Orthosiphon stamineus water extracts inhibit human herpes virus 1 KOS-1 and acyclovir-resistant strains by virucidal activity and suppressing virus early infection

Aim@#To determine the efficacy and mode of action of hot and cold water extracts of Orthosiphon stamineus leaves against two strains of human herpes virus 1 (HHV-1) i.e. KOS-1 and acyclovir (ACV)-resistant UKM-1 (UKM-1) strains. @*Methodology and results@#Hot and cold water extracts of O. stamineus were not cytotoxic to vero cells as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) assay with 50% cytotoxicity concentration (CC50) values of 3.4 and 3.3 mg/mL respectively. Antiviral activity was determined by plaque reduction assay in post-treatment, pre-treatment and virucidal assays followed by time-addition and time removal assay to relate with the stages during the viral infection cycle. Both extracts displayed antiviral activity against HHV-1 KOS-1 and HHV-1 UKM-1 strains with 50% effective concentration (EC50) values between 0.12-0.15 mg/mL in reducing plaque formation. The calculated selectivity indices (SI) were 23 and 28 for hot and cold water extract respectively, indicating that they have good potential as antiviral agent. The extracts were virucidal towards both HHV-1 KOS-1 and HHV-1 UKM-1 strains which may directly affects the virus structure. This is supported with the fact that exposure of the extracts inhibit viral attachment and penetration to the vero cells. In time-of addition assay, both extracts were effective during the early stage of virus infection cycle for HHV-1 KOS-1 strain which is in parallel with the results from the attachment and penetration studies. For HHV-1 UKM-1 strain, contact to the extracts at any time during post-infection inhibits virus replication and also progeny release. @*Conclusion, significance and impact of study@#Cold and hot water extracts of O. stamineus have good potential as antiviral agent against HHV-1 strain KOS-1 and more importantly against UKM-1 strain which is ACV-resistant. The extracts displayed virucidal effect and inhibition of early virus replication cycle involving viral attachment and penetration to cells.

Growth, Leaf Gas Exchange and Secondary Metabolites of Orthosiphon stamineus as Affected by Multiwall Carbon Nanotubes Application

Aims: This study was conducted to study the growth, leaf gas exchange and secondary metabolites of Orthosiphon stamineus as affected by Multiwalled carbon nanotubes application (MWCNT). Study Design: Orthosiphon stamineus were exposed to four different multi-walled carbon nanotubes (MWCNTs) concentration (0, 700, 1400 and 2100 mg L-1). The experiment was organized in a randomized complete block (RCBD) design with three replications. Each experimental unit consisted of twelve plants, and there were a total of 144 plants used in the experiment. Place and Duration of Study: Department of Biology, Faculty of Science Universiti Putra Malaysia between November 2016 to March 2017. Methodology: Each plant was watered with 50 mL of MWCNTs solution in week 2 and 9. The leaves number were counted manually and the total plant biomass was taken by calculating the dry weight of root, stem, and leaf per seedling The total chlorophyll content in the leaves was measured using a SPAD chlorophyll meter. The leaf gas exchange was determined using LI-6400XT portable photosynthesis system. Total phenolics and flavonoid were determined using Folin-Ciocalteu reagent. Results: It was found that application of MWCNTs would reduce the growth characteristics of this plant that was shown by decreased leaf numbers, total biomass and total chlorophyll content (TCC). As MWCNTs concentration increases from 0 > 2100 mg L-1, the leaf gas exchange parameter also shows reduced patterns. Generally, as the rate of MWCNTs increased from 700 > 2100 mg L-1 MWCNTs reduce the net photosynthesis (A), stomatal conductance (gs), transpiration rate (E) and increased the plant water use efficiency. The production of the secondary metabolites was directly dependable on MWCNTs concentration. As the rate was enhanced from 0 > 700 > 1400 > 2100 mg L-1 the production of total phenolics and flavonoids was enhanced. Conclusion: The current study revealed that the high application of MWCNTs concentration reduce the growth rate of O. stamineus, leaf gas exchange and simultaneously increase the production of secondary metabolites.

Hexane Extract of Orthosiphon stamineus Induces Insulin Expression and Prevents Glucotoxicity in INS-1 Cells

BACKGROUND: Hyperglycemia, a characteristic feature of diabetes, induces glucotoxicity in pancreatic beta-cells, resulting in further impairment of insulin secretion and worsening glycemic control. Thus, preservation of insulin secretory capacity is essential for the management of type 2 diabetes. In this study, we evaluated the ability of an Orthosiphon stamineus (OS) extract to prevent glucotoxicity in insulin-producing cells. METHODS: We measured insulin mRNA expression and glucose-stimulated insulin secretion (GSIS) in OS-treated INS-1 cells after exposure to a high glucose (HG; 30 mM) concentration. RESULTS: The hexane extract of OS elevated mRNA expression of insulin as well as pancreatic and duodenal homeobox-1 of INS-1 cells in a dose-dependent manner. The hexane OS extract also increased the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) in a concentration-dependent manner. Additionally, Akt phosphorylation was elevated by treatment with 100 and 200 micromol of the hexane OS extract. Three days of HG exposure suppressed insulin mRNA expression and GSIS; these expressions were restored by treatment with the hexane OS extract. HG elevated peroxide levels in the INS-1 cells. These levels were unaffected by OS treatment under both normal and hyperglycemic conditions. CONCLUSION: Our results suggested that the hexane extract of OS elevates insulin mRNA expression and prevents glucotoxicity induced by a 3-day treatment with HG. This was associated with the activation of PI-3K and Akt.

Determination of Three Active Flavones in Orthosiphon stamineus by HPLC / 医药导报

Objective To develop a method for content determination of sinensetin,eupatorin,and 3′-hydroxy-5,6,7, 4′-tetramethoxyflavone in Orthosiphon stamineus. Methods The determination was carried out on a Symmetry C18 column (4.6 mm×250 mm,5 μm) by HPLC.The mobile consisted of acetonitrile and water containing 0.05% H3 PO4 in gradient elution. The flow rate was 1 mL?min-1 ,the column temperature was 30 ℃ ,the detected wavelength was set at 365 nm, and the injection volume was 10 μL. Results The peak areas and the sample quantity of the three components presented good linear relationship in the range of 0. 50 - 5. 00 μg for sinensetin,0. 50 - 5. 00 μg for eupatorin, and 0. 05 - 0. 50 μg for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone.The average recoveries were 101.26%,100.28% and 99.66%,respectively. RSD were 1.73%, 0.82% and 1.67%, respectively. Conclusion The method is proved to be simple,accurate and can be used for the quality evaluation of Orthosiphon stamineus.

Identifying the developmental stages and optimizing the sample preparation for anatomical study of Orthosiphon stamineus.

This study focuses on the characterization of morphological and anatomical traits of Orthosiphon stamineus which belongs to the Lamiaceae family. Orthosiphon stamineus, better known as “Misai Kucing” or “cats whiskers” by the locals, contained active phenolics compounds such as flavanoids. Despite its wide usage as a medicinal plant, information regarding Orthosiphon stamineus specific developmental stages is relatively scarce. Furthermore, to date, no anatomical data of this plant is available. Therefore, this study aims to systematically identify the developmental stages and its anatomy which may provide more insight to its medical application. The result showed some distinct morphological and anatomical characteristics. In the morphological study, it was observed that Orthosiphon stamineus is a herbal shrub with well-developed creeping rootstock. The leaves are simple, green, and arranged in opposite pairs. The stem is approximately 54 cm in height at the oldest stage (62- 64 days). In order to proceed with the anatomical study of Orthosiphon stamineus, the cross sections of different developmental stages of leaves and stems were examined. Several critical steps prior to viewing the prepared slides, which include dehydration, sectioning and staining, were optimized accordingly.

Preliminary morphological and anatomical study of Orthosiphon stamineus.

This study focuses on the characterization of morphological and anatomical traits of Orthosiphon stamineus which belongs to the Lamiaceae family. Orthosiphon stamineus, better known as “Misai Kucing” or “cats whiskers” by the locals, contained active phenolics compounds such as flavanoids. Despite its wide usage as a medicinal plant, information regarding Orthosiphon stamineus specific developmental stages is relatively scarce. Furthermore, to date, no anatomical data of this plant is available. Therefore, this study aims to systematically identify the developmental stages and its anatomy which may provide more insight to its medical application. The result showed some distinct morphological and anatomical characteristics. In the morphological study, it was observed that Orthosiphon stamineus is a herbal shrub with well-developed creeping rootstock. The leaves are simple, green, and arranged in opposite pairs. The stem is approximately 28 cm in height at the stage (12 days). The flowers have long wispy stamens shaped with pale purple color. In anatomical study, the cross sections of the stem for tow stage (32) and (62) days of this plant were examined. All the detailed systematic study of this plant has not worked earlier.

Study on the developmental toxicity of a standardized extract of Orthosiphon stamineus in rats

Infusions of Orthosiphon stamineus Benth., Lamiaceae, leaves are widely used in Southeastern Asia to treat different illnesses. Nonetheless, no data is available on the safety of O. stamineus for pregnant women and their babies. This study was undertaken to evaluate the developmental toxicity of O. stamineus standardized aqueous extract in female Sprague Dawley rats (n=21) at 0, 250, 500, 1000 and 2000 mg/kg/day, by gavage on gestation days 6-20. Clinical signs of maternal toxicity, body weight gain, and food and water consumption were recorded. Caesarean sections were performed on gestation day 21; resorptions and living and dead fetuses were counted. Fetuses were weighed and examined for external abnormalities. Half of the fetuses from each litter were cleared and stained with Alizarin red S for skeleton evaluation. O. stamineus standardized aqueous extract did not alter pregnancy body weight gain and food and water consumption and caused no other sign of maternal toxicity. Embryolethality and prenatal growth retardation were not observed either. O. stamineus standardized aqueous extract increased a few skeleton variations and a skull bone malformation (hyoid bone absent) in a non-dose dependent manner. Anogenital distance was increased in male and female fetuses exposed to the highest O. stamineus standardized aqueous extract dose, an indication that the extract could possibly contain androgenic compounds.

HPLC-TOF/MS profile and nitric oxide scavenging activity of Orthosiphon stamineus leaf extracts

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.