Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Anti-Uterine Fibroid Effect of Standardized Labisia Pumila Var. Alata Extracts In Vitro and in Human Uterine Fibroid Cancer Xenograft Model.

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.
.

Application of high performance liquid chromatography and Fourier-transform infrared spectroscopy techniques for evaluating the stability of Orthosiphon aristatus ethanolic extract and its nano liposomes

ABSTRACT 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.

Orthosiphon stamineus Extracts Inhibits Proliferation and Induces Apoptosis in Uterine Fibroid Cells

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-ß1 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.

Analysis of Free Amino Acids in Different Extracts of Orthosiphon stamineus Leaves by High-Performance Liquid Chromatography Combined with Solid-Phase Extraction.

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.

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

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 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.

High-performance Thin-layer Chromatography Method Development, Validation, and Simultaneous Quantification of Four Compounds Identified in Standardized Extracts of Orthosiphon stamineus.

CONTEXT: Orthosiphon stamineus is a medicinal herb widely grown in Southeast Asia and tropical countries. It has been used traditionally as a diuretic, abdominal pain, kidney and bladder inflammation, gout, and hypertension. AIMS: This study aims to develop and validate the high-performance thin layer chromatography (HPTLC) method for quantification of rosmarinic acid (RA), 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF), sinensitin (SIN) and eupatorin (EUP) found in ethanol, 50% ethanol and water extract of O. stamineus leaves. MATERIALS AND METHODS: HPTLC method was conducted using an HPTLC system with a developed mobile phase system of toluene: ethyl acetate: formic acid (3:7:0.1) performed on precoated silica gel 60 F254 TLC plates. The method was validated based on linearity, accuracy, precision, limit of detection, limit of quantification (LOQ), and specificity, respectively. The detection of spots was observed at ultraviolet 254 nm and 366 nm. RESULTS: The linearity of RA, TMF, SIN, and EUP were obtained between 10 and 100 ng/spot with high correlation coefficient value (R2) of more than 0.986. The limit of detection was found to be 122.47 ± 3.95 (RA), 43.38 ± 0.79 (SIN), 17.26 ± 1.16 (TMF), and 46.80 ± 1.33 ng/spot (EUP), respectively. Whereas the LOQ was found to be 376.44 ± 6.70 (RA), 131.45 ± 2.39 (SIN), 52.30 ± 2.01 (TMF), and 141.82 ± 1.58 ng/spot (EUP), respectively. CONCLUSION: The proposed method showed good linearity, precision, accuracy, and high sensitivity. Hence, it may be applied in a routine quantification of RA, SIN, TMF, and EUP found in ethanol, 50% of ethanol and water extract of O. stamineus leaves. SUMMARY: HPTLC method provides rapid estimation of the marker compound for routine quality control analysis.The established HPTLC method is rapid for qualitative and quantitative fingerprinting of Orthosiphon stamineus extract used for commercial product.Four identified markers (RA, SIN, EUP and TMF) found in three a different type of O. stamineus extracts specifically ethanol, 50% ethanol and water extract were successfully quantified using HPTLC method. Abbreviations Used: HPTLC: High-performance thin layer chromatography; RA: Rosmarinic acid; TMF: 3'-hydroxy-5,6,7,4'-tetramethoxyflavone; SIN: Sinensitin; EUP: Eupatorin; E: Ethanol; EW: 50% ethanol; W: Water; BK: Batu Kurau; KB: Kepala Batas; S: Sik; CJ: Changkat Jering; SB: Sungai Buloh.

A comparative study of conventional and supercritical fluid extraction methods for the recovery of secondary metabolites from Syzygium campanulatum Korth.

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%).

Mutagenicity and preclinical safety assessment of the aqueous extract of Clinacanthus nutans leaves.

CONTEXT: Clinacanthus nutans (CN) is used traditionally for treating various illnesses. Robust safety data to support its use is lacking. OBJECTIVE: To evaluate the adverse effects of aqueous extract of CN leaves (AECNL). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: The median lethal dose (LD50) of the AECNL is >5000 mg/kg and the no-observed-adverse-effect level is identified to be greater than 2000 mg/kg/day in 90-d study.

Genotoxicity, acute and subchronic toxicity studies of nano liposomes of Orthosiphon stamineus ethanolic extract in Sprague Dawley rats.

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.

Selected metabolites profiling of Orthosiphon stamineus Benth leaves extracts combined with chemometrics analysis and correlation with biological activities.

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.

Isolation, Characterization, Crystal Structure Elucidation of Two Flavanones and Simultaneous RP-HPLC Determination of Five Major Compounds from Syzygium campanulatum Korth.

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)-2',4'- dihydroxy-6'-methoxy-3',5'-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.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts.

BACKGROUND: 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. OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Analysis of L-citrulline and L-arginine in Ficus deltoidea leaf extracts by reverse phase high performance liquid chromatography.

BACKGROUND: Ficus deltoidea (FD) is one of the native plants widely distributed in several countries in Southeast Asia. Previous studies have shown that FD leaf possess antinociceptive, wound healing and antioxidant properties. These beneficial effects have been attributed to the presence of primary and secondary metabolites such as polyphenols, amino acids and flavonoids. OBJECTIVE: The aim was to develop a reverse phase high-performance liquid chromatography method with ultraviolet detection that involves precolumn derivatisation with O-phthaladehyde for simultaneous analysis of two amino acids L-citrulline and L-arginine in FD leaf extracts. MATERIALS AND METHODS: An isocratic elution program consisting of methanol: acetonitrile: Water at 45:45:10 v/v (solvent A) and 0.1 M phosphate buffer pH 7.5 (solvent B) at A: B v/v ratio of 80:20 on Zorbax Eclipse C18 SB-Aq column (250 × 4.6 mm, 5 µm) were used. The flow rate was set at 1 ml/min and detection was carried out at 338 nm with 30 min separation time. RESULTS: Good linearity for L-citrulline and L-arginine was obtained in the range 0.1-1000 µg/ml at R (2) ≥ 0.998. The limit of detection and limit of quantification values for both L-citrulline and L-arginine were 1 and 5 µg/ml, respectively. The average of recoveries was in the range 94.94-101.95%, with relative standard deviation (%RSD) less than 3%. Intra- and inter-day precision was in the range 96.36-102.43% with RSD less than 2%. CONCLUSION: All validation parameters of the developed method indicate the method is reliable and efficient for simultaneous determination of L-citrulline and L-arginine for routine analysis of FD.

Anti-Obesity Effects of Melastoma malabathricum var Alba Linn in Rats Fed with a High-Fat Diet.

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.

Isolation, Characterization, Crystal Structure Elucidation, and Anticancer Study of Dimethyl Cardamonin, Isolated from Syzygium campanulatum Korth.

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.

Crystal structure elucidation and anticancer studies of (-)-pseudosemiglabrin: a flavanone isolated from the aerial parts of Tephrosia apollinea.

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.

Preparation and characterization of nano liposomes of Orthosiphon stamineus ethanolic extract in soybean phospholipids.

BACKGROUND: O. stamineus is a medicinal herb with remarkable pharmacological properties. However, poor solubility of the active principles limits its medicinal value. This study sought to prepare nano liposomes of OS ethanolic extract in unpurified soybean phospholipids in order to improve its solubility and permeability. OS liposomes were prepared by the conventional film method, and were characterized for solubility, entrapment efficiency, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), particle size and zeta potential, release, absorption in everted rat intestinal sacs, and DPPH scavenging effect. RESULTS: OS liposomes showed substantial enhancement of extract's solubility from 956 ± 34 to 3979 ± 139 µg/ml, with entrapment efficiency of 66.2 ± 0.9%. FTIR study indicates interaction between soybean phospholipids and OS extract. TEM and dynamic light scattering showed presence of round anionic nano liposomes with particle size and zeta potential of 152.5 ± 1.1 nm and -49.8 ± 1.0 mV, respectively. A study using the fluorescent probe pyrene showed the critical micellar concentration is 9.2 ± 2.9 µg/ml. Release studies showed 94 ± 0.1% release in non-formulated extract and 62.4 ± 0.1% in OS liposomes. Released extract from OS liposomes showed improvement in DPPH scavenging effect, IC50 = 23.5 ± 1.1 µg/ml compared to 32.4 ± 0.5 µg/ml in non-formulated extract. OS liposomes were stable at pH 5.5 and 7.4, but showed reversible agglomeration at pH 1.6. Absorption in everted rat intestinal sacs showed substantial improvement in permeability of 3'-hydroxy-5, 6, 7, 4″-tetramethoxyflavone, sinensetin, eupatorin, and 3 other unknown compounds. CONCLUSIONS: Enhanced solubility, absorption and antioxidant effect may improve the overall pharmacological effects and medicinal value of OS ethanolic extract.

Proapoptotic and antimetastatic properties of supercritical CO2 extract of Nigella sativa Linn. against breast 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.