Ursolic Acids Research Articles

New hybrids between triterpenoid acids and nucleoside HIV-RT inhibitors

One-pot synthesis of ester-linked conjugates of betulinic, ursolic or oleanolic acids with anti-HIV drugs AZT, derivatives of AZT and 3TC was performed, the resulting conjugates having shown high anti-HIV activities.

Pharmacogenomic Characterization of Cytotoxic Compounds from Salvia officinalis in Cancer Cells.

Salvia officinalis is used as a dietary supplement with diverse medicinal activity (e.g. antidiabetic and antiatherosclerotic effects). The plant also exerts profound cytotoxicity toward cancer cells. Here, we investigated possible modes of action to explain its activity toward drug-resistant tumor cells. Log10IC50 values of two constituents of S. officinalis (ursolic acid, pomolic acid) were correlated to the expression of ATP-binding cassette (ABC) transporters (P-glycoprotein/ABCB1/MDR1, MRP1/ABCC1, BCRP/ABCG2) and epidermal growth factor receptor (EGFR) or mutations in RAS oncogenes and the tumor suppressor gene TP53 of the NCI panel of cell lines. Gene expression profiles predicting sensitivity and resistance of tumor cells to these compounds were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Furthermore, the binding of both plant acids to key molecules of the NF-κB pathway (NF-κB, I-κB, NEMO) was analyzed by molecular docking. Neither expression nor mutation of ABC transporters, oncogenes, or tumor suppressor genes correlated with log10IC50 values for ursolic acid or pomolic acid. In microarray analyses, many genes involved in signal transduction processes correlated with cellular responsiveness to these compounds. Molecular docking indicated that the two plant acids strongly bound to target proteins of the NF-κB pathway with even lower free binding energies than the known NF-κB inhibitor MG-132. They interacted more strongly with DNA-bound NF-κB than free NF-κB, pointing to inhibition of DNA binding by these compounds. In conclusion, the lack of cross-resistance to classical drug resistance mechanisms (ABC-transporters, oncogenes, tumor suppressors) may indicate a promising role of the both plant acids for cancer chemotherapy. Genes involved in signal transduction may contribute to the sensitivity or resistance of tumor cells to ursolic and pomolic acids. Ursolic and pomolic acid may target different steps of the NF-κB pathway to inhibit NF-κB-mediated functions.

Simultaneous Determination of Oleanolic and Ursolic Acids in Rat Plasma by HPLC-MS: Application to a Pharmacokinetic Study After Oral Administration of Different Combinations of QingGanSanJie Decoction Extracts.

A liquid chromatography-mass spectrometry method has been developed and validated for rapid simultaneous determination of the oleanolic and ursolic acid contents in rat plasma with betulinic acid as the internal standard (IS). The plasma samples were prepared by a liquid-liquid extraction procedure. Chromatographic separation was performed with a Chromasil-C18 column (250 mm × 4.6 mm, i.d. 5 μm) with methanol-water as mobile phase at 1 mL/min. The detection was accomplished under selected-ion-monitoring mode with a negative electrospray ionization interface. Linear calibration curves were obtained between the range of 0.86-421.2 and 0.94-462.0 ng/mL for oleanolic and ursolic acids, with lower limits of quantification at 0.43 and 0.47 ng/mL, respectively. The extraction recovery exceeded 70% in plasma. The intra- and interday precision values were <9.8% with the accuracy as -7.0 to 9.9% at three different QC levels in both cases. The pharmacokinetic behaviors of oral dosage of QingGanSanJie decoctions were then studied in rats following the developed approach. The t1/2 values of the oleanolic and ursolic acids after oral administration of the monarch medicine extract were significantly different (P < 0.05) from other prescription extracts containing different herb pieces with different compatibilities. Bimodal phenomena appeared in every concentration-time curve for the oleanolic and ursolic acids at 3-8 h after administration. The minister, assistant and guide medicines in the formula could prolong the metabolism of the oleanolic and ursolic acids in vivo, providing an experimental basis for the slow onset and long action of the Traditional Chinese Medicine compound.

Selective and cost-effective protocol to separate bioactive triterpene acids from plant matrices using alkalinized ethanol: Application to leaves of Myrtaceae species.

Background:Triterpenes as betulinic (BA), oleanolic (OA) and ursolic acids (UA) have increasingly gained therapeutic relevance due to their wide scope of pharmacological activities. To fit large-scale demands, exploitable sources of these compounds have to be found and simple, cost-effective methods to extract them developed. Leaf material represents the best plant sustainable raw material. To obtain triterpene acid-rich extracts from leaves of Eugenia, Psidium and Syzygium species (Myrtaceae) by directly treating the dry plant material with alkalinized hydrated ethanol. This procedure was adapted from earlier methods to effect depolymerization of the leaf cutin.Materials and Methods:Extracts were prepared by shaking the milled dry leaves in freshly prepared 2% NaOH in 95% EtOH solution (1:4 w/v) at room temperature for 6 h. Working up the product in acidic aqueous medium led to clear precipitates in which BA, OA and UA were quantified by gas chromatography.Results:Pigment-free and low-polyphenol content extracts (1.2–2.8%) containing 6–50% of total triterpene acids were obtained for the six species assayed. UA (7–20%) predominated in most extracts, but BA preponderated in Eugenia florida (39%). Carried out in parallel, n-hexane defatted leaves led to up to 9% enhancement of total acids in the extracts. The hydroalcoholate treatment of Myrtaceae species dry leaves proved to be a cost-effective and environmentally friendly method to obtain triterpene acids, providing them be resistant to alkaline medium. These combined techniques might be applicable to other plant species and tissues.

Antimicrobial activity of oleanolic and ursolic acids: an update.

Triterpenoids are the most representative group of phytochemicals, as they comprise more than 20,000 recognized molecules. These compounds are biosynthesized in plants via squalene cyclization, a C30 hydrocarbon that is considered to be the precursor of all steroids. Due to their low hydrophilicity, triterpenes were considered to be inactive for a long period of time; however, evidence regarding their wide range of pharmacological activities is emerging, and elegant studies have highlighted these activities. Several triterpenic skeletons have been described, including some that have presented with pentacyclic features, such as oleanolic and ursolic acids. These compounds have displayed incontestable biological activity, such as antibacterial, antiviral, and antiprotozoal effects, which were not included in a single review until now. Thus, the present review investigates the potential use of these triterpenes against human pathogens, including their mechanisms of action, via in vivo studies, and the future perspectives about the use of compounds for human or even animal health are also discussed.

Voltammetric procedure for the determination of oleanolic and ursolic acids in plant extracts

In this work, a novel method based on square wave voltammetric determination of pentacyclic triterpenes (oleanolic and ursolic acids, OA and UA) at an unmodified glassy carbon electrode in plant material after chromatographic separation was developed.

Determination of Oleanolic and Ursolic Acids in Hedyotis diffusa Using Hyphenated Ultrasound-Assisted Supercritical Carbon Dioxide Extraction and Chromatography.

Oleanolic acid (OA) and ursolic acid (UA) were extracted from Hedyotis diffusa using a hyphenated procedure of ultrasound-assisted and supercritical carbon dioxide (HSC–CO2) extraction at different temperatures, pressures, cosolvent percentages, and SC–CO2 flow rates. The results indicated that these parameters significantly affected the extraction yield. The maximal yields of OA (0.917 mg/g of dry plant) and UA (3.540 mg/g of dry plant) were obtained at a dynamic extraction time of 110 min, a static extraction time of 15 min, 28.2 MPa, and 56°C with a 12.5% (v/v) cosolvent (ethanol/water = 82/18, v/v) and SC–CO2 flowing at 2.3 mL/min (STP). The extracted yields were then analyzed by high performance liquid chromatography (HPLC) to quantify the OA and UA. The present findings revealed that H. diffusa is a potential source of OA and UA. In addition, using the hyphenated procedure for extraction is a promising and alternative process for recovering OA and UA from H. diffusa at high concentrations.

Sustainable Utilization of TCM Resources.

Sustainable development aims to maintain utilization of finite resources without exhausting them and meet the demand of present and future generations [1]. Sustainability can only be achieved by balancing production and consumption in a reasonable dimension. It offers a vision of progress that integrates immediate and longer-term objectives. However, the nature of our current resource use endangers their future survival. Moreover, the consequences of our resource use in terms of impacts on the environment may induce serious damage to the environment. Sustainable utilization of natural resources should combine market demand of raw materials, ecological stability, and social benefits [2] and regard them as inseparable and interdependent components in the whole progress of industrial production. Traditional Chinese Medicine (TCM) has a long history of application and has attracted more and more world-wide attention for its significant effect in prevention and treatment of diseases. Wild medicinal resource is the basis of TCM and accounts for nearly 80% of all the sources of Chinese herbs. With the development of the modern TCM industry and gradual degradation of the natural environment, natural reserves of medicinal resources face a tremendous amount of pressure. Many medicinal plants are at risk of extinction and some adulterants have appeared on the market bringing to attention the need to ensure sustainable utilization of medicinal resources. More and more measures have been taken to guarantee the supply [3] and identification [4–6] as well as quality control [7, 8] and new drug development [9]. In this special issue, a total of 15 papers have been devoted to sustainable utilization of TCM resources. Identification of Radix Astragali using DNA barcoding, DNA barcode-based PCR-RFLP, and diagnostic PCR for authentication of Jinqian Baihua She (Bungarus Parvus), using ITS2 to identify Ferula species and indirubin-containing medicinal plants, are included. Rapid identification of plants in the Asteraceae with improved RBF-ANN classification models based on MOS-sensor E-nose is also provided. Introduction and cultivation can supply raw medicinal materials in short supply and play an important role in the sustainable utilization of TCM. Prevention and cure of diseases and insect pests and characteristics of soil microbial community in ginseng cultivation are offered. A systematic review on the production mode of Chinese herbal medicines is also presented. This special issue also reports natural resource monitoring of Rheum tanguticum by multilevel remote sensing. Traceability based on chemical two-dimensional barcode for controlling quality in the whole process of the industrial chain of TCM is included. Finally, measuring Agarwood formation ratio quantitatively by fluorescence spectral imaging technique and determining oleanolic and ursolic acids using hyphenated ultrasound-assisted supercritical carbon dioxide extraction and chromatography are introduced. Readers of this special issue will find not only different identification methods and updated reviews on cultivation, but also some important issues influencing quality control. The industrial chain of TCM, from the production of raw materials to the sale of the finished products, is a complicated multilink process. There are still some key questions to be resolved in the whole process including but not limited to breeding, prevention and cure of diseases and insect pests, quality traceability, exploiting new medicinal resources, and the mechanism of drug metabolism. Shilin Chen Yitao Wang Zhongzhen Zhao Christine J. Leon Robert J. Henry

Investigations on some metabolites of Tecoma stans Juss. callus tissue. Part III. Chromatographical search for iridoids, phenolic acids, terpenoids and sugars

Tissus cultures of &lt;i&gt;Tecoma stans&lt;/i&gt; Juss. cultivated on modified Murashige-Skoog medium (RT-k) were phytochemically analysed by means of chromatographical methods (PC, TLC). The following products were found as metabolites: phenolic acids - chlorogenics, caffeic, ferulic, vanillic, o-coumaric and sinapic; steroids - β-sitosterol; triterpenes - ursolic and oleanolic acids, α-amyrine; sugars - glucose, fructose, sucrose, xylose. Meso-inositol was isolated in 0.8% yield. In intact plant leaves, some differences concerning the content and/or number of individual compounds were observed, namely: lack of sinapic acid and occurrence of p-coumaric acid, lower content of β-sitosterol, lack of oleanolic acid, occurrence of β-amyrine and of one unidentified triterpenoid, lack of xylose, occurrence of maltose, raffinose, and stachiose. The level of mesoinositol inn leaves was distincly lower than in the callus tissues. Neither in callus tissues nor in leaves iridoid glycosides were found.

Synthesis of Substituted Esters of Ursolic, Betulonic, and Betulinic Acids Containing the Nitroxyl Radical 4-Amino-2,2,6,6-Tetramethylpiperidine-1-Oxyl

Synthesis of Substituted Esters of Ursolic, Betulonic, and Betulinic Acids Containing the Nitroxyl Radical 4-Amino-2,2,6,6-Tetramethylpiperidine-1-Oxyl

Supercritical fluid extraction of heather (Calluna vulgaris) and evaluation of anti-hepatitis C virus activity of the extracts

Previous studies using lipid extracts of heather (Calluna vulgaris) leaves showed the presence of high concentrations of ursolic and oleanolic acid. These two compounds have been reported to present antiviral activity against hepatitis C virus (HCV). In this work, the supercritical fluid extraction of heather was studied with the aim of assessing a potential anti-HCV activity of the extracts owing to their triterpenic acid content. Supercritical extraction assays were carried out exploring the pressure range of 20–50MPa, temperatures of 40–70°C and 0–15% of ethanol cosolvent. The content of oleanolic and ursolic acid in the extracts were determined, and different samples were screened for cellular cytotoxicity and virus inhibition using a HCV cell culture infection system. Antiviral activity was observed in most extracts. In general, superior anti-HCV activity was observed for higher contents of oleanolic and ursolic acids in the extracts.

Aortic relaxant activity of Crataegus gracilior Phipps and identification of some of its chemical constituents.

This study focused on the assessment of the vasorelaxant activity of the organic and aqueous extracts obtained from leaves and fruits of a Mexican hawthorn (Crataegus gracilior) on isolated rat aorta, and on the purification and identification of some of their secondary metabolites by the use of chromatographic and spectroscopic techniques. The results obtained showed that the methanol extract has a significantly more potent and effective vasorelaxant effect than the other tested extracts, with an EC50 = 8.69 ± 4.34 µg/mL and an Emax = 94.6% ± 11.30%, values that are close to that of acetylcholine, the positive control. From the same extract, two major triterpenes were isolated and identified as ursolic and corosolic acids by comparison of their experimental NMR spectroscopic data with those reported in the literature. Chlorogenic acid, rutin, quercetin, kaempferol and (+)-catechin were also identified using HPLC coupled with PDAD. All these compounds have already been proven to possess on their own antihypertensive effect and other benefits on cardiovascular diseases and they can support, at least in part, the traditional use of this plant species.

Rapid, Selective, and Sensitive Analysis of Triterpenic Acids in Hippophae rhamnoides L. Using HPLC with Pre-Column Fluorescent Derivatization and Identification with Post-Column APCI-MS

Hippophae rhamnoides L., a fruit which is rich in natural chemical ingredients, has become an important raw material of health food, medicine, and cosmetics. In the present work, a method using 2-(5-benzoacridine)ethyl-p-toluenesulfonate as pre-column derivatization reagent with high sensitivity, selectivity, and short separation time was developed to analyze triterpenic acids (TTAs) in H. rhamnoides L. by HPLC with fluorescence detection and simultaneously confirmed by post-column atmospheric pressure chemical ionization-mass spectrometry. The method was validated by linearity, limits of detection (LODs) and lower limits of quantification (LLOQs), precision, and accuracy. Good linear correlations were observed for all TTAs with correlation coefficients greater than 0.9996. LODs and LLOQs were in the range of 1.71–2.14 and 7.45–8.23 ng mL−1, respectively. The method was successfully employed to analyze TTAs in H. rhamnoides L. from various origins in the Qinghai–Tibetan plateau with trace amount of samples within 15 min. A systemic mapping of the TTAs in H. rhamnoides L. of different origins was established. The results indicated that H. rhamnoides L. is rich in TTAs, especially in oleanolic and ursolic acids, and the content of TTAs varies across origins. This work will support the research on pharmaceutical applications of H. rhamnoides L. and provide information for quality control.

Role of triterpenic acids on thrombin regulation and PAR1 signaling

The premalignant role of Thrombin (Thr) in tumor adhesion, growth, metastasis, and angiogensis is well recognized [1], being correlated with overexpression and activation of protease activated receptor (PAR-1) in primary breast tissues [2] and breast cancer (BC) cell lines [3 – 4]. Anti-coagulant therapies have been tried for cancer patients, however with several concerns regarding multiple drug interactions and risk of thrombocytopenia. Triterpenic acids (TAs), in particularly, the lupane, oleanane and ursane-type structures, have been associated to a broad spectrum of pharmacological activities including anti-angiogenic, as well as inhibitors of human platelets aggregation, sparking renewed interest with regard to their potential in cancer treatment, as well as new anti-coagulant therapies. The present work aims to determine anti-Thr activity of four TAs (ursolic, betulinic, betulonic and oleanolic (OA)), in comparison to hirudin (a natural Thr inhibitor). The amidolytic Thr activity (using a chromogenic bioassay), as well as a Thr-induced fibrinogen polymerization assay were performed in the presence of different TAs concentrations. Furthermore, the role of pure TAs upon PAR-1 protein expression, using a human BC cell model (MDA-MB-231), was also assessed by western blot. According to the results, the four TAs present the capacity to inhibit Thr activity, being ursolic and betulinic acids, the most potent ones. Regarding the effect of TAs upon PAR-1 protein expression, BC cells MDA-MB-231 were incubated for 48h with IC50 values, previously determined for each TAs. The results demonstrated the capacity of certain TAs to donwregulated PAR-1 protein expression, along with a significant decrease on cellular viability. The results bring new insights on dual effect of TAs as modulators of Thr activation as well as inhibitors of PAR1 signaling, underlying their therapeutic potential as new anti-coagulant compounds with anti-tumor properties. Keywords: Triterpenic acids, thrombin, protease activated receptor (PAR-1), breast cancer

Sesqui- and triterpenoids from the liverwort Lepidozia chordulifera inhibitors of bacterial biofilm and elastase activity of human pathogenic bacteria

Five dammarane-type triterpenoids, five pentacyclic triterpenoids (three of them carrying a carboxylic acid group), and two aromadendrane-type sesquiterpenoids were isolated from an Argentinian collection of the liverwort Lepidozia chordulifera. Compounds were characterized by comparison of their spectral data with those previously reported and tested in their ability to control bacterial growth, biofilm formation, bacterial Quorum Sensing process (QS), and elastase activity of Pseudomonas aeruginosa, as well as bacterial growth and biofilm formation of Staphylococcus aureus. The key role played by biofilm and elastase activity in bacterial virulence make them a potential target for the development of antibacterial agents. The aromadendrane-type sesquiterpenoid viridiflorol was the most potent biofilm formation inhibitor, producing 60% inhibition in P. aeruginosa and 40% in S. aureus at 50µg/ml. Ursolic and betulinic acids (two of the pentacyclic triterpenoids isolated) were able to reduce 96 and 92% the elastase activity of P. aeruginosa at 50µg/ml, respectively. Among the analyzed triterpenoids, those that carry a dammarane skeleton were the most potent inhibitors of the P. aeruginosa biofilm formation and were active against both P. aeruginosa and S. aureus. Subsequently, a computer-assisted study of the triterpenoid compounds was carried out for a better understanding of the structure-activity relationships.

&lt;i&gt;In ovo&lt;/i&gt; antiviral potency of the leaf constituents of Tanzanian &lt;i&gt;Toussaintia&lt;/i&gt; species against Infectious Bursal Disease Virus and Newcastle Disease Virus

The chemical constituents of Toussantia orientalis and T. patriciae (Annonaceae) leaf extracts were evaluated for their antiviral activities in ovo against Infectious Bursal Disease Virus (IBDV) and Newcastle Disease Virus (NDV). The nine-day-old embryonated eggs in a set of five were used for testing through the allantoic route inoculation assay for each tested compound and controls. For NDV assay, the allantoic fluids from the specimens were further harvested to determine viral contagion. The tested compounds exhibited potency with varying levels of significance at a screening concentration of 360 µg/ml against the two viral strains. Embryos infected with IBDV survived, grew to normal size with complete organ formation and had mean weights comparable to those of the uninfected ones when treated with the aminocinnamoyl tetraketides 1, 2, and 4, glucosylflavonoid 7 from T. orientalis and ursolic acid derivatives 9 and 10 from T. patriciae demonstrating high efficacy against IBDV. The compounds also exhibited antiviral activity against NDV, showing viral titre reduction ranging from 1:16 - 1:256 in the haemagglutination test, with compound 11 having the lowest titre value (1:16) followed by compound 7 (1:32). The compounds that exhibited significant antiviral efficacy could be considered potential leads for the development of antiviral agents. Keywords : Aminocinnamoyl tetraketides, ursolic acids, Toussaintia orientalis, Toussaintia patriciae, Annonaceae.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

Triterpenes as uncompetitive inhibitors of α-glucosidase from flowers of Punica granatum L.

The α-glucosidase and maltase inhibitory effects of Punica granatum L. flowers (PGF) were investigated. The methanol extract (PGFMe), n-hexane extract (PGFH), chloroform extract (PGFC) and the remaining water fraction (PGFW) were assayed for their α-glucosidase and maltase inhibitory effects. PGFW showed potent α-glucosidase inhibition with IC50 of 0.8 μg/mL followed by PGFMe (IC50 of 4.0 μg/mL) then PGFC (IC50 of 5.21 μg/mL) in comparison to acarbose (0.9 μM). Due to its selectivity towards α-glucosidase, PGFC was subjected to bioactivity-guided isolation of its main active constituents. Five known compounds (1–5) were identified as β-sitosterol (1), oleanolic acid (2), ursolic acid (3), p-coumaric acid (4) and apigenin (5). Ursolic and oleanolic acids showed potent α-glucosidase inhibition (IC50 of 39.0 and 35.0 μM, respectively), while they did not show significant maltase inhibition. Kinetic study using the double Lineweaver–Burk plot revealed that ursolic acid uncompetitively inhibited α-glucosidase in comparison with acarbose as a competitive inhibitor.

Anti-Trichomonas vaginalis activity from triterpenoid derivatives.

Trichomonas vaginalis is a flagellated parasite that causes trichomonosis, the most common non-viral sexually transmitted disease (STD) in the world. Worryingly, trichomonosis is associated to increased transmission of HIV. Due to high frequency of the infection during pregnancy and the development of metronidazole-resistant isolates, therapeutic alternatives to 5-nitroimidazole are being searched. Triterpenes are natural products presenting several biological activities such as anti-protozoal activity. The aim of this study was to evaluate the in vitro anti-T. vaginalis activity from betulinic and ursolic acids, as well as semisynthetic derivatives obtained. Compounds obtained from betulinic acid presented better activity than those from ursolic acid. Piperazine derivatived from betulinic acid presented minimum inhibitory concentration (MIC) value of 91.2 μM, and the kinetic growth curve performed with parasites treated with this most active compound revealed complete inhibition of trophozoite proliferation at 2 h of incubation and total abolition of trophozoite growth in 24 h, revealing that the piperazine derivative is an efficient trichomonacidal molecule. The same compound promoted total erythrocyte lysis and lactate dehydrogenase (LDH) liberation of 83 and 100% (at 45.6 and 91.2 μM, respectively), indicating parasite membrane damage. The piperazine derivative demonstrated cytotoxic effect against the HMVII and HeLa cell lineages at the MIC value. This is the first report of semisynthetic triterpenoid derivatives with anti-T. vaginalis activity, revealing the high potential of these compounds as trichomonacidal agents.

Extraction characteristics and kinetic studies of oleanolic and ursolic acids from Hedyotis diffusa under ultrasound-assisted extraction conditions

We report a hyphenated procedure for the extraction of oleanolic acid (OA) and ursolic acid (UA) from Hedyotis diffusa. The hyphenated ultrasound-assisted extraction (HUAE) procedure consists of heat-reflux extraction (HRE) followed by ultrasound-assisted extraction (UAE). This hyphenated technique uses a lower temperature, consumes less solvent and is faster than previous methods. Compared to heat-reflux and traditional ultrasound-assisted extractions, HUAE gave a 12% higher yield of OA and UA from H. diffusa and required less severe operating conditions. Thus, mild operating conditions can be used, which results in a higher quality product. HUAE is an effective and selective method for the extraction of OA and UA. Additionally, the HUAE of OA and UA from H. diffusa were interpreted using a second-order kinetic model to predict the extraction parameters at various temperatures. Furthermore, the activation energies for the extractions of OA and UA were 9.073 and 10.575kJ/mol, respectively, based on the second-order extraction rate constants used to build the models. By fitting the main parameters of the model, we could compare the effects of the extraction parameters on the H. diffusa material and better understand the extraction process.