An improved classification of Orthosiphon stamineus using a data fusion technique is presented. Five different commercial sources along with freshly prepared samples were discriminated using an electronic nose (e-nose) and an electronic tongue (e-tongue). Samples from the different commercial brands were evaluated by the e-tongue and then followed by the e-nose. Applying Principal Component Analysis (PCA) separately on the respective e-tongue and e-nose data, only five distinct groups were projected. However, by employing a low level data fusion technique, six distinct groupings were achieved. Hence, this technique can enhance the ability of PCA to analyze the complex samples of Orthosiphon stamineus. Linear Discriminant Analysis (LDA) was then used to further validate and classify the samples. It was found that the LDA performance was also improved when the responses from the e-nose and e-tongue were fused together.
This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anti-candidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.
Pancreatic cancer has the highest mortality rate among cancers due to its aggressive biology and lack of effective treatment. Gemcitabine, the first line anticancer drug has reduced efficacy due to acquired resistance. The current study evaluates the toxicological effects of Orthosiphon stamineus (O.s) and its marker compound (rosmarinic acid) in combination with gemcitabine. O.s (200 or 400 mg/kg/day) and rosmarinic acid (32 mg/kg/day) were administered orally and gemcitabine (10 mg/kg/3 days) intraperitoneally either alone or in combination treatment for fourteen days. Parameters including blood serum biochemistry, hematology, myeloid-erythroid ratio, incident of lethality, and histopathological analysis of liver, kidney, and spleen tissues were studied. Neither, individual drugs/extract nor chemo-herbal combinations at tested doses induced any toxicity and damage to organs in nude mice when compared to control group. Toxicological data obtained from this study will help to select the best doses of chemo-herbal combination for future pancreatic xenograft tumor studies.
The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000 mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000 mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between both male and female treated rats in any doses tested. No abnormality of internal organs was observed between treatment and control groups. The oral lethal dose determined was more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000 mg/kg per day.
Orthosiphon stamineus extracts contain three flavonoids (3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20mM phosphate buffer (NaH(2)PO(4)) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052-250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048-0.368, 0.025-0.135, and 0.05-0.476 for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333-1.688, 0.722-1.055, and 0.548-1.819, respectively. The accuracy for intraday were 91.25%-103.38%, 94.32%-109.56%, and 92.85%-109.70% for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%-103.92%, 103.58%-104.57%, and 103.9%-105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.
The aim of the present study was to verify the anti-inflammatory activity of Orthosiphon stamineus leaf extracts and to identify the active compound(s) contributing to its anti-inflammatory activity using a developed HPLC method. Active chloroform extract of O. stamineus was fractionated into three fractions using a dry flash column chromatography method. These three fractions were investigated for anti-peritoneal capillary permeability, in vitro nitric oxide scavenging activity, anti-inflammatory and nitric oxide (NO) inhibition using carrageenan-induced hind paw edema method. The flavonoid rich chloroform extract fraction (CF2) [containing sinensetin (2.86% w/w), eupatorin (5.05% w/w) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (1.101% w/w)], significantly reduced rat hind paw edema, NO and decreased dye leakage to peritoneal cavity at p < 0.05. IC(50) of in vitro NO scavenging of CF2 was 0.3 mg/mL. These results suggest that the anti-inflammatory properties of these CF2 may possibly be due to the presence of flavonoid compounds capable of affecting the NO pathway.
Orthosiphon stamineus Benth., which is used as a gastroprotective herbal remedy in Malaysia, was assessed for its anti-ulcerogenic activity against ethanol-induced ulcers in rats. Fifty percent methanol was used to extract the oven-dried O. stamineus leaves. The extract was then lyophilized with a rotary evaporator and freeze-dried. Oral administration of O. stamineus methanolic extract (OSME) (125, 250, 500, and 1,000 mg/kg) was found to significantly decrease the ulcer index (P < .01, P < .001, P < .001, and P < .001, respectively). Histological study of a section of the rat stomach also showed a marked improvement in the gastric mucosal damage in groups receiving OSME. In order to further investigate the gastroprotective mechanism of OSME, mucus secretion and lipid peroxidation level were estimated in vitro and ex vivo. OSME exhibited dose-dependent stimulation of mucus secretion (r = 0.718, P < .001) and inhibition of lipid peroxidation in rat gastric mucosal homogenates (both in vitro [r = 0.819, P < .05] and ex vivo [r = 0.981, P < .05]). It was concluded that the gastroprotective mechanism of OSME was partly due to its ability to inhibit lipid peroxidation and stimulate gastric mucus secretion.
The anti-pyretic activity of a standardized methanol/water (50/50) extract of Orthosiphon stamineus Benth. (SEOS) was investigated for its effect on normal body temperature and yeast-induced pyrexia in Sprague Dawley (SD) rats. The SEOS showed no effect on normal body temperature. Doses of 500 and 1000 mg/kg body weight of SEOS significantly reduced the yeast-induced elevation in body temperature. This effect persisted up to 4 h following the administration of the extract. The anti-pyretic effect of SEOS was comparable with that of paracetamol (acetaminophen in U.S) (150 mg/kg p.o.), a standard anti-pyretic agent. HPLC study revealed that rosmarinic acid, sinensetin, eupatorin and tetramethoxyflavone were present in SEOS in the amounts of 7.58%, 0.2%, 0.34% and 0.24% respectively. The LD(50) of the extract in rats was higher than 5000 mg/kg body weight. Therefore, the present study ascertained that SEOS possesses a significant anti-pyretic activity.
Anti-inflammatory and analgesic activities of a standardized Orthosiphon stamineus methanol:water (50:50 vol/vol) leaf extract (SEOS) were evaluated in animal models. Oral administration of SEOS at doses of 500 and 1,000 mg/kg significantly reduced the hind paw edema in rats at 3 and 5 hours after carrageenan administration (P < .01 and P < .01; P < .01 and P < .05, respectively). SEOS (1,000 mg/kg, p.o.) also produced significant (P < .05) analgesic activity in both the acetic acid-induced writhing test and the formalin-induced licking test (late phase) in mice and rats, respectively. However, SEOS showed no effect on the tail flick and hot plate tests in mice. The results of the present study support the proposal that O. stamineus has anti-inflammatory and non-narcotic analgesic activities. These findings justify the traditional use of the plant for treating pain and inflammation.
Orthosiphon stamineus Benth. (Lamiaceae) is an important plant in traditional folk medicine that is used to treat hypertension and kidney stones. In humans, this plant has been tested as an addition regiment for antihypertensive treatment. Among the treatments for hypertension, O. stamineus had been to have diuretic and vasorelaxant effects in animal models. There is still very little information regarding the vasorelaxant effect of O. stamineus. Therefore, the present study was designed to investigate the vasorelaxant activity and mechanism of action of the fractions of O. stamineus. The vasorelaxant activity and the underlying mechanisms of the chloroform fraction of the 50% methanolic extract of O. stamineus (CF) was evaluated on thoracic aortic rings isolated from Sprague Dawley rats. CF caused relaxation of the aortic ring pre-contracted with phenylephrine in the presence and absence of endothelium, and pre-contracted with potassium chloride in endothelium-intact aortic ring. In the presence of endothelium, both indomethacin (a nonselective cyclooxygenase inhibitor) and [Formula: see text]-[1,2,4]Oxadiazolo[4,3-[Formula: see text]]quinoxalin-1-one (ODQ, selective soluble guanylate cyclase inhibitor) had a small effect on the vasorelaxation response. On the other hand, in the presence of Nω-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor), methylene blue (cyclic guanosine monophosphate lowering agent), tetraethylammonium ([Formula: see text], nonselective calcium activator [Formula: see text] channel blocker), 4-aminopyridine (4-AP, voltage-dependent [Formula: see text] channel blocker), barium chloride ([Formula: see text], inwardly rectifying [Formula: see text] channel blocker), glibenclamide (nonspecific ATP-sensitive [Formula: see text] channel blocker), atropine (muscarinic receptor blocker) and propranolol (β-adrenergic receptor blocker), the vasorelaxant effect significantly reduced the relaxation stimulated by CF. CF was also found to be active in reducing [Formula: see text] release from the sarcoplasmic reticulum and blocking calcium channels.
Orthosiphon stamineus (OS), Benth. (Lamiaceae) is widely used in Malaysia for treatments of various kidney and liver ailments. In the experiment, DPPH* radicals scavenging, Fe(3+)-induced lipid peroxidation inhibiting activities and trolox equivalent antioxidant capacity (TEAC) of methanol/water extract of Orthosiphon stamineus (SEOS) were determined. The results indicated that SEOS exhibited antioxidant, lipid peroxidation inhibition and free radical scavenging activities. The hepatoprotective activity of the SEOS was studied using CCl(4)-induced liver toxicity in rats. The activity was assessed by monitoring liver function tests through the measurement of alanine transaminase (ALT) and aspartate transaminase (AST). Furthermore, hepatic tissues were also subjected to histopathological studies. Pretreatment of SEOS (125, 250, 500 and 1000 mg/kg p.o.) dose-dependently reduced the necrotic changes in rat liver and inhibited the increase of serum ALT and AST activities. The results of the present study indicated that the hepatoprotective effect of Orthosiphon stamineus might be ascribable to its antioxidant and free radical scavenging property.
Primarily, optimization of ultrasonic-assisted extraction (UAE) conditions of Orthospihon stamineus was evaluated and verified using a central composite design (CCD) based on three factors including extraction time (minutes), ultrasound amplitude (A), and solvent concentration (%). The response surface methodology (RSM) was performed to develop an extraction method with maximum yield and high rosmarinic acid content. The optimal UAE conditions were as follows: extraction time 21 min, ultrasound amplitudes 62 A, and solvent composition 70% ethanol in water. The crude extract was further fractionated using solid-phase extraction (SPE), where six sequential fractions that varied in polarity (0-100% Acetonitrile in water) were obtained. Next, the six fractions were evaluated for their antioxidant and anti-cancer properties. This study found that Fraction 2 (F2) contained the highest rosmarinic acid content and showed the strongest antioxidant activity. Additionally, F2 showed an anti-proliferative effect against prostate cancer (DU145) with no harmful effect on normal cells.
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).
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.
The present study investigated the antiobesity and lipid lowering effects of an ethanolic extract of leaves obtained from Orthosiphon stamineus (200 and 400 mg/kg) and its major compound (rosmarinic acid, 10 mg/kg) in obese mice (C57BL/6) induced by a high-fat diet. Continuous supplementation with O. stamineus extract (200 and 400 mg/kg) for 8 weeks significantly decreased body weight gain (p
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.
This paper describes the first reported attempt to isolate DNA sequences containing repeat motifs in Eurycoma longifolia and Orthosiphon stamineus. A library enriched for genomic repeat motifs was developed using novel oligonucleotides designed with inosine residues incorporated at predetermined positions. A total of eight and twelve specific molecular markers were developed for O. stamineus and E. longifolia respectively. These markers have a potential application in estimating population diversity levels and QTL mapping in these two medicinal plants, which are widely used in the Malaysian herbal industry.
Alzheimer's disease (AD) is a chronic neurodegenerative brain disease which is characterized by impairment in cognitive functioning. Orthosiphon stamineus (OS) Benth. (Lamiaceae) is a medicinal plant found around Southeast Asia that has been employed as treatments for various diseases. OS extract contains many active compounds that have been shown to possess various pharmacological properties whereby in vitro studies have demonstrated neuroprotective as well as cholinesterase inhibitory effects. This study, therefore aimed at determining whether this Malaysian plant derived flavonoid can reverse scopolamine induced learning and memory dysfunction in the novel object recognition (NOR) test and the elevated plus maze (EPM) test. In the present study, rats were treated once daily with OS 50 mg/kg, 100 mg/kg, 200 mg/kg and donepezil 1 mg/kg via oral dosing and were given intraperitoneal (ip) injection of scopolamine 1 mg/kg daily to induce cognitive deficits. Rats were subjected to behavioral analysis to assess learning and memory functions and hippocampal tissues were extracted for gene expression and immunohistochemistry studies. All the three doses demonstrated improved scopolamine-induced impairment by showing shortened transfer latency as well as the higher inflexion ratio when compared to the negative control group. OS extract also exhibited memory-enhancing activity against chronic scopolamine-induced memory deficits in the long-term memory novel object recognition performance as indicated by an increase in the recognition index. OS extract was observed to have modulated the mRNA expression of CREB1, BDNF, and TRKB genes and pretreatment with OS extract were observed to have increased the immature neurons against hippocampal neurogenesis suppressed by scopolamine, which was confirmed by the DCX-positive stained cells. These research findings suggest that the OS ethanolic extract demonstrated an improving effect on memory and hence could serve as a potential therapeutic target for the treatment of neurodegenerative diseases like AD.
One species of lace bug Cochlochila bullita Stål (Heteroptera: Tingidae) was found heavily infested Orthosiphon aristatus Blume Miq., an important medicinal plant in Malaysia. A morphological re-description of C. bullita was done in order to facilitate the identification of this oligophagous insect pest. Five variables, body length and width, antenna length, tibia length and head width were measured from 15 samples from each stage. Among these variables, body length and width were used to construct the ratio for species identification; while body lengths with the other three variables were used to distinguish the nymphs from each developmental stage. The measurements of four traits except the antenna length showed significant differences between the development stages. And thus suggest the body width, tibia length and head width were suitable parameters used to distinguish the nymphal stages. However, the result on the growth factor showed only the sizes of the head followed a more constant growth rate with growth ratios (1.21-1.39) lie between the Dyar's ratio. Body length and width ratio for the adult female and male was 1.51 +/- 0.00 and 1.59 +/- 0.01, respectively. These data are pertinent for identifying developmental stages and to distinguish the species of the lace bug.
Objectives: The effects of water and 50% ethanolic-water extracts of Orthosiphon stamineus Benth (OS) on cell proliferation and apoptotic activity against uterine leiomyosarcoma (SK-UT-1) cells were investigated. Methods: Anti-proliferation effect was evaluated through cell cycle analysis whereas apoptotic activity was determined via screening and quantifying using fluorescence microscopy and flow cytometric analysis, respectively. The effect of extracts on molecular mechanism was studied using real-time reverse transcription polymerase chain reaction and Western blotting. Results: Cell cycle flow cytometric analysis showed the induction of cell cycle arrests were behaves in a p53-independent manner. The examination using fluorescence microscopy and Annexin V flow cytometry revealed the presence of morphological features of apoptotic bodies. Downregulation of anti-apoptotic gene (Bcl-2) supports the apoptotic activity of OS extracts although poorly induce PARP-1 cleavage in Western blot analysis. The extracts also inhibit the SK-UT-1 growth by suppressing VEGF-A, TGF-β1 and PCNA genes, which involved in angiogenesis and cell proliferation. Conclusion: This study demonstrates that O. stamineus extracts are able to inhibit proliferation and induced apoptosis of uterine fibroid cells and is worth further investigation.