Orthosiphon Stamineus Research Articles

POTENTIALS OF THE CAT’S WHISKER PLANTS (Orthosiphon aristatus) FOR KIDNEY HEALTH

The cat's whisker plant (Orthosiphon stamineus) can be useful as an antioxidant, anti-inflammatory, antihypertensive, antidiabetic, and anti-microbial activity and has a diuretic effect. The leaves of the cat's whiskers are medicinal plants that contain flavonoid compounds. Cat whiskers plant can also be used to treat various types of diseases both empirically and clinically, including kidney stones. Kidney stones are a clinical disorder due to the blockage of the components of crystal stones that inhibit the work of the kidneys and can cause abnormalities in the urinary tract. Some constituent compounds that are usually found in kidney stones are calcium oxalate, calcium phosphate (brusit), uric acid, cystine, struvite (MgNH4PO4), and wedelit (Bakta, 1995). Efforts to treat kidney stones by utilizing a variety of herbs as medicine, in this context by using a cat whiskers plant called Alternative Medicine, this is because herbal plants have minimal side effects in long-term use when used with the right indications and proportions, the price is also more affordable and easy to obtain. The purpose of this paper is to discuss and provide information on morphology, secondary metabolite content, and pharmacological activity of cat whiskers plants so that they can be used as a support for kidney health. This research is a study of literature or literature review is a type of research that collects, manages, uses, and reviews research data sourced from scientific research journals, previous research manuscripts, and textbooks that have relevance to the theme. The results of a literature search indicate that the cat's whisker plant can be beneficial for kidney health because the extract has diuretic activity and can modulate the excretion of sodium and potassium ions in the urine. The presence of flavonoids, saponins, and phenolic compounds that play a role in diuresis activity, so this cat whiskers plant is efficacious in treating kidney stone disease because it has the effect of destroying kidney stones, reducing urine (diuretic) and anti-inflammatory (anti-inflammatory).

Toxicity Test of Cat Whiskers (Orthosiphon stamineus) and Miana (Coleus artropurpureus) Leaves Against Artemia salina Leach Using BSLT

Medicinal plants are a good source of therapeutic drugs because they contain phytochemical compounds. Cat's whiskers (Orthosiphon stamineus) and miana (Coleus artropurpureus) are plants that are widely used as herbal medicines. However, there are no studies related to toxicology to ensure its safety. The aim of this study was to determine the toxicity of 96% ethanol extract of cat whiskers and miana leaves to shrimp (Artemia salina Leach) larvae. Toxicity test was carried out using the Brine Shrimp Lethality Test (BSLT) method with 48 hours old Artemia salina shrimp larvae. The concentration of the extract used was 1 ppm, 2 ppm, 3 ppm, 4 ppm and 5 ppm. The results of the toxicity test showed that the 96% ethanol extract of cat's whiskers leaves had an LC50 value of 8.0855 ppm, while the 96% ethanol extract of miana leaves had an LC50 value of 4.7067 ppm. Based on the results of the study, it can be concluded that cat's whiskers and miana leaves have the potential to be developed as candidates for anticancer drugs.

Polymolecular botanical drug of Orthosiphon stamineus extract (C5OSEW5050ESA) as a complementary therapy to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine remains the cornerstone of pancreatic cancer treatment, despite exhibiting a modest effect on patient survival due to the development of drug resistance. Nuvastatic™ polymolecular botanical drug Orthosiphon stamineus (O.stamineus) is a folklore Asian herbal medicine that is used for the treatment of a variety of ailments. However, little is known about the mechanism of actions of the Nuvastatic™ polymolecular botanical drug of O.stamineus as a complementary therapy in resistant pancreatic cancer. It is postulated that the proprietary O.stamineus extract formulation (ID: C5EOSEW5050ESA) in Nuvastatic™ may sensitise resistant pancreatic cancer cells to gemcitabine. This study was conducted to assess the cytotoxic activity and synergistic effects of C5EOSEW5050ESA in gemcitabine-resistant pancreatic cancer cells. The effects of C5EOSEW5050ESA treatment on cell viability, multidrug-resistant genes, epithelial-mesenchymal transition, cellular senescence, cell death, and Notch signalling pathway were evaluated in gemcitabine-resistant Panc-1cells. C5EOSEW5050ESA sensitised gemcitabine resistant cells towards C5EOSEW5050ESA-gemcitabine combination treatment by reducing the expression of multidrug-resistant genes and epithelial-mesenchymal transition markers in gemcitabine-resistant cells compared to the control group, possibly through the inhibition of Notch signalling. This study provides valuable insight into using C5EOSEW5050ESA as a potential complementary treatment for resistant pancreatic cancer.

In silico analysis of Potential Phytochemical Constituents of Orthosiphon stamineus as Significant Inhibitors Against Urolithiasis

Naturally occurring bioactives from plant sources have played a significant role in developing multi-targeted drugs towards treatment of various human ailments. Urolithiasis is the process of stone formation in the kidney, urinary tract or in bladder. It occurs to 1 in 20 people at different phases of their life causing pain in the abdomen, flank or groin and bloody urine at times. The current study focuses on pythochemical activity of Orthosiphon stamineus(Java tea) that can act against kidney stone. The target proteins responsible for stone formation was identified through literature search and the three dimensional structures available was retrieved from Protein Data Bank and structures unavailable was modelled through computational methods. These proteins were docked against the phytochemicals and certain synthetic compounds which are commercially used for Urolithiasis. The best docked compounds and their interaction have been studied and it was identified that when compared to the synthetic compounds, there is high Volume 110|10-12 competence for phytochemicals in binding to the target proteins which suggests that these compounds can be used further for the process of designing a drug effectively against urolithiasis.

Identification of Java Tea Adulteration by Babadotan and Tekelan using Machine Learning

Java Tea (Orthosiphon aristatus) is a common herbal medicinal plant that functions as a health treatment and treats various diseases. The high demand for Java Tea causes high prices and a decrease in the amount of medicinal plant raw materials, causing various quality control problems such as the content of various bioactive components and adulteration from babadotan and tekelan. So far, the detection of adulteration has been carried out by various analyzes, including chemical analysis and statistical methods to process data. The data used is of high dimension with a very high-density level, thus causing difficulties in classification. The mixed data of Orthosiphon aristatus consists of 1201 features with a total sample of 216. This study uses a Random Forest (RF) method with a total of 100 trees, and the RF method is combined with the Recursive Feature Elimination (RFE) method. In the RF and RFE that can be produced, the optimum value for the number of features is 244. The experimental evaluation results revealed that the proposed method could achieve a high accuracy of 81.82% in identifying Orthosiphon aristatus.

Antioxidant Capacity, Total Phenolic, and Flavonoid Content from Java Tea (Orthosiphon aristatus) Extracts

Orthosiphon aristatus, known as kumis kucing in Indonesia, has some biological activity, i.e., as an anti-inflammatory, fever, rheumatism, diabetes, and kidney stone. This plant is also known for being rich in phenolic compounds such as flavonoids. In this study, we would like to compare the antioxidant activity, total phenolics, and flavonoid content from the stems and leaves of Orthosiphon aristatus extracted with water and ethanol by maceration. The yield stem and leaves of the water extract are 12.98-31.28%, and ethanol extract 3.58 - 9.82% (dry basis). The stem and leaves water extract contains a phenolic of 7.82-26.43 (mg GAE/g) and ethanol extract of 2.27-7.84 (mg GAE/g). Water extract Orthosiphon aristatus flavonoid content is 1.23-2.86 (mg EK/g), and ethanol extract is 1.09-3.95 (mg EK/g). The antioxidant activity of ethanol (IC50), extract from leaves was 123.91 (µg/mL) and from stems was 125.50 (µg/mL), while IC50 of water extract from leaves was 114.70 (µg/mL) and from stems was 125.03 (µg/mL). The correlation test result of active compounds using linear regression is phenolic compounds with antioxidants stronger than flavonoids. The qualitative thin-layer chromatography fingerprint analysis showed that ethanol and water extract contained natural sinensetin compounds.

Poloxamer and Chitosan-Based <i>In Situ</i> Gels Loaded with <i>Orthosiphon stamineus</i> Benth. Extracts Containing Rosmarinic Acid for the Treatment of Ocular Infections

Orthosiphon stamineus Benth. (OS) is a commonly used medicinal plant for curbing bacterial infections globally. This work aimed to fabricate poloxamer and chitosan-based in situ gels loaded with standardized aqueous-ethanolic OS leaf extracts and investigate their antimicrobial efficacy as a potential remedy against ocular infections. In situ gels containing 0.5% w/v OS extract prepared using cold dispersion method were subjected to physicochemical characterization, including in vitro-release studies. Antimicrobial efficacy was tested against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Pseudomonas aeruginosa using agar diffusion method. Thin layer chromatography and high performance liquid chromatography chromatograms confirmed the presence of rosmarinic acid (RA) and sinensitin in OS extracts with same retention factor (0.26 and 0.49) and retention times (12.2 and 20.7 min) against reference standards. A homogenous brown coloured in situ gel exhibited low viscosity as a solution and increased viscosity in gel form at ocular temperature. The optimized formulations, P7 (21% P407/4% P188), P8 (21% P407/5% P188) and F5 (1.5% chitosan and 45% β-glycerophosphate) exhibited ideal ocular pH (7.27-7.46), phase transition at ocular temperature (33-37°C) and prolonged RA release up to 12 h. Formulation F5 showed an inhibition zone of 4.3 mm against M. luteus. Among all, formulation F5 alone exhibited modest antimicrobial activity against M. luteus. OS extracts at 5% and 10% were most active against tested bacteria however, loading them into in situ gels resulted in sedimentation. Hence, isolation of RA from OS extract is suggested before loading into formulations for a better antimicrobial activity.

Optimization Method and Stability Test to Determinate Luteolin, Quercetin, Apigenin, and Sinensetin Levels in Herbal Medicines Using TLC-Densitometry

Background: Nephrolithiasis is a condition in which there are one or more kidney stones in the pelvis or calyces. Luteolin, quercetin, apigenin, and sinensetin are marker compounds in the extracts of Plantago major, Sonchus arvensis, Strobilanthes crispus and Orthosiphon stamineus which have nephrolithiasis activity. To control the quality of herbal medicines, a TLC-Densitometry method was developed in this study using luteolin, quercetin, apigenin, and sinensetin as phytochemical markers. Objective: The present work aimed to develop optimal conditions for analyzing luteolin, quercetin, apigenin, and sinensetin. Methods: Determination of optimal conditions for analysis is carried out by determining the composition of the mobile phase, chamber saturation time, and analysis wavelength. Silica gel 60 F254 was used as the stationary phase. Stability tests were carried out by analyzing standards and samples at 0, 4, 8, and 24 hours. Results: The best separation that produces symmetrical peaks of herbal medicine was achieved under isocratic conditions using the composition of the mobile phase chloroform : acetone: dichloromethane : acetonitrile : formic acid (6 : 2: 2 : 0,05 : 0.05 v/v/v/v/ v) with a wavelength of 335 nm with a saturation time of 30 minutes. Conclusion: In this study, the optimal conditions for the analysis of luteolin, quercetin, apigenin, and sinensetin. Luteolin, quercetin, apigenin, and sinensetin are unstable during 8 hours of storage. Therefore, standard solutions and samples must be made fresh to maintain stability.

Mechanism of action of Orthosiphon stamineus against non-alcoholic fatty liver disease: Insights from systems pharmacology and molecular docking approaches

Non-alcoholic fatty liver disease (NAFLD) is one of the most common complications of a metabolic syndrome caused by excessive accumulation of fat in the liver. Orthosiphon stamineus also known as Orthosiphon aristatus is a medicinal plant with possible potential beneficial effects on various metabolic disorders. This study aims to investigate the in vitro inhibitory effects of O. stamineus on hepatic fat accumulation and to further use the computational systems pharmacology approach to identify the pharmacokinetic properties of the bioactive compounds of O. stamineus and to predict their molecular mechanisms against NAFLD. Methods: The effects of an ethanolic extract of O. stamineus leaves on cytotoxicity, fat accumulation and antioxidant activity were assessed using HepG2 cells. The bioactive compounds of O. stamineus were identified using LC/MS and two bioinformatics databases, namely the Traditional Chinese Medicine Integrated Database (TCMID) and the Bioinformatics Analysis Tool for the Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM). Pathway enrichment analysis was performed on the predicted targets of the bioactive compounds to provide a systematic overview of the molecular mechanism of action, while molecular docking was used to validate the predicted targets. Results: A total of 27 bioactive compounds corresponding to 50 potential NAFLD-related targets were identified. O. stamineus exerts its anti-NAFLD effects by modulating a variety of cellular processes, including oxidative stress, mitochondrial β-oxidation, inflammatory signalling pathways, insulin signalling, and fatty acid homeostasis pathways. O. stamineus is significantly targeting many oxidative stress regulators, including JNK, mammalian target of rapamycin (mTOR), NFKB1, PPAR, and AKT1. Molecular docking analysis confirmed the expected high affinity for the potential targets, while the in vitro assay indicates the ability of O. stamineus to inhibit hepatic fat accumulation. Conclusion: Using the computational systems pharmacology approach, the potentially beneficial effect of O. stamineus in NAFLD was indicated through the combination of multiple compounds, multiple targets, and multicellular components.

Therapeutic Effectiveness of Sinensetin Against Cancer and Other Human Complications: A Review of Biological Potential and Pharmacological Activities.

Plant and their active phytoproducts have been used in modern medicine and playing an important role in the health sectors since a very early age. Human beings need a considerable amount of these plant-based phytochemicals for their health. The flavonoidal class phytochemical is an important class of natural products in modern healthcare because of their different pharmacological activities and health benefits. Flavonoidal class phytochemicals have been used to treat diabetes and related secondary complications in humans. Flavonoids have antiapoptotic, anti-hyperlipidemic, anti-inflammatory, and anti-oxidant potential in the health sectors. Sinensetin, also called 3',4',5,6,7-pentametoksiflavon is a colorless compound with a molecular weight 372.37g/mol and is found to be present in the Orthosiphon stamineus. In the present investigation, we aim to collect scientific information on sinensetin and analyze it for its biological potential and therapeutic benefits against various types of disorders and complications. Medicinal importance and pharmacological activities data have been collected and analyzed in the present work for sinensetin through literature data analysis of different research works. Google Science Direct, PubMed, Scopus, and Google Scholar were mainly searched to collect the scientific information in the present work. The present work analyzed sinensetin biological potential, pharmacological activities, and analytical aspects. Literature data analysis of different scientific research works revealed the biological potential of phytochemicals in medicine, including flavonoids. Sinensetin has anti-tumor, antiinflammatory, anti-oxidant, anti-diabetic, and antibacterial activities through their testing in different in vitro and in vivo models. Sinensetin has physiological functions, including anti-oxidant, antiinflammation, and anti-cancer potential in medicine. Scientific data analysis signified the biological importance of sinensetin against tumors, gastric cancer, colorectal cancer, breast cancer, diabetes, influenza H1N1 infection, obesity, inflammation, colitis, brain disorders, and microbial infections. Further biological potential of sinensetin on enzymes and angiogenesis has been analyzed in the present work. Sinensetin was isolated through different analytical and extraction techniques, including chromatographic techniques. Literature data analysis signified sinensetin's biological potential and pharmacological activities in medicine.

Anti-tumour activity and toxicological studies of combination treatment of Orthosiphon stamineus and gemcitabine on pancreatic xenograft model.

BACKGROUNDPancreatic cancer is the most aggressive cancer type. Gemcitabine is the first line chemo-drug used for pancreatic cancer but exerts a broad spectrum of organ toxicities and adverse effects in patients. AIMTo evaluate the anti-tumour activity and toxicological effects of Orthosiphon stamineus extract formulation (ID: C5EOSEW5050ESA trademarked as Nuva-staticTM), and gemcitabine combination on pancreatic xenograft model.METHODSMice were randomly divided into six groups of 6 mice each (n = 6) and given different treatments for 28 d. The study design consisted of a 2 x 3 factorial treatment structure, with gemcitabine (yes/no) by oral (at 1200 and 400 mg/kg per day). Human pancreatic cancer cells were injected subcutaneously into the flanks of athymic nude mice. C5EOSEW5050ESA (200 or 400 mg/kg per day) was administered orally, while gemcitabine (10 mg/kg per 3 d) was given intraperitoneally either alone or in combination treatment. Histopathological analyses of vital organs, tumour tissues, and incidence of lethality were analysed. Analyses of tumour necrosis and proliferation were determined by haematoxylin-eosin staining and immunohistochemistry for Ki-67, respectively. RESULTSNo signs of toxicity or damage to vital organs were observed in all treatment groups compared to the untreated group. C5EOSEW5050ESA at 200 mg/kg and gemcitabine combination had no additive antitumor effects compared to a single treatment. Remarkably, a comparably greater response in a reduction in tumour growth, Ki-67 protein expression, and necrosis was demonstrated by 400 mg/kg of C5EOSEW5050ESA and gemcitabine combination than that of the individual agents. CONCLUSIONThese results highlighted the synergistic activity of C5EOSEW5050ESA with gemcitabine to reduce pancreatic tumour growth in mice compared to a single treatment. Thus, this study provides valuable insights into using C5EOSEW5050ESA as a complementary treatment with gemcitabine for pancreatic cancer.

Effect of Supplementing Ground Leaf of Misai (Orthosiphon stamineus) in Diet on Growth Performance of Broiler Chickens

The use of herbs in animal nutrition is one of the important approaches in overcoming the disadvantages of excessive use of artificial chemicals in animal nutrition. The present study was done to evaluate response of broilers feeding on a diet supplemented with the ground leaf of misai (Orthosiphon stamineus). The birds in this, study were 160 one-day-old male broiler chickens, given ad libitum water and feed for up to 20 days. Treatments were given to 21-day-old male broiler chickens. Data was collected and evaluated after slaughtering 42-day- male broiler chickens. It was shown that supplementing broiler diets with O. stamineus powdered leaf at a rate of 8 g/kg resulted in growth performance comparable to tetracycline and Vitamin E supplementation. It was also shown that supplementing the diet with 8 g/kg O. stamineus had a blood enzyme-lowering effect. In broilers receiving tetracycline supplementation, however, significant serum enzyme activity was observed. Results also showed that 8 g/kg of ground O. stamineus leaf in the diet was equivalent to 200 mg/kg Vitamin E supplementation. Therefore, O. stamineus leaf powder can promote organic, safe, and sustainable broiler chicken production, and as diet supplement.

Solubility of Rosmarinic Acid in Supercritical Carbon Dioxide Extraction from Orthosiphon stamineus Leaves

Rosmarinic acid (RA) is present in a broad variety of plants, including those in the Lamiaceae family, and has a wide range of pharmacological effects, particularly antioxidant activity. To extract RA from Orthosiphon stamineus (OS) leaves, a Lamiaceae plant, a suitable extraction process is necessary. The present study used a green extraction method of supercritical carbon dioxide (SCCO2) extraction with the addition of ethanol as a modifier to objectively measure and correlate the solubility of RA from OS leaves. The solubility of RA in SCCO2 was determined using a dynamic extraction approach, and the solubility data were correlated using three density-based semi-empirical models developed by Chrastil, del Valle-Aguilera, and Gonzalez. Temperatures of 40, 60, and 80 °C and pressures of 10, 20, and 30 MPa were used in the experiments. The maximum RA solubility was found at 80 °C and 10 MPa with 2.004 mg of rosmarinic acid/L solvent. The RA solubility data correlated strongly with the three semi-empirical models with less than 10% AARD. Furthermore, the fastest RA extraction rate of 0.0061 mg/g min−1 was recorded at 80 °C and 10 MPa, and the correlation using the Patricelli model was in strong agreement with experimental results with less than 15% AARD.

Pulsed UV Integrated Infrared Dryer System for Orthosiphon Stamineus Herb.

The drying system is an important part for food and herbs preservation. Therefore, an effective drying system are needed to ensure the increase the shelf life of the food and herb without degrade its good nutrient and bioactive compound. In this study, Orthosiphon stamineus herb was used to dry using an Infrared dryer and combination of Pulsed UV light treatment + Infrared drying. Infrared drying involves transferring heat by radiation from a hot source to a lower-temperature substance that has to be heated or dried, meanwhile the pulsed UV light is a non-thermal treatment. The temperature of the heated element determines the peak wavelength of the radiation. The objective of this study is to design an infrared dryer system and analyze the quality of the dried herb. The Orthosiphon Stamineus have been dried using a 200W Infrared dryer system at 60°C. Total phenolic compounds and antioxidant capacity were determined using the Folin-Ciocalteu method and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay measured using a UV/VIS Spectrophotometer. A moisture analyzer was used to look at the changes in moisture content, and a colorimeter was used to look at the colour changes. The result showed that drying Orthosiphon stamineus by using an Infrared dryer and combination of Pulsed UV light treatment + Infrared drying under 60°C has significantly affected the herbal leaves quality in terms of moisture content, colour properties, Antioxidant capacity, and Total phenolic content.

Preferensi Konsumen terhadap Minuman Fungsional Berbasis Ekstrak Kumis Kucing (Orthosiphon aristatus) sebagai Jamu

The development of Java tea extract-based functional drink as a new concept of “Jamu” drink has been carried out since 2006. However, there was no consumer preference survey, hence this study was conducted. This research aimed to identify the factors that influence consumer preferences on buying “Jamu” specify the intensity of sensory attributes of ideal “Jamu” accordingly in the Bogor area. This would allow pursuing the final target to determine the consumer acceptance towards concentrate Java tea extract-based functional drink as a Jamu. A chi-square analysis was performed to determine the relationship between the respondent’s demographic factors and consumer preferences for Jamu. The demographic factors showed a significant association with consumer preferences for Jamu at a significance level of 5 %. Each demographic factor had a different influence on consumer preferences. The intensity of the sensory attributes of concentrated Java tea extract-based functional drinks was significantly incompatible with an ideal Jamu, according to Wilcoxon test results. This result showed that consumers did not consider the developed concentrate having Jamu criteria sensorically. The intensity of sweetness and astringency that met the consumer preference, orange aroma, and freshness attribute at high intensity, plus the unpleasant sensory characteristics of Jamu which could not be detected in the product might provide the product’s strengths that make the developed product potentially acceptable by market.

Medicinal plants used by the community of Lipulalongo Village, Banggai Laut District, Central Sulawesi, Indonesia

Abstract. Haruna MF, Kenta AM, Herawati. 2022. Medicinal plants used by the community of Lipulalongo Village, Banggai Laut District, Central Sulawesi, Indonesia. Asian J Ethnobiol 5: 62-68. Plants have been traditionally used for medicinal purposes to cure diseases or maintain body wellness and health. However, the utilization of medicinal plants by a particular community is unique and specific to the context of area, thus it might differ from one community to another. This study aimed to document the diversity of plant species used by the community in Lipulalongo Village, Labobo Sub-district, Banggai Laut District, Central Sulawesi, Indonesia, as well as to reveal the method of use of such plants in their medication practices. The research used a cruising survey method, and data collection used interviews, field observation, and identification. The study recorded 21 species of medicinal plants, namely Phaleria macrocarpa Boerl, Andrographis paniculata Burm.f., Orthosiphon stamineus Bent, Coleus scutellarioides (L.) Benth., Averrhoa bilimbi L., Psidium guajava L., Capsicum annum L., Annona squamosa L., Syzygium aromaticum (L.) Merr. & L.M.Perry, Morinda citrifolia L., Gossypium arboreum L., Sesbania grandiflora (L) Pers., Cymbopogon citratus (DC.) Stapf, Curcuma domestica Valeton, Curcuma zedoria (Christm.) Roscoe), Areca catechu L, Alpinia galangal (L.) Willd), Carica papaya L., Theobroma cacao L., Chamaedaphne sp., and Blumea balsamifera (L.) DC. The leaf was the most utilized plant organ rather than fruit, stem, bark, rhizomes, or tubers. Boiling and drinking was the most common method of preparation and mode of application, respectively. Diseases that can be prevented and treated using medicinal plants are fever, diarrhea, colds, coughs, sore teeth, skin diseases (e.g., boils, tinea versicolor, and ringworm), digestive tract diseases in the stomach, and therapy after giving birth.

Protein Profiling of Orthosiphon stamineus Embryogenic Callus using Liquid Chromatography Coupled with Tandem-Mass Spectrometry (LC-MS/MS)

Orthosiphon stamineus is one of the most prominent medicinal herbs widely grown in Southeast Asia. Propagation of O. stamineus using tissue culture technique helps to rapidly produce samples for research purposes, increase the production of secondary metabolites and is considered as one of the strategies in plant genetic improvement. Despite vast information on O. stamineus pharmacological properties, the protein profile of this species is currently underexplored. In the present study, proteins expressed in embryogenic callus developed from O. stamineus leaves in Murashige and Skoog medium supplemented with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were identified via Liquid chromatography- tandem mass spectrometry (LC-MS/MS). The presence of embryogenic callus was confirmed with microscope observation and Evan Blue assay. Using this proteomic approach, we identified peptides that represent 22 proteins localized to different cell compartments and involved in various biological and metabolic functions in plants. This finding is useful to improve our understanding of protein functions, thus their future applications could be further explored. Keywords: Embryogenic, Callus, Herb, Orthosiphon stamineus, Proteins

HPLC Method Optimization for Simultaneous Determination of Quercetin, Luteolin, Sinensetin, and Stigmasterol in Herbal Medicines

Background: Quercetin, luteolin, sinensetin and stigmasterol each is the main marker compound in extracts of Sonchus arvensis, Plantago major, Orthosiphon stamineus, and Strobilanthes crispus, respectively. These extracts show nephrolithiasis activity. For quality control of herbal medicines, a high performance liquid chromatography (HPLC) method has been developed in this study using quercetin, luteolin, sinensetin and stigmasterol as phytochemical markers. Objective: to show optimal conditions of analysis and evaluate the stability of quercetin, luteolin, sinensetin and stigmasterol. Methods: The optimal conditions for analysis were carried out by determining the composition of the mobile phase, the flow rate, and the detector's wavelength. Zorbax Eclipse Plus C18 150 x 4.6 mm, 5 μm was used as the column. The stability test was done by analyzing the standard and samples stored at 4oC for 0, 3, 6 and 24 hours. Results: The best separation of the extract was achieved under isocratic conditions using a mixture of water: methanol: phosphoric acid: acetic acid : acetonitrile (50: 30: 0.05: 0.05: 20 v/v/v/ v/v) as mobile phase with detector wavelength of 352 nm, a mobile phase flow rate of 1 mL/min, and a sample injection volume of 10 μL. Conclusion: In this study, the optimal condition for analysis of quercetin, luteolin, sinensetin and stigmasterol. Quercetin, luteolin, sinensetin and stigmasterol were not stable during 6 hours storage, therefore, standard solutions and samples should be made fresh to maintain the stability.

Diuresis Activity of A Combination of Alang-Alang Rhyme Extract (Imperata cylindrical L.) and Cat's Whistle Leaves (Orthosiphon stamineus L.) in Male Mice (Mus musculus).

Diuretics are substances that can increase the rate of excretion of urine volume and indicate the amount of expenditure (loss) of solutes and water. The Alang-Alang rhizome and the Cat's Whiskers plant are one of the many plants that have many advantages and have been used for a long time in an effort to maintain health. This study aims to determine the combined activity of Alang-alang (Imperata cylindrical L.) Rhizome Extract and Kumis Leaf Extract. Cat (Orthosiphon stamineus L.) as a diuretic in mice (Mus musculus). The test animals were divided into 5 groups, each group consisted of 3 test animals. Group I was given a 1% w/v Na-CMC colloidal solution as a control. Groups II, III and IV were given a suspension of a combination of Alang-Alang rhizome extract and Kumis Kucing Leaf extract with each dose of 100 mg/Kg BW ratio (1 : 1), (1 : 2) and (2 : 1) as the treatment group. The highest to lowest diuretic activity sequentially with the average value being furosemide (3.67), combination (Alang-Alang rhizome: Daun Kumis Kucing) 2 : 1 (2.67), combination (Rimpang Alang-Alang: Daun Kumis Kucing) ) 1 : 2 (1,33), and a combination (Alang-Alang Rhizome: Cat's Whisker Leaf) 1 : 1 (1,33). The largest combination dose that gave an increase in urinary frequency was at a combination dose of 2: 1 (Rhimpang Alang-Alang: Daun Kumis Kucing) with an average value of 2.67.

Suppression of Melanoma Growth in a Murine Tumour Model Using Orthosiphon stamineus Benth. Extract Loaded in Ethanolic Phospholipid Vesicles (Spherosome)

Orthosiphon stamineus Benth (O.S) is a traditional south-east Asian herb. The extract of O.S is used in the formulation of ethanolic nanolipid vesicle system to have considerable potential for tumour therapeutics. The research objective is to develop and characterise the anticancer and antiangiogenic effect of O.S extract in the form of nano-ethanolic spherosomes (ESP) using phospholipids in melanoma. Spherosomes formulation of O.S was developed using the thin-film re-hydration method and converted to gel using Acrypol 1%. The formulations were subjected to optimisation and physical-chemical characterisations like particle size, surface charge, DSC, FTIR, and TEM. Cytotoxicity of O.S and ESP was studied using an endothelial cell line (EA. hy926). Furthermore, anti-melanoma effect of O.S spherosome gel was studied in albino mice after topical administration. ESP-6 with the ratio of extract (O.S): cholesterol: phospholipid (1: 6: 0.5) showed the highest entrapment efficiency (80.56 ± 0.84%) using ultraviolet spectroscopy. In-vivo permeation/penetration studies revealed deeper absorption of ESP-6 compared to a hydroethanolic gel of O.S. In-vitro and in vivo anti-melanoma studies demonstrated the significant tumour-suppressing effect of ESP-6 on murine melanoma. Percentage inhibition of tumour growth by O.S and ESP-6 at 3000 mg/kg showed to be 63.98 ± 7.86% and 87.76 ± 7.90%, respectively. Spherosome vesicles were developed with a smooth surface. The results demonstrated that O.S extract showed no toxicity when tested on the endothelial cell line. O.S loaded in spherosomes has the potential to lower the growth of melanoma in mice. The spherosomes loaded with O.S do not promote tumour growth or act as antiangiogenetic in melanoma.