Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and ß-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7-9 expression and downregulation of Bcl-2 and full-length caspase-7-9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis.

Chemical-Genetic Interactions of Bacopa monnieri Constituents in Cells Deficient for the DNA Repair Endonuclease RAD1 Appear Linked to Vacuolar Disruption.

Colorectal cancer is a common cancer worldwide and reduced expression of the DNA repair endonuclease XPF (xeroderma pigmentosum complementation group F) is associated with colorectal cancer. Bacopa monnieri extracts were previously found to exhibit chemical-genetic synthetic lethal effects in a Saccharomyces cerevisiae model of colorectal cancer lacking Rad1p, a structural and functional homologue of human XPF. However, the mechanisms for B. monnieri extracts to limit proliferation and promote an apoptosis-like event in RAD1 deleted yeast was not elucidated. Our current analysis has revealed that B. monnieri extracts have the capacity to promote mutations in rad1∆ cells. In addition, the effects of B. monnieri extracts on rad1∆ yeast is linked to disruption of the vacuole, similar to the mammalian lysosome. The absence of RAD1 in yeast sensitizes cells to the effects of vacuole disruption and the release of proteases. The combined effect of increased DNA mutations and release of vacuolar contents appears to induce an apoptosis-like event that is dependent on the meta-caspase Yca1p. The toxicity of B. monnieri extracts is linked to sterol content, suggesting saponins may be involved in limiting the proliferation of yeast cells. Analysis of major constituents from B. monnieri identified a chemical-genetic interaction between bacopasaponin C and rad1∆ yeast. Bacopasaponin C may have potential as a drug candidate or serve as a model for the development of analogs for the treatment of colorectal cancer.

DNA barcoding of species of Bacopa coupled with high-resolution melting analysis.

In Thailand, there are three species of Bacopa, namely, B. monnieri, B. caroliniana, and B. floribunda. Among these species of Bacopa, B. monnieri is the only medicinal species, used for the treatment of cognitive impairment and improvement of cognitive abilities because of its bioactive constituents, bacoside A and B. However, because of the similar characteristics of these species, it is difficult to differentiate among related species, resulting in confusion during identification. For this reason, and to ensure therapeutic quality for consumers, authentication is important. In this study, the three abovementioned species of Bacopa were evaluated using barcoding coupled with high-resolution melting (Bar-HRM) analysis based on primers designed for the trnL-F sequences of the three species. The melting profiles of the trnL-F amplicons of B. caroliniana and B. floribunda were clearly different from the melting profile of the trnL-F amplicon from B. monnieri; thus, the species could be discriminated by Bar-HRM analysis. Bar-HRM was then used to authenticate commercial products in various forms. The melting curves of the six commercial samples indicated that all the tested products contained genuine B. monnieri species. This method provides an efficient and reliable authentication system for future commercial herbal products and offers a reference system for quality control.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Bioassay-Guided Isolation of Two Flavonoids from Derris scandens with Topoisomerase II Poison Activity.

Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4'-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment.

Cytotoxic stilbenes from the roots of Paphiopedilum godefroyae.

Two new stilbenes, 2-(3',5'-dimethoxyphenyl)-6-hydroxy-5-methoxybenzofuran (1) and 3'-hydroxy-2,5'-dimethoxystilbene (2), together with seven known stilbenes (3, 5-10) and one flavanone (4), were isolated from the roots of Paphiopedilum godefroyae. Their chemical structures were determined on the basis of their spectroscopic data. These isolated compounds were evaluated for their cytotoxicity against human small cell lung cancer (NCI-H187) cell lines and an arylbenzofuran derivative, 5,6-dimethoxy-2-(3-hydroxy-5-methoxyphenyl)benzofuran (6), was shown to be strongly cytotoxic with an IC50 value of 5.10 µM.

Anticancer activity of selected Colocasia gigantia fractions.

The objective of this study is to investigate the anticancer potential of the extract of Colocasia gigantea C. gigantea), a plant member of the Araceae family. In the present study, we investigated the cytotoxic activity of C. gigantea extract on cervical cancer (Hela) and human white blood cells (WBC) in vitro. The authors then identified the bioactive ingredients that demonstrated cytotoxicity on tested cells and evaluated those bioactive ingredients using the bioassay-guided fractionation method. The results showed that not all parts of C. gigantea promote cytotoxic activity. The dichloromethane leaf fraction showed significant cell proliferation effect on Hela cells, but not on WBCs. Only the n-hexane tuber fraction (Fr. 1T) exhibited significant cytotoxicity on Hela cells (IC50 = 585 µg/ml) and encouraged WBC cell proliferation. From GC-Mass spectrometry, 4,22-Stigmastadiene-3-one, Diazoprogesterone, 9-Octadecenoic acid (Z)-, hexyl ester and Oleic Acid were the components of Fr 1T that demonstrated cytotoxic potential. In conclusion, C. gigantea's Fr 1T shows potential for cervical cancer treatment.

Characterization and phylogenetic analysis of the complete chloroplast genome of Curcuma comosa and C. latifolia.

Members of the Curcuma genus, a crop in the Zingiberaceae, are widely utilized rhizomatous herbs globally. There are two distinct species, C. comosa Roxb. and C. latifolia Roscoe, referred to the same vernacular name "Wan Chak Motluk" in Thai. C. comosa holds economic importance and is extensively used as a Thai traditional medicine due to its phytoestrogenic properties. However, its morphology closely resembles that of C. latifolia, which contains zederone, a compound known for its hepatotoxic effects. They are often confused, which may affect the quality, efficacy and safety of the derived herbal materials. Thus, DNA markers were developed for discriminating C. comosa from C. latifolia. This study focused on analyzing core DNA barcode regions, including rbcL, matK, psbA-trnH spacer and ITS2, of the authentic C. comosa and C. latifolia species. As a result, no variable nucleotides in core DNA barcode regions were observed. The complete chloroplast (cp) genome was introduced to differentiate between the two species. The comparison revealed that the cp genomes of C. comosa and C. latifolia were 162,272 and 162,289 bp, respectively, with a total of 133 identified genes. The phylogenetic analysis revealed that C. comosa and C. latifolia exhibited a very close relationship with other Curcuma species. The cp genome of C. comosa and C. latifolia were identified for the first time, providing valuable insights for species identification and evolutionary research within the Zingiberaceae family.

A new benzoquinone and a new stilbenoid from Paphiopedilum exul (Ridl.) Rolfe.

One new alkyl benzoquinone, paphionone (1), one new trans-stilbenoid, (E)-6,5'-dihydroxy-2,3'-dimethoxystilbene (2), and eight known stilbenoids and flavonoids (3-10) were isolated from the leaves and roots of Paphiopedilum exul (Orchidaceae). Their chemical structures were determined based on IR, ECD, MS and NMR analyses. Cytotoxicity of all isolated compounds towards human hepatocellular carcinoma (HepG2) cell line was examined in vitro by MTT assay. The para-hydroxybenzyl substituted stilbene 10 was potently cytotoxic to the cancer cells, with an IC50 value of 4.80 ± 1.10 µM (selectivity index = 20.83). All compounds were non-toxic to normal human embryo fibroblast (OUMS-36) cell line.

A comprehensive review on Saraca asoca (Fabaceae) - Historical perspective, traditional uses, biological activities, and conservation.

ETHNOPHARMACOLOGICAL RELEVANCE: Saraca asoca (Roxb.)W.J.de Wilde, (Fabaceae) is a plant of significant medicinal value in traditional Indian medicine, with a long history of use in the treatment of gynaecological disorders and other ailments, and is held in high esteem. This plant has long existed in Indian tradition and is revered as sacred. AIM OF THE STUDY: This work aimed to explore the taxonomic revision of Saraca asoca from ancient times to the present and to evaluate the ethnobotanical, phytochemical and pharmacological information associated with traditional use and develop a roadmap for conservative strategies of species. MATERIALS AND METHODS: The study draws on a comprehensive range of herbal, traditional, ethnobotanical, and ethnopharmacological information, including ancient Ayurvedic textbooks and various databases, using a single keyword or a combination of multiple keywords. RESULTS: This review establishes a roadmap for understanding the traditional history of medicinal plants, particularly Saraca, and highlights the transfer of traditional knowledge from pharmacopoeias, materia medica, and classical textbooks over many centuries. The study also emphasises the importance of conservation strategies to protect Saraca as a valuable resource for healthcare and suggests that more research is needed to systematically evaluate its phytochemical, pharmacological, and clinical properties, as well as to develop safety, pharmacology, and toxicology reports for traditional formulations. CONCLUSIONS: In light of this study, S. asoca could be considered an important source of potential herbal drugs. The review concludes with a call for further research and conservation efforts to protect Saraca and other traditional medicinal plants for the benefit of current and future generations.

Antimicrobial Activity against Foodborne Pathogens and Antioxidant Activity of Plant Leaves Traditionally Used as Food Packaging.

In accordance with Thai wisdom, indigenous plant leaves have been used as food packaging to preserve freshness. Many studies have demonstrated that both antioxidant and antimicrobial activities contribute to protecting food from spoilage. Hence, the ethanolic extracts of leaves from selected plants traditionally used as food packaging, including Nelumbo nucifera (1), Cocos nucifera (2), Nypa fruticans (3), Nepenthes mirabilis (4), Dendrocalamus asper (5), Cephalostachyum pergracile (6), Musa balbisiana (7), and Piper sarmentosum (8), were investigated to determine whether they have antioxidant and antimicrobial activities against spoilage microorganisms and foodborne pathogens that might be beneficial for food quality. Extracts 1-4 exhibited high phenolic content at 82.18-115.15 mg GAE/g and high antioxidant capacity on DPPH, FRAP and SRSA assay at 14.71-34.28 µg/mL, 342.92-551.38 µmol Fe2+/g, and 11.19-38.97 µg/mL, respectively, while leaf extracts 5-8 showed lower phenolic content at 34.43-50.08 mg GAE/g and lower antioxidant capacity on DPPH, FRAP, and SRSA at 46.70-142.16 µg/mL, 54.57-191.78 µmol Fe2+/g, and 69.05->120 µg/mL, respectively. Extracts 1-4 possessed antimicrobial activities against food-relevant bacteria, including Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, and Escherichia coli. Only N. mirabilis extract (4) showed antimicrobial activities against Salmonella enterica subsp. enterica serovar Abony and Candida albicans. Extracts 5-8 showed slight antimicrobial activities against B. cereus and E. coli. As the growth and activity of microorganisms are the main cause of food spoilage, N. fruticans (3) was selected for bioassay-guided isolation to obtain 3-O-caffeoyl shikimic acid (I), isoorientin (II) and isovitexin (III), which are responsible for its antimicrobial activity against foodborne pathogens. N. fruticans was identified as a new source of natural antimicrobial compounds I-III, among which 3-O-caffeoyl shikimic acid was proven to show antimicrobial activity for the first time. These findings support the use of leaves for wrapping food and protecting food against oxidation and foodborne pathogens through their antioxidant and antimicrobial activities, respectively. Thus, leaves could be used as a natural packaging material and natural preservative.

HPLC and DNA barcoding profiles for identification of the selected twelve Mucuna species and its application for detecting prohibited aphrodisiac Mucuna products.

Aphrodisiac herbal products originated from various plants including Mucuna species. In Thai folklore, Mucuna macrocarpa Wall. and M. pruriens (L.) DC. have long been consumed and utilized for their aphrodisiac properties. Consumption of these plants can lead to serious adverse effects caused by l-dopa. The plants have been legally banned for use as foods, dietary supplements, or nutraceuticals by the FDA of several countries. To protect consumers, methods for the identification of illicit plants or herbal products are needed. This study aimed to identify the selected twelve Mucuna species and examine the aphrodisiac herbal products containing M. macrocarpa and M. pruriens by using HPLC analysis of l-dopa coupled with DNA barcoding profiles of ITS, matK, rbcL, and trnH-psbA. The results showed that l-dopa could be found not only in the seeds of M. macrocarpa and M. pruriens but also in associated allied Mucuna species. Then, a DNA barcode was introduced to support in HPLC profiling to identify the plants. DNA barcodes of twelve Mucuna species found in Thailand were established and used to reconstruct a phylogenetic tree. In this study, ITS2 sequences showed the highest interspecific variability and could be used to differentiate all Mucuna species. The results of ITS2 sequence coupled with HPLC analysis revealed that all the purchased aphrodisiac products originated from M. pruriens only. Therefore, the integration of HPLC analysis and DNA barcoding profile was an efficient method for the identification of prohibited Mucuna species for safety monitoring of herbal supplements and protecting customer safety. Regulatory agencies should raise awareness and restrain the use of these commercial products.

Antinociceptive efficacy of Clerodendrum petasites S. Moore, a Thai medicinal plant, and its CNS safety profiles.

Background: Clerodendrum petasites, an herbal plant in Thailand, has been used for many years in folk medicine. However, scientific evidence regarding CNS safety pharmacology and antinociceptive activity of C. petasites (CP) has not yet been well characterized. Purpose: The present study aimed to assess the CNS safety pharmacology and antinociceptive and antiinflammatory effects of CP extract. Methods: The effect of CP extract on CNS safety pharmacology was assessed using LABORAS automated home cage monitoring and rotarod test. Its pharmacological activity was evaluated both in-vitro, and in-vivo using hot-plate, acetic acid-induced writhing, formalin, and carrageenan-induced paw edema models. Results and conclusion: CP extract significantly improved thermal and chemical nociceptive behaviors and acute inflammatory pain at all doses: 300, 600, and 1200 mg/kg, p.o. The antiinflammatory effect of CP extract in inflammatory pain models was comparable to the effect of positive control: indomethacin 10 mg/kg at all dose levels tested. Further, the CP extract at 600 mg/kg dose significantly inhibited 82.3% of carrageenan-induced total edema. In-vitro, CP extract at 12.5, 25, and 50 µg/mL concentrations significantly reduced the expression of LPS-induced nitric oxide, IL-6, and TNF-α expression in both RAW 264.7 macrophage and BV-2 microglial cell lines. In addition, CP extract did not show any potential effects on the CNS, indicated by no significant effects on motor coordination, spontaneous locomotor activity, general behaviors, and well-being compared to vehicle-treated mice (p > 0.05). Overall, the present study evidences the potential antinociceptive, antiinflammatory efficacies of CP extract with a favorable CNS safety profile.

Essential oil composition of Curcuma species and drugs from Asia analyzed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry.

Essential oils (EOs) comprised of various bioactive compounds have been widely detected in the Curcuma species. Due to the widespread distribution and misidentification of Curcuma species and differences in processing methods, inconsistent reports on major compounds in rhizomes of the same species from different geographical regions are not uncommon. This inconsistency leads to confusion and inaccuracy in compound detection of each species and also hinders comparative study based on EO compositions. The present study aimed to characterize EO compositions of 12 Curcuma species, as well as to detect the compositional variation among different species, and between the plant specimens and their related genetically validated crude drug samples using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The plant specimens of the same species showed similar EO patterns, regardless of introducing from different geographical sources. Based on the similarity of EO compositions, all the specimens and samples were separated into eight main groups: C. longa; C. phaeocaulis, C. aeruginosa and C. zedoaria; C. zanthorrhiza; C. aromatica and C. wenyujin; C. kwangsiensis; C. amada and C. mangga; C. petiolata; C. comosa. From EOs of all the specimens and samples, 54 major compounds were identified, and the eight groups were chemically characterized. Most of the major compounds detected in plant specimens were also observed in crude drug samples, although a few compounds converted or degraded due to processing procedures or over time. Orthogonal partial least squares-discriminant analysis allowed the marker compounds to discriminate each group or each species to be identified.

Unraveling the Adsorption Behavior of Thymol on Carbon and Silica Nanospheres for Prolonged Antibacterial Activity: Experimental and DFT Studies.

Functionalization of thymol (Thy) on nanocarriers is a key step in achieving prolonged antimicrobial activity. This requires nanomaterials with uniform particle diameters and suitable thymol sorption. Herein, hollow carbon (HC) and SiO2-carbon core-shell (SiO2@C) were investigated due to their diverse morphologies and ease of surface modification. HC (14 ± 1 nm size) and SiO2@C (10 ± 1.5 nm size) were synthesized by the Stöber method before thymol was loaded by incipient wetness impregnation. Nanoparticle physicochemical properties were characterized by advanced techniques, including X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS). Adsorption energies of thymol on the carbon and SiO2 surfaces were elucidated by density functional theory (DFT) simulations. Moreover, the in vitro thymol release profiles and antibacterial activity were evaluated. The experimental results indicated that the oxy-carbon surface species of HC led to longer thymol release profiles than the -OH group of SiO2@C. The DFT calculations revealed that the weaker physical interaction of thymol on HC was better for drug release than that on SiO2@C. Thus, a longer thymol release profile of HC with hollow structures showed better antibacterial performance against Gram-positive bacteria Staphylococcus aureus than that of SiO2@C with core-shell structures. This work confirms the important role of carbon morphology and specific functional groups in thymol release profiles for the further development of inhibition products.

PCR combined with lateral flow immunochromatographic assay to differentiate the narcotic Mitragyna speciosa from related species and detect it in forensic evidence.

Plants in the genus Mitragyna (Rubiaceae) are used in traditional medicine because of their broad therapeutic activity. Four Mitragyna species, M. speciosa (Roxb.) Korth. (MS), M. rotundifolia (Roxb.) Kuntze (MR), M. diversifolia (Wall. ex G. Don) Havil. (MD), and M. hirsuta Havil. (MH), occur in Thailand. M. speciosa, commonly known as 'Kratom' in Thai, is the only narcotic species for which buying, selling, importing or possessing has been prohibited by law in Thailand and some other countries. Mitragynine and 7-hydroxymitragynine, the major psychoactive compounds, are important in the treatment of opioid withdrawal. However, this species is used in traditional medicine to relieve pain and inflammation. Consequently, a rapid and easy technique for differentiating M. speciosa from closely related species is needed for routine forensic analysis. In this study, polymerase chain reaction coupled with lateral flow immunochromatographic assay (PCR-LFA) based on matK was developed for the detection of M. speciosa in forensic specimens. Duplex primers (MS-F-FAM, Ctrl-F-DIG and Ctrl-R-Biotin) were designed based on species-specific nucleotide indels observed exclusively in the matK sequences of M. speciosa. Positive results for M. speciosa are indicated by the clear presence of three black lines on the lateral flow cassette. Forensic samples were investigated, and the three black test lines indicating M. speciosa were observed for seven of eight specimens. PCR-LFA has been proven to be fast, easy and efficient for detecting the narcotic M. speciosa and could be developed as a rapid forensic diagnostic technique for other plants.

A PCR-lateral flow immunochromatographic assay (PCR-LFA) for detecting Aristolochia species, the plants responsible for aristolochic acid nephropathy.

Aristolochic acids (AAs), which are strong carcinogens, have caused dietary supplements with Aristolochia plants to be discontinued worldwide. Therefore, the development of a method to identify these herbs is critical for customer safety. To support the regulation of Aristolochia-free products, a PCR coupled with lateral flow immunochromatographic assay (PCR-LFA) that is specific to the nucleotide signature in plastid rbcL gene region of Aristolochia species was developed to detect Aristolochia plants and related herbal products. Triplex primers (A397F, C357F and R502) were designed based on specific nucleotides observed exclusively in the rbcL sequences of Aristolochia. Positive results for Aristolochia occur when the three pink lines are clearly developed on the developed lateral flow strip and can be seen by the naked eye. In this study, the lateral flow strip has sensitivity for detecting amplicons amplified from genomic DNA at the concentrations as low as 0.01 ng. Various kinds of samples, including purchased crude drugs and polyherbal samples, have been investigated, and the results showed that Aristolochia crude drugs and Aristolochia-containing products are still present in dispensaries. In conclusion, with the goal of protecting consumers from the health risks associated with Aristolochia contamination, PCR-LFA was developed and demonstrated to be efficient for detecting plants belonging to Aristolochia in various kinds of samples.

Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells.

Background: Triple-negative breast cancer (TNBC) responds poorly to the available drugs; thus, the mortality rate associated with TNBC remains high. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells. Methods: 7HF uptake by MDA-MB-468 cells was assessed using spectrophotometry. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. Then, the effect of 7HF treatment (6, 12, and 24 µM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. Polymerization of α- and ß-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs. Results: We observed that 7HF was able to enter the MDA-MB-468 cells. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. 7HF reduced polymerization of α-, ß-tubulin in dose-dependent manner. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. According to molecular docking analysis, 7HF preferred to bind to ß-tubulin over α-tubulin. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF. Conclusions: This is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. The findings will be useful for developing 7HF-based treatment for patients with TNBC.

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, "Kho-Khlan".

The pain relief formula "Ya Pa Som Kho-Khlan (YPSKK)" or "ยาผสมโคคลาน" in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: "Kho-Khlan". Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products.

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.