Transferosomes stabilized hydrogel incorporated rhodomyrtone-rich extract from Rhodomyrtus tomentosa leaf fortified with phosphatidylcholine for the management of skin and soft-tissue infections.

Rhodomyrtus tomentosa leaf (RT)-incorporated transferosomes were developed with lecithin and cholesterol blends with edge activators at different ratios. RT-transferosomes were characterized and employed in transferosomal gel formulations for the management of skin and soft-tissue infections. The optimized formulation entrapped up to 81.90 ± 0.31% of RT with spherical vesicles (405.3 ± 2.0 nm), polydispersity index value of 0.16 ± 0.08, and zeta potential of - 61.62 ± 0.86 mV. Total phenolic and flavonoid contents of RT-transferosomes were 15.65 ± 0.04 µg GAE/g extract and 43.13 ± 0.91 µg QE/g extract, respectively. RT-transferosomes demonstrated minimum inhibitory and minimum bactericidal concentrations at 8-256 and 64-1024 µg/mL, respectively. Free radical scavenging assay showed RT-transferosomes with high scavenging activity against DPPH and ABTS radicals. Moreover, RT-transferosomes demonstrated moderate activity against mushroom tyrosinase, with IC50 values of 245.32 ± 1.32 µg/mL. The biocompatibility results against L929 fibroblast and Vero cells demonstrated IC50 at 7.05 ± 0.17 and 4.73 ± 0.13 µg/mL, respectively. In addition, nitric oxide production significantly decreased by 6.78-88.25% following the treatment with 31.2-500 ng/mL RT-transferosomes (p < 0.001). Furthermore, the freeze-thaw stability study displayed no significant change in stability in the sedimentation and pH of gel fortified with RT-transferosomes. The results suggested that RT-transferosome formulation can be effectively employed as natural biomedicines for scar prevention and the management of skin soft-tissue infections.

Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection.

BACKGROUND: This study aimed to evaluate the efficacy of combinations of steroidal alkaloids and conessine from the Thai medicinal plant Holarrhena antidysenterica with antibiotics against Pseudomonas aeruginosa strains possessing different efflux-pump-mediated multidrug-resistant (MDR) phenotypes in a Galleria mellonella infection model. METHODS: P. aeruginosa strains with defined mutations that result in the overexpression of the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps, and a strain with all three of these pumps deleted, were used. In vitro, the effect of combinations of steroidal alkaloids and conessine with antibiotics was compared with antibiotic treatment alone via MIC determination and time-kill assays. Efficacy of combinations of the steroidal alkaloids and conessine with levofloxacin were compared with monotherapies against infections in G. mellonella larvae by measuring larval mortality and bacterial burden. RESULTS: Combination therapies of conessine or steroidal alkaloids with levofloxacin enhanced bacterial inhibition in vitro and restored antibiotic efficacy in vivo compared to the constituent monotherapies. Neither conessine nor the steroidal alkaloids induced any detectable toxicity in G. mellonella larvae. The enhanced efficacy of the combination treatments was most pronounced with conessine and correlated with reduced larval burden of infecting P. aeruginosa. Notably, the enhanced efficacy of conessine/levofloxacin combinations was only detected in the parent strain and strains that overexpressed the MexAB-OprM or MexEF-OprN efflux systems. CONCLUSIONS: Steroidal alkaloids from Holarrhena antidysenterica, and particularly the principal active ingredient conessine, restored levofloxacin efficacy against resistant P. aeruginosa strains possessing efflux-mediated MDR phenotypes. The compounds should be investigated further as a potential novel therapy.

Conessine as a novel inhibitor of multidrug efflux pump systems in Pseudomonas aeruginosa.

BACKGROUND: Holarrhena antidysenterica has been employed as an ethnobotanical plant for the treatment of dysentery, diarrhoea, fever, and bacterial infections. Biological activities of the principle compound, conessine including anti-diarrhoea and anti-plasmodial effects were documented. Our previous study reported potency of Holarrhena antidysenterica extract and conessine as resistance modifying agents against extensively drug-resistant Acinetobacter baumannii. This study aimed to investigate (i) whether conessine, a steroidal alkaloid compound, could act as a resistance modifying agent against multidrug-resistant Pseudomonas aeruginosa, and (ii) whether MexAB-OprM efflux pump involved in the mechanism. METHODS: Conessine combined with various antibiotics were determined for synergistic activity against P. aeruginosa PAO1 strain K767 (wild-type), K1455 (MexAB-OprM overexpressed), and K1523 (MexB deletion). H33342 accumulation assay was used to evaluate efflux pump inhibition while NPN uptake assay was assessed membrane permeabilization. RESULTS: Conessine significantly reduced MICs of all antibiotics by at least 8-fold in MexAB-OprM overexpressed strain. The levels were comparable to those obtained in wild-type strain for cefotaxime, levofloxacin, and tetracycline. With erythromycin, novobiocin, and rifampicin, MICs were 4- to 8-fold less than MICs of the wild-type strain. Loss of MexAB-OprM due to deletion of mexB affected susceptibility to almost all antibiotics, except novobiocin. Synergistic activities between other antibiotics (except novobiocin) and conessine observed in MexB deletion strain suggested that conessine might inhibit other efflux systems present in P. aeruginosa. Inhibition of H33342 efflux in the tested strains clearly demonstrated that conessine inhibited MexAB-OprM pump. In contrast, the mode of action as a membrane permeabilizer was not observed after treatment with conessine as evidenced by no accumulation of 1-N-phenylnaphthylamine. CONCLUSIONS: The results suggested that conessine could be applied as a novel efflux pump inhibitor to restore antibiotic activity by inhibiting efflux pump systems in P. aeruginosa. The findings speculated that conessine may also have a potential to be active against homologous resistance-nodulation-division (RND) family in other Gram-negative pathogens.

Successful treatment of refractory erythrodermic psoriasis with traditional Thai herbal medicine.

BACKGROUND: Thai herbal formulations have been used traditionally in Thailand for the treatment of psoriasis. However, there is still a lack of scientific data supporting the effects of Thai herbal formulations in psoriasis treatment. OBJECTIVES: This study aimed to demonstrate the therapeutic effects of Thai herbal formulations for the treatment of erythrodermic psoriasis. MATERIALS AND METHODS: All Thai herbal formulations (haematic tonic, lymphatic treatment, skin treatment) were obtained from a traditional Thai medicine doctor, Mr. Somporn Chanwanitsakul. The effects of Thai herbal formulations in a patient with erythrodermic psoriasis were assessed for four weeks. Primary outcome, psoriasis area and severity index (PASI) and secondary outcome, safety data and dermatology life quality index (DLQI) measurements were evaluated at baseline and four weeks. Then, in vitro biological activities (antioxidant, anti-microbial, cytotoxic effects, and anti-inflammatory) of Thai herbal formulations were determined to promote the therapeutic effects. RESULTS: Thai herbal formulations were safe and effective in the treatment of erythrodermic psoriasis and had a modest positive impact on the DQLI of the patient. In addition, in vitro studies have shown that all Thai herbal formulations revealed remarkable anti-oxidant and anti-inflammatory potential to support their therapeutic activities. However, the Thai herbal formulations possessed weak intrinsic antibacterial activities against all tested bacterial strains (MIC and MBC E. coli, S. aureus, S. pyogenes, P. aeruginosa: > 256 µg/ml). CONCLUSION: The findings indicated that successful treatment of erythrodermic psoriasis with Thai herbal formulations was involved in their anti-oxidant and anti-inflammatory activities. They could be considered as an alternative treatment for refractory erythrodermic psoriasis.

Atomistic insight and modeled elucidation of conessine towards Pseudomonas aeruginosa efflux pump.

Drug-resistant Pseudomonas aeruginosa efflux pump extrudes antibiotics from cells for survival. Efflux pump inhibitor (EPI) thus becomes an interesting alternative to handle the drug-resistant bacteria. Conessine, a natural steroidal alkaloid from Holarrhena antidysenterica, previously exhibited efflux pump inhibitory potential. Our molecular docking and molecular dynamics (MD) studies provided atomistic information as well as the interaction of conessine with bacterial MexB efflux pump in phospholipid bilayer membrane to further the previous experimental report. Herein, the binding site and proposed mode of action of conessine were identified compared to known/commercial EPIs such as PAßN or designed-synthetic P9D. Our results explained conessine binding mode of action as an effective agent against the MexB efflux pump. The MD simulation also suggested that conessine was able to affect glycine loop (G-loop) flexibility, and the reduced G-loop flexibility due to conessine could hinder an antibiotics extrusion. In addition, our study suggested the conessine core structure buried in a hydrophobic region in the efflux pump similar to other known EPIs. Our finding could cope as a key for the design and development of the conessine derivative as novel EPI against P. aeruginosa.Communicated by Ramaswamy H. Sarma.

Synergistic Antibacterial Effects of Meropenem in Combination with Aminoglycosides against Carbapenem-Resistant Escherichia coli Harboring blaNDM-1 and blaNDM-5.

Infections due to carbapenem-resistant Escherichia coli (CREC) are problematic due to limitation in treatment options. Combination therapies of existing antimicrobial agents have become a reliable strategy to control these infections. In this study, the synergistic effects of meropenem in combination with aminoglycosides were assessed by checkerboard and time-kill assays. Of the 35 isolates, 19 isolates (54.3%) were resistant to carbapenems (imipenem and meropenem) with the MIC ranges from 16 to 128 µg/mL. These isolates were resistant to almost all antibiotic classes. Molecular characteristics revealed co-harboring of carbapenemase (blaNDM-1, blaNDM-5 and blaOXA-48) and extended-spectrum ß-lactamases (ESBL) genes (blaCTX-M, blaSHV and blaTEM). The checkerboard assay displayed synergistic effects of meropenem and several aminoglycosides against most CREC isolates. Time-kill assays further demonstrated strong synergistic effects of meropenem in combination with either amikacin, gentamicin, kanamycin, streptomycin, and tobramycin. The results suggested that meropenem in combination with aminoglycoside therapy might be an efficient optional treatment for infections cause by CREC.

Therapeutic effects of traditional Thai herbal blood and wind tonic formulations for treatment of menopausal symptoms.

INTRODUCTION: Traditional Thai herbal medicine formulations have been used as alternative therapies for menopausal symptoms due to concerns from adverse effects associated with hormone therapy. This study aimed to demonstrate the effects of traditional Thai herbal blood and wind tonic formulations used by a traditional Thai medicine doctor, Mr. Somporn Chanwanitsakul, in postmenopausal women. MATERIALS AND METHODS: A pilot clinical study was conducted on thirty-five postmenopausal women, referring to Tambon Thung Tam Sao Health Promotion Hospital, Hat Yai, Songkhla, from October 2019 to March 2020. The participants consumed combined Thai herbal formulations including blood tonic and wind tonic thrice daily for four weeks. Outcomes were assessed at baseline, end of treatment (4 weeks), and follow-up (8 weeks). Pre- and post-treatment measures included menopause rating scale, sleep quality, and quality of life questionnaire. All data were analyzed using SPSS software at the significance level of 0.05. RESULTS: Therapeutic effects of Thai herbal medicine formulations on menopausal symptoms intensity were assessed by modified Menopause Rating Scale (MRS). Severity of women's total menopausal symptoms decreased significantly (p < 0.05) at end of treatment and follow-up. Analysis of changes in specific symptoms indicated significantly less moderate headache, mild hot flashes, sweating, emotional instability, irritability, anxiety, sleep problem, lethargy, back pain, joint pain, muscular discomfort, dry skin, dryness of vagina, boring sex, and frequent urination (MRS score 0). In addition, subjective analysis of sleep quality using Pittsburgh Sleep Quality Index (PSQI) data revealed significant post-treatment improvements in subjective sleep quality and daytime dysfunction over the last month (PSQI score 0). Furthermore, subjective analysis of quality of life using World Health Organization Quality of Life Brief showed significant post-treatment improvement in psychological health (score 23). CONCLUSION: The findings demonstrate that Thai herbal medicine formulations used by a traditional Thai medicine doctor, Mr. Somporn Chanwanitsakul, are effective for treating menopausal symptoms and improve sleep quality and quality of life in postmenopausal women.

In vivo safety assessment of rhodomyrtone, a potent compound, from Rhodomyrtus tomentosa leaf extract.

BACKGROUND: Rhodomyrtus tomentosa (Aiton) Hassk. has been traditionally used to relieve various diseases. Rhodomyrtone, a bioactive acylphloroglucinol compound isolated from the leaves of Rhodomyrtus tomentosa, has been scientifically evidenced as a potential antibacterial agent. This study aimed to assess safety of rhodomyrtone in both invertebrate and vertebrate models. MATERIAL AND METHODS: Safety of rhodomyrtone was determined in an invertebrate model, Galleria mellonella as well as vertebrate models including zebrafish (Danio rerio) and murine. In addition, toxicity to human erythrocytes was also measured. RESULTS: Treatment of Galleria mellonella with rhodomyrtone at 100 mg/kg body weight up to four days showed no visible toxic effects (100 % survival). In zebrafish embryo model, at least 80 % survival of embryos was demonstrated when treated with rhodomyrtone at 0.5 µg/mL for three days. Prior to clinical trial, it is a prerequisite that rhodomyrtone has to be evaluated for its biocompatibility with human blood components. The results displayed that rhodomyrtone at 256 µg/mL did not cause any observable human erythrocyte haemolysis. Furthermore, preclinical assessment of rhodomyrtone formulation justified potential applications of rhodomyrtone in humans. Oral toxicity testing in a mouse model indicated the absence of systemic toxicity when the animals received up to 5000 mg/kg body weight of rhodomyrtone formulation for a period of fourteen days. CONCLUSIONS: As the minimal inhibitory concentration of rhodomyrtone against most Gram-positive pathogens is 0.5-1 µg/mL, the results suggest that it should produce no toxic effects at concentrations used in human, thus support further development in pharmaceutical industries and public health applications.

Traditional Thai herbal medicine as an alternative treatment for refractory chronic eczema.

INTRODUCTION: Traditional Thai herbal medicine has been an important part of health care in Thailand for centuries due to perceived efficacy with fewer side effects. However, limited clinical evidence of the effectiveness of traditional Thai herbal medicine for eczema therapy has been documented. This case report aimed to demonstrate the efficacy of traditional Thai herbal formulations used by a traditional Thai medicine doctor, Mr. Somporn Chanwanitsakul, for refractory chronic eczema. MATERIALS AND METHODS: In this case, an eight-year-old girl with chronic eczema was treated daily with traditional Thai herbal medicines combining two oral formulations: (i) haematic tonic and (ii) lymphatic and skin treatment with two external formulations: (i) bath formulation and (ii) topical therapy for a period of four weeks. Therapeutic effects were assessed weekly using photographic comparison. RESULTS: After two weeks of treatment, itching decreased and skin lesions became darker and thinner. All skin lesions completely disappeared and only post-inflammatory pigmentation was noticed after four weeks. In addition, no recurrence was observed at a subsequent three month follow-up examination following the end of treatment. CONCLUSIONS: The findings indicate that combined traditional Thai herbal formulations can be used to successfully treat chronic eczema and could be considered as an alternative treatment for refractory chronic eczema.

Dual ß-lactam combination therapy for multi-drug resistant Pseudomonas aeruginosa infection: enhanced efficacy in vivo and comparison with monotherapies of penicillin-binding protein inhibition.

The aim of the study was to determine the efficacy of dual ß-lactam combination treatments derived from eight approved drugs against Galleria mellonella larvae infected with MDR strains of P. aeruginosa. Carbapenem-resistant P. aeruginosa NCTC 13437 and an unrelated clinical isolate were used to infect G. mellonella larvae and the efficacy of twenty-eight dual ß-lactam combination therapies were compared to their constituent monotherapies. For the most potent combinations identified, penicillin-binding protein (PBP) inhibition profiles were measured and compared with each constituent antibiotic. Five of the dual ß-lactam combinations resulted in greater than 70% survival of infected G. mellonella. Two combinations showed potent, enhanced efficacy versus both strains - ceftazidime + meropenem and aztreonam + meropenem. Comparison of PBP inhibition profiles revealed that the enhanced efficacy of these two dual ß-lactam combinations could not be explained by more potent inhibition of PBPs or inhibition of a broader range of PBPs. A possible contribution to the enhanced efficacy of the combinations could be stimulation of innate immunity via increased haemocyte numbers compared to their constituent monotherapies. Combinations of ß-lactam antibiotics show promise in overcoming MDR P. aeruginosa and are worthy of additional study and development.

Holarrhena antidysenterica Extract and Its Steroidal Alkaloid, Conessine, as Resistance-Modifying Agents Against Extensively Drug-Resistant Acinetobacter baumannii.

Emergence and spread of antibiotic-resistant Acinetobacter baumannii have become a major public health concern. This study was designed to investigate the efficacy of Holarrhena antidysenterica extract and its major steroidal alkaloid conessine as resistance-modifying agents (RMAs) on the susceptibility of A. baumannii to novobiocin and rifampicin. A significant synergistic activity of both the extract and conessine in combination with either novobiocin or rifampicin with fractional inhibitory concentration index ≤0.5 was demonstrated. Fluorescent dyes and different efflux pump inhibitors were used to further investigate the synergism. Increase in the uptake of 1-N-phenylnaphthylamine in the bacterial cells treated with the extract and conessine was not observed indicating that both substances did not act as permeabilizers. With regard to efflux pump inhibition, no accumulation in ethidium bromide (EtBr) was noticed suggesting that the AdeABC pump was not involved. In contrast, accumulation in Pyronin Y was significantly increased (p < 0.05) demonstrating that the synergism was due to interference with the AdeIJK pump. Study on frequencies of the spontaneous mutational resistance to the extract in combination with antibiotics demonstrated attenuation in drug-resistant organisms. Thus, H. antidysenterica extract and conessine as RMAs may offer a combinatory therapy to restore antibiotic susceptibility in the extensively drug-resistant A. baumannii.

Synergistic effects of ethnomedicinal plants of Apocynaceae family and antibiotics against clinical isolates of Acinetobacter baumannii.

OBJECTIVE: To investigate the efficacy of 17 ethnomedicinal plants belonging to Apocynaceae family used in combination with 16 conventional antibiotics against non-multidrug resistant-, multidrug resistant (MDR)-, and extensive drug resistant (XDR) Acinetobacter baumannii (A. baumannii). METHODS: Antibacterial activity and resistance modifying ability of 272 combinations were determined by growth inhibition assays and further confirmed by time-kill assay. RESULTS: Among the combinations of the antibiotics with Apocynaceae ethanol extracts on this pathogen, 15 (5%) had synergistic effects, 23 (8%) had partial synergistic effects and 234 (86%) had no effects. Synergistic activity was observed mostly when the Apocynaceae extracts were combined with rifampicin or cefazolin. Interestingly, 10 out of 17 combinations between the extracts and rifampicin displayed synergistic or partial synergistic behaviors. Holarrhena antidysenterica extract was additionally tested to restore rifampicin activity against clinical isolates of MDR and XDR A. baumannii. With respect to total or partial synergy, 70% was XDR A. baumannii isolates and 66% was MDR A. baumannii isolates. CONCLUSIONS: Holarrhena antidysenterica extract clearly demonstrated the ability to restore rifampicin activity against both A. baumannii ATCC19606 and clinically isolated A. baumannii. Additional studies examining its active principles as well as mechanisms of actions such as the effects on efflux pumps and outer membrane permeability alterations are recommended.

Holarrhena antidysenterica as a resistance modifying agent against Acinetobacter baumannii: Its effects on bacterial outer membrane permeability and efflux pumps.

Increasing rates of infections caused by multidrug resistant Acinetobacter baumannii (MDRAB) and extensively drug resistant A. baumannii (XDRAB) have caused the need for searching alternative agents. The purposed of this project was to search plant-derived natural products that act as resistant modifying agents (RMAs) against A. baumannii. In this study, we further evaluated the activity of Holarrhena antidysenterica that has been previously proposed as RMA of novobiocin for a model strain, A. baumannii ATCC 19606 on clinically isolated non-MDRAB, MDRAB, and XDRAB. Effects of H. antidysenterica on outer membrane permeability and efflux pumps of the pathogen were conducted to preliminary elucidate mechanisms of this resistant modifier. Novobiocin was selected as a model antibiotic because it is well-established as an effective agent against Gram-positive pathogens. But, it possessed low level of antibacterial activity against Gram-negative pathogens due to an effective permeability barrier of these pathogens. H. antidysenterica ethanol extract possessed weak intrinsic antibacterial activity with minimum inhibitory concentration (MIC) more than 1000 µg/mL. The extract, at concentrations of 250, 125, and 62.5 µg/mL, remarkably enhanced the inhibitory effects of novobiocin (1/4 × MIC; 1-4 µg/mL) against XDRAB isolates. Synergistic effects of novobiocin at 1/4 × MIC and 1/8 × MIC in combination with H. antidysenterica either at 31.2, 15.6, or 7.8 µg/mL against clinical isolates non-MDRAB, MDRAB, and XDRAB were evidenced for 80% of the combinations (189 out of 234 combinations). Although, no enhancement of the accumulation of ethidium bromide was observed after treated with H. antidysenterica, this plant extract weakened the outer membrane of the pathogen as indicated by an increase in the N-phenyl-1-naphthylamine uptake. Our results suggested that H. antidysenterica which primarily interrupts membrane permeability should be further investigated as a promising resistant modifier for A. baumannii.