. The method was validated for selectivity, linearity, precision, accuracy and limits of detection and quantification. Selectivity was confirmed based on retention time and ultraviolet-visible spectra; linearity was in the range 0.5-500 μg/ml at R2=1.0; the intraday and interday precision was determined as the relative standard deviation of peak area and retention time. Percentage recovery, limits of detection and the limits of quantification of rutin, quercitrin and quercetin were also determined. The reported method was applied for the quantification of the 3 marker compounds in 15 extracts of C....

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%).

Clinacanthus nutans (CN) is used traditionally for treating various illnesses. Robust safety data to support its use is lacking.To evaluate the adverse effects of aqueous extract of CN leaves (AECNL).The oral toxicity of the AECNL was tested following Organisation for Economic Co-operation and Development (OECD) guidelines. Mutagenicity (Ames test) of AECNL was evaluated using TA98 and TA100 Salmonella typhimurium strains.No mortality or morbidity was found in the animals upon single and repeated dose administration. However, significant body weight loss was observed at 2000 mg/kg during sub-chronic (90 d) exposure. In addition, increased eosinophil at 500 mg/kg and decreased serum alkaline phosphatase levels at 2000 mg/kg were observed in male rats. Variations in glucose and lipid profiles in treated groups were also observed compared to control. Ames test revealed no evidence of mutagenic or carcinogenic effects at 500 μg/well of AECNL.The median lethal dose (LD50) of the AECNL is5000 mg/kg and the no-observed-adverse-effect level is identified to be greater than 2000 mg/kg/day in 90-d study.

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.

The topic of halal has become a global issue among the Muslim communities throughout the world as Muslims have specific requirements in consumption of food including pharmaceutical products. There is an increase in demand for halal pharmaceuticals and hence this study was aimed to assess the halal status of nutritional and dietary supplements available at two government hospitals in Klang Valley based on their active pharmaceuticals ingredients (APIs) and excipients. This descriptive and exploratory study was done by collecting data from product leaflets and approaching manufacturers to gather relevant information on the products. A data collection form was used to record the required particulars of the products. The products were then classified into halal, mushbooh, or haram by referring to standard pharmaceutical references with respect to the ingredients’ sources, and feedback from the manufacturers. In general for both hospitals, a total of 61 nutritional and dietary supplements were reviewed and it was found that 47.5% (N = 29) were halal, 49.2% (N = 30) were mushbooh, 0% haram, and 3.3% (N = 2) were with unknown status. Apart from that, the sources and halal status of the APIs and excipients contained in the products were also determined. A total of 148 API were analysed and 92.6% (n = 137) were found halal, 7.4% (n = 11) mushbooh, and 0% haram. From a total of 154 excipients found, 83.1% (n = 128) were halal, 16.9% (n = 26) were mushbooh, and 0% haram. Ingredients such as ethanol, gelatin, and magnesium stearate were found to be the most common ingredients which caused mushbooh status. The results showed that there were no haram products found in this therapeutic class. Hence, producing halal nutritional and dietary supplements should be highly possible by changing the sources of the common mushbooh ingredients to halal certified sources. Cooperation from individuals and all responsible parties are crucial in achieving the aim of using halal pharmaceuticals.

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.

Catharanthus roseus (C. roseus) is an important medicinal plant contains a lot of useful alkaloids. The objective of this study is to determine the effect of Catharanthus roseus leaves extract on the MDA-MB-231 cancer cell invasion potential and matrix metalloproteinases activity (MMP-2 and MMP-9) in breast cancer cell lines, MDA-MB-231 and MCF7. MTS cytotoxicity assay was used to evaluate the cytotoxic effect of the extract against the two tumour cell lines. Transwell invasion assay was used to investigate the invasive activity of MDA-MB-231 cancer cell line and gelatin zymography was used to determine the effect of C. roseus extract on the activity of MMP-2 and MMP-9 on the two tumour cell lines. The result showed that C. roseus extract was found to be not cytotoxic for both tumor cell line, MDA-MB-231 and MCF-7, with IC50 values more than 200µg/ml. However the extract showed significant inhibitory effect on MDA-MB-231 invasion ability (p

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.

Morinda citrifolia L. has been used for the treatment of a wide variety of diseases, including cancer. This study was undertaken to evaluate the anti-angiogenic effect of M. citrifolia fruits and leaves. Anti-angiogenic activity was evaluated in vivo using the chick chorioallantoic membrane assay. Bioactivity-guided fractionation and isolation were performed to identify the active constituent, and high-performance liquid chromatography analysis was then used to quantify the amount of this active constituent in the active extracts and fraction. The methanol extracts of fruits and leaves of M. citrifolia and the subsequent chloroform fraction of the fruit methanolic extract were found to have potential anti-angiogenic activity and were more potent compared to suramin. Scopoletin was identified as one of the chemical constituents that may be partly responsible for the anti-angiogenic activity of M. citrifolia fruits. The present findings further support the use of M. citrifolia in cancer or other pathological conditions related to angiogenesis.

Orthosiphon stamineus Benth. (Family: Lamiaceae), a famous traditional medicinal plant in tropical and sub-tropical region, is being used for treating various ailments including inflammatory conditions. Since anti-inflammation and antiangiogenesis involve cyclooxygenase-II pathway, the present study is aimed to carry out antiangiogenic studies and bioassay-guided isolation on aqueous extract of the plant. After antiangiogenic activity studies on the extract, fractions were prepared by partitioning the extract with hexane, chloroform and ethyl acetate, which were then evaluated for antiangiogenic activity. The most active, hexane fraction (HF) upon activity-guided isolation gave three compounds, 1 (betulinic acid), 2 (oleanolic acid) and 3 (ursolic acid). The total contents of these three compounds were determined in the extract and its fractions using HPLC. The extract and HF exhibited 20% and 80% antiangiogenic activity, respectively, whereas, chloroform and ethyl acetate fractions were inactive. The three isolated compounds exhibited 100% antiangiogenic activity. Median effective concentrations (EC50s) of HF, 1, 2 and 3 were found to be 84, 32, 34 and 38 μg/mL, respectively. The results of this study indicate that hexane fraction and the isolated compounds have promising antiangiogenic activity. Additionally, the isolated compounds may be sued as analytical markers to standardize extracts and formulations prepared from O. stamineus.

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.

α-Mangostin is an oxygenated heterocyclic xanthone with remarkable pharmacological properties, but poor aqueous solubility and low oral bioavailability hinder its therapeutic application. This study sought to improve the compound's solubility and study the mechanism underlying solubility enhancement. Solid dispersions of α-mangostin were prepared in polyvinylpyrrolidone (PVP) by solvent evaporation method and showed substantial enhancement of α-mangostin's solubility from 0.2 ± 0.2 μg/mL to 2743 ± 11 μg/mL. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated interaction between α-mangostin and PVP. Transmission electron microscopy and dynamic light scattering showed self-assembly of round anionic nanomicelles with particle size in the range 99-127 nm. Powder X-ray diffraction indicated conversion of α-mangostin from crystalline into amorphous state, and scanning electron microscopy showed the presence of highly porous powder. Studies using the fluorescent probe pyrene showed that the critical micellar concentration is about 77.4 ± 4 μg/mL. Cellular uptake of nanomicelles was found to be mediated via endocytosis and indicated intracellular delivery of α-mangostin associated with potent cytotoxicity (median inhibitory concentration of 8.9 ± 0.2 μg/mL). Improved solubility, self-assembly of nanomicelles, and intracellular delivery through endocytosis may enhance the pharmacological properties of α-mangostin, particularly antitumor efficacy.

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

Aim of the study The present study was aimed to investigate the pharmacological basis for the use of Loranthus ferrugineus in hypertension. Materials and methods Loranthus ferrugineus methanol extract (LFME) was obtained using Soxhelt extractor and then successively fractionated using chloroform, ethyl acetate and n-butanol. The n-butanol fraction of LFME (NBF-LFME) was studied using isolated rat thoracic aorta. Results NBF-LFME (1.0 × 10−5 to 3.0 mg/ml) was found to be the most potent to concentration-dependently relax the endothelium-intact phenyephrine (PE, 1 μM)- and high K+ (80 mM)-precontracted rat aortic rings. Removal of the endothelium completely abolished the vascular relaxing properties of NBF-LFME. Pretreatment with atropine (1 μM), l -NAME (10 μM), indomethacin (10 μM) and methylene blue (10 μM) significantly blocked NBF-LFME-mediated relaxation. Endothelium-dependent and -independent relaxations induced by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were significantly enhanced in aortic rings pretreated with NBF-LFME when compared to those observed in control aortic rings. On the contrary, glibenclamide (10 μM), propranolol (1 μM) and prazosin (0.01 μM) did not alter NBF-LFME-induced relaxation. Conclusions The results suggest that NBF-LFME induced vascular relaxation by stimulating muscarinic receptors, activating the endothelium-derived nitric oxide-cGMP-relaxant pathway, promoting prostacyclin release and/or possibly through its ability to lengthen the released nitric oxide half-life. The present data further supports previous in vivo findings and explain the traditional use of Loranthus ferrugineus as an anti-hypertensive agent.