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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%).

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.

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.

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.

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.

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

This paper presents the studies performed on extraction of Orthosiphon stamineus, Benth by using different solvent for the identification and quantification of the caffeic acid derivatives such as caffeic acid and rosmarinic acid which confers to the leaves of this plant with remarkable pharmaceutical properties. High performance thin-layer chromatographic (HPTLC) allows the identification and the quantification of more than 20 samples in the same chromatographic run. The analysis of the samples requires 15-30 min compared with more than 2 h using a typical HPLC method. Using the techniques of the HPTLC and the UV-VIS spectra we have found that the extraction of this herb plant contain, the caffeic acid and rosmarinic acid ranging between 0.029% up to 0.506% and up to 0.24% to 2.24% respectively. Keywords: Caffice acid derivatives, quantification, Malaysian Orthosiphon stamineus, HPTLC

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.

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.

The hydrodistilled essential leaves and stems oils of Orthosiphon stamineus Benth were analysed by GC–MS/MS. Sixty nine compounds representing 97.6 and 97.4% of the total leaves and stems oils, respectively were identified, of which -caryophyllene (24.0 and 35.1%), -humulene (14.2 and 18.4%), -elemene (11.1 and 8.5%), 1-octen-3-ol (8.2 and 7.0%), bourbonene (3.4 and 3.0%), -pinene (2.1 and 1.7%), caryophyllene oxide (1.6 and 2.2%), camphene (1.6 and 1.3%) and limonene (1.2 and 1.1%) were the major compounds. Thus, the monoterpenes and sesquiterpenes were the predominant portions of the oils. Essential oils and methanol extract of O. stamineus and the derived fractions of hexane, chloroform, and ethyl acetate were tested for anti-fungal activity, which was determined by disc diffusion and minimum inhibitory concentration (MIC) determination methods. The oils, methanol extract and derived fractions of methanol extract displayed great potential of anti-fungal activity as a mycelial growth inhibitor against the tested phytopathogenic fungi such as Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Colletotricum capsici and Phytophthora capsici, in the range of 49.3–70.3% and minimum inhibitory concentration ranging from 500 to 1000g/ml.

A disposable screen printed array sensor strip based on self-plasticized lipid membranes combined with chemometric algorithm has been developed and applied for quantification of Orthosiphon stamineus Benth extracts. Sinensetin, a pharmacologically active flavonoid in Orthosiphon stamineus Benth, was quantified with the sensor system using standard addition method. The method was compared with high performance thin layer chromatography (HPTLC). Partial least square (PLS) and principal component regression (PCR) were applied to the array sensor output to determine the sinensetin in O. stamineus samples from different suppliers. Comparison between the PLS and PCR models presented in the quantitative analysis showed that PLS have substantially better predictive capability than PCR. The root mean square error (RMSE) of Prediction for PLS and PCR were 0.17 ppm and 0.19 ppm, respectively. The concentration of sinensetin by PLS fell within the range of 0.25%–0.30% in six different batches of extracts that were supplied by Hovid Sdn Bhd (HV) while a range 0.18%–0.24% was obtained in ten different batches of extracts supplied by Nusantara Herbs Sdn Bhd (NH). The array sensor showed good correlation (0.9902) with the HPTLC method.

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.

ABSTRACT This study was undertaken to investigate the antiangiogenesis activity of standardized extracts/fractions of the leaf of Piper sarmentosum, using rat aorta model. The pulverized leaf was extracted sequentially and methanol extract was further fractionated with hexane, chloroform and ethylacetate. Both extracts and fractions were standardized by reverse phase HPLC with UV detection at 260 nm, using two markers, sarmentine and sarmentosine. Chloroform and methanol extracts have exhibited antiangiogenesis activity of 100% and 20% respectively. Antiangiogenesis activity of hexane and chloroform fractions was found to be 10% and 90% respectively, while ethylacetate fraction was found to be inactive. The analysis of most active extract and fraction has exhibited different profile by HPLC on the basis of amides. This study indicates that chloroform extract and fraction have promising antiangiogenesis activity and have potential for diseases involving angiogenesis. Keywords : antiangiogenesis activity, Piper sarmentosum Roxb.

Most herbs have their own characteristic smell due to the presence of volatile compounds. Traditionally, volatiles are analyzed by using sophisticated and expensive gas chromatography (GC) in tandem with a selective mass spectrometric (MS) detector. An alternative approach is described based on the use of an electronic nose. The approach is much simpler than the traditional GC–MS counterparts, but providing key information of the samples analyzed. Using lipids and gas chromatography stationary phase materials with different polarities as sensing membranes, a quartz crystal microbalance smell sensor array has been developed for the analysis of traditional medicinal plants. The headspace vapors of different types of Tongkat Ali (Eurycoma longifolia) extracts were analyzed by the smell sensor and GC–MS. Correlation between the sensor response and the identified compounds were studied using principal component analysis. Some of the identified compounds exhibited good correlation with the quartz crystal microbalance (QCM) sensor array data.

Quality control of herbal medicines remain a challenging issue towards integrating phytomedicine into the primary health care system. As medicinal plants is a complicated system of mixtures, a rapid and cost-effective evaluation method to characterize the chemical fingerprint of the plant without performing laborious sample preparation procedure is reported. A novel research methodology based on an in-house fabricated multichannel sensor incorporating an array of artificial lipid-polymer membrane as a fingerprinting device for quality evaluation of a highly sought after herbal medicine in the Asean Region namely Eurycoma longifolia (Tongkat Ali). The sensor array is based on the principle of the bioelectronic tongue that mimics the human gustatory system through the incorporation of artificial lipid material as sensing element. The eight non-specific sensors have partially overlapping selectivity and cross-sensitivity towards the targeted analyte. Hence, electrical potential response represented by radar plot is used to characterize extracts from different parts of plant, age, batch-to-batch variation and mode of extraction of E. longifolia through the obtained potentiometric fingerprint profile. Classification model was also developed classifying various E. longifolia extracts with the aid of chemometric pattern recognition tools namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). The sensor seems to be a promising analytical device for quality control based on potentiometric fingerprint analysis of phytomedicine.

This preliminary work investigates whether Fourier transform infrared spectroscopy (FT-IR), in combination with multivariate analysis can be used to distinguish Phyllanthus niruri Linn. samples from different locations. A FT-IR spectrometer used in this work is equipped with a transmittance accessory. All samples were scanned in the mid-infrared range of 400–4000 cm −1 but only the region of 400–2000 cm −1 that contained the most spectral information was analyzed with multivariate techniques of principal component analysis (PCA), genetic algorithm (GA), linear discriminant analysis (LDA) and SIMCA. PCA was used to reduce the spectra to three principal components, which explained 96.2% of the whole variance and a plot of principal components two and three showed groupings of the samples according to origin. Secondly, the spectra compressed with PCA were used to develop a LDA. The GA applied for 50 or 100 generations used concept of maximizing the ratio of between-groups variance to within-groups variance and identified the most discriminating variable for subsequent LDA. The SIMCA technique consisted of constructing an enclosure for each location using separate principal component models and using these models to classify the whole data. The best discriminatory approach was the technique using GA with LDA, where using just six wavenumber variables obtained from application of GA for 100 generations, gave 100% correct prediction for both originally grouped and cross-validated data. However, both PCA–LDA and SIMCA also gave very good classification. Basically, FT-IR analysis is rapid and has the power to discriminate samples in terms of geographical origin with the aid of multivariate analyses.

The modified Schneider's gel slide method is used to monitor the inhibitory effect of 50% methanol extract of Orthosiphon stamineus on the growth of calcium oxalate crystals. The images of crystals grown in the gel were captured at 1, 2, 3, 4 and 24 h with digital camera and image analysis was carried out. A total of nine variables relating to size and shape parameters were calculated and six were used for further analysis. Principal component analysis (PCA) and self-organizing map (SOM) were applied in the visualization of the size and shape distribution of the produced crystals using the following six variables: area, convex area, aspect ratio, equivalent diameter, roundness and full ratio. The results indicate that the modified gel slide method with the use of PCA and SOM to reduce the dimensionality of the data allowed an intuitive presentation of the differences in the studied inhibitors. The decrease in crystal growth for the extract and positive control of sodium citrate was clearly evident in the principal component scores and also on the location in the SOM. Evidence about the decrease in growth was also provided by an inhibition index of 0.290 for the extract and 0.255 for the positive control.

Phytochemical investigation on the leaves of Blumea balsamifera DC resulted in the isolation of 11 flavonoids. Their chemical structures were elucidated by means of elemental analyses and different spectroscopic methods, such as UV, IR, NMR and MS. The free radical scavenging activity of organic extracts of B. balsamifera DC leaves and that of pure flavonoids isolated from the leaves was evaluated using 1,1-diphenyl-2-picrylhydrazyl radical. A dose response curve was plotted for determining SC50 values (the concentrations required to inhibit radical formation by 50%). The antioxidant activities of crude extracts decreased in the order: methanol extract > chloroform extract > pet-ether extract. The antioxidant activities of all compounds tested decreased in the order: quercetin > rhamnetin > luteolin > luteolin-7-methylether > l -ascorbic acid > blumeatin > butylated hydroxyanisole > 5,7,3′,5′-tetrahydroxyflavanone > tamarixetin > butylated hydroxytoluene > α-tocopherol > dihydroquercetin-4′-methylether > dihydroquercetin-7,4′-dimethylether. This result indicates that flavonoid contents of different solvent extracts of B. balsamifera DC leaves were responsible for their antioxidant properties.

With the proliferation of numerous herbal remedies on the market, the ability to verify the actual presence of the active herbal ingredient in a product would be very useful. In this paper, a microcontroller-based electronic taste sensing system capable of discriminating between liquid samples containing Eurycoma longifolia and those that do not, is described. The ‘taste’ of the liquid sample is sensed using specially fabricated disposable screen-printed array of non-specific lipid-membrane sensors and classified by means of artificial neural network (ANN). The overall system is controlled by an embedded microcontroller, which performed the data acquisition, the ANN-based pattern recognition and the user interface tasks. From the extensive tests that were performed, excellent recognition results have been obtained. The system is flexible and could easily be trained for other herbal samples.

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.

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.

Recently, there has been increasing interest in Ficus deltoidea (Moracea) due to its chemical composition and the potential health benefits. The leaves of the plant have been suggested to have potential antidiabetic effects. Inhibition of carbohydrate-hydrolysing enzymes, such as α-glucosidase and α-amylase is one of the therapeutic approaches to control postprandial hyperglycemia. In this study, enzymes inhibitory effect and antioxidant properties of different fractions of methanolic extract obtained from F. deltoidea leaves was evaluated. Further, the possible relationship between pharmacological properties and phytochemical content of fractions was investigated. The n-butanol fraction showed significant α-glucosidase and α-amylase inhibitory effects (IC 50 values 15.1 and 39.42 μg/ml, respectively) along with the remarkable antioxidant activity when compared to the other fractions. High performance liquid chromatography (HPLC) chemical profiling of the n-butanol fraction revealed that the contents of isovitexin (24.63 mg/g) and vitexin (8.3 mg/g) were found to be significantly higher than the other fractions. These results indicate that F. deltoidea could be the potential source of promising anti-diabetic drug. Key words : Ficus deltoidea, enzymes, phytochemical, high performance liquid chromatography (HPLC), isovitexin, vitexin.

OBJECTIVE: Ficus deltoidea leaves have been used in traditional medicine in Southeast Asia to treat diabetes, inflammation, diarrhea, and infections. The present study was conducted to assess the genotoxicity and acute and subchronic toxicity of a standardized methanol extract of F. deltoidea leaves. METHODS: Sprague Dawley rats were orally treated with five different single doses of the extract and screened for signs of toxicity for two weeks after administration. In the subchronic study, three different doses of the extract 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. Phytochemical standardization was performed using a colorimeter and high-performance liquid chromatography. 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 subchronic toxicity study, there were no significant adverse effects on food consumption, body weight, organ weights, mortality, clinical chemistry, hematology, gross pathology, or histopathology. However, a dose-dependent increase in the serum urea level was observed. The Ames test revealed that the extract did not have any potential to induce gene mutations in S. typhimurium, either in the presence or absence of S9 activation. Phytochemical analysis of the extract revealed high contents of phenolics, flavonoids, and tannins. High-performance liquid chromatography analysis revealed high levels of vitexin and isovitexin in the extract, and the levels of heavy metals were below the toxic levels. CONCLUSION: The no-observed adverse effect level of F. deltoidea in rats was determined to be 2500 mg/kg.

Orthosiphon stamineusBenth has been traditionally used to treat hypertension. The study aimed to investigate the vascular reactivity of water extract (WOS) and water : methanolic (1 : 1) extract (WMOS) ofOrthosiphon stamineusBenth and AT1receptors blocker in the mechanisms of antihypertensive mediated byα1-adrenergic receptor and EDNO and PGI2releases in the SHR aortic rings. SHR (230–280 g) were divided into four groups: control, WOS, WMOS, and losartan. After being fed orally for 14 days, the aorta was harvested and subjected to PE (10−9to 10−5 M) and ACh (10−9to 10−5 M) with and without L-NAME (100 µM) and indomethacin (10 µM), respectively. WOS, WMOS, and losartan significantly reduced the contractile responses to PE intact suggesting the importance of endothelium in vasorelaxation. Losartan significantly enhanced the ACh-induced vasorelaxation. L-NAME significantly inhibited the ACh-induced relaxation in all groups. Indomethacin enhanced ACh-induced vasorelaxation in WMOS. Collectively,Orthosiphon stamineusleaves extract reduced vasoconstriction responses by the alteration ofα1-adrenergic and AT1receptors activities. The involvement of EDNO releases was clearly observed in this plant. In WOS, PGI2releases might not participate in the ACh-induced vasorelaxation. However, in WMOS, enhancement of vasorelaxation possibly due to continuous release of PGI2.

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. 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.

Objective: This study aimed to perform pharmacokinetic profile of rosmarinic acid (RA), sinensitin (SIN), eupatorin (EUP) and 3΄-hydroxy-5,6,7,4΄-tetramethoxyflavone (TMF) in Orthosiphon stamineus ethanolic extract (OS-E) and its nanoliposomes (OS-EL) after oral and intravenous administration in Sprague-Dawley rat’s plasma by developing and validating a high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection.Methods: An isocratic elution program consisting of methanol: tetrahydrofuran: water (0.1% H3PO4) mixture in the volume ratio 55: 5: 40 on Nucleosil C18 column (250 × 4.6 mm internal diameter × 5 µm particles size) was applied. The current study followed a two-ways crossover study design. OS-E and OS-EL were administered orally at 1000 and 500 mg/kg, respectively. They were also administered intravenously at 250 mg/kg via the tail vein.Results: The HPLC-UV method was successfully developed and validated for simultaneous determination of major chemical constituent from OS-E and OS-EL in rat’s plasma. The method recorded the mean recoveries from extraction were between 91.39 and 100.32%. With regards to the intravenous administration of OS-EL, all four marker compounds appeared to be poorly distributed and cleared slowly from the body compared to OS-E. Whilst in oral administration of OS-EL, the bioavailability of all marker compounds were higher than OS-E due to higher solubility of encapsulation in phospholipids.Conclusion: The higher solubility and bioavailability of OS-EL may contribute to encapsulation in phospholipids.

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