Effectiveness of Ya-Samarn-Phlae in diabetic wound healing: Evidence from in vitro studies and a multicenter randomized controlled clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Ya-Samarn-Phlae (YaSP) has traditionally been widely used in southern Thailand for treating chronic and infected wounds, including diabetic foot ulcers. However, there are only a limited number of clinical studies supporting the use of this polyherbal formulation. Therefore, the present work aims to provide clinical evidence to support the application of YaSP, prepared according to a standardized traditional procedure (T-YaSP). Additionally, its potential chemical markers and wound healing-related biological activities were examined. MATERIALS AND METHODS: The in vitro wound healing-related biological activities of YaSP ethanol extract and T-YaSP, including antibacterial activity against Staphylococcus epidermidis, inhibition and eradication of staphylococcal biofilm, anti-inflammatory effects, and enhancement of human dermal fibroblast migration in scratch wounds, were examined using well-established protocols. The chemical profiles of the ethanol extract of YaSP and T-YaSP were compared, and with promising chemical markers, arecoline, alpha-mangostin, and curcumin were selected and quantified using the HPLC method. A prospective, multicenter, randomized, controlled, parallel-group study was conducted over 12 weeks to evaluate the efficacy of the YaSP solution as an adjunct therapy, combined with standard wound care, for diabetic ulcers compared to standard treatment. RESULTS: The YaSP extract reduces NO production and can scavenge NO radicals in LPS-induced RAW 264.7 macrophage cells. Additionally, in a scratch assay, this extract and one of its herbal components, Curcuma longa, enhance the migration of human dermal fibroblasts. T-YaSP, containing 2.412 ± 0.002 mg/g of arecoline, 2.399 ± 0.005 mg/g of curcumin, and 0.017 ± 0.000 mg/g of α-mangostin, has shown the ability to inhibit the development and eradicate the mature biofilm of S. epidermidis. The use of T-YaSP as an adjunct therapy led to a significantly higher proportion of patients achieving healing within six weeks compared to the standard treatment group (36%/9 patients vs. 4%/1 patient; p = 0.013). After 12 weeks, 19 out of 25 patients in the T-YaSP group experienced complete healing, whereas only four patients in the standard treatment group achieved complete wound healing (76% in the T-YaSP group vs. 16% in the control group; p < 0.001). CONCLUSION: The results presented here represent the first randomized controlled trial to demonstrate the effectiveness of the traditional polyherbal solution, T-YaSP, which exhibits a wide range of wound healing-related activities. Utilizing T-YaSP as an adjunctive treatment resulted in a significant improvement in the number of type 2 diabetic patients achieving complete healing. However, to explore and utilize YaSP further, conducting a double-blind, randomized controlled trial with a larger population is necessary.

Safety Evaluation of the Polyherbal Formulation NawaTab: Acute and Subacute Oral Toxicity Studies in Rats.

NawaTab is a tablet formulation developed from the Nawametho polyherbal formula used in Surat Thani province, Southern Thailand, for the treatment of hyperlipidemia. This study aims at evaluating the acute and subacute toxicity of NawaTab in rats. In the acute toxicity study, NawaTab was evaluated in female rats following the OECD Guideline No. 423. In the subacute toxicity study, NawaTab was tested in both male and female rats following the OECD Guideline No. 407. In the acute toxicity study, no lethal effects or toxic signs were observed during the duration of the study. In the subacute toxicity study, there was no mortality and no abnormality in clinical signs, body weight, food consumption, relative organ weight, and hematological parameters of NawaTab-treated rats. Significantly increased water consumption by male rats (500 mg/kg BW) and female rats (250, 500, and 1000 mg/kg BW) was observed. In addition, globulin and total cholesterol of female rats (1000 mg/kg BW) significantly increased. These alterations were within normal physiological ranges. Moreover, necropsy and histopathological findings of NawaTab-treated rats demonstrated no obvious alterations attributable to NawaTab administration. The present study revealed that NawaTab has no significant acute oral toxicological effects. The lethal dose with a 50% mortality rate (LD50) was higher than 5000 mg/kg BW in rats. The subacute oral administration of NawaTab for 28 days did not have any major toxicological effects. Based on this study, NawaTab could be safe to use with caution pending its chronic toxicity study.

Supplementation with the Traditional Thai Polyherbal Medicine NawaTab Ameliorates Lipid Profiles in High-Fat Diet-Induced Hyperlipidemic Rats.

A tablet formulation developed from Nawametho decoction (NawaTab), a traditional Thai herbal mixture described in the Worayokasan scripture, has been used clinically for several years in the management of borderline hyperlipidemic individuals. Nevertheless, scientific evidence supporting its claims has never been identified. This study aimed to describe the antihyperlipidemic properties of NawaTab in a well-described model of high-fat diet (HFD)-induced hyperlipidemic rats. Decoction of Nawametho containing equal quantities of Aegle marmelos (L.), Carthamus tinctorius L., Hibiscus sabdariffa Linn., Phyllanthus emblica L., Piper longum L., Piper nigrum L., Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula Retz., and Zingiber officinale Roscoe were prepared. The HFD-fed rats were administered NawaTab for 4 consecutive weeks starting from the ninth week of HFD treatment at a dose of 125 mg/kg (p.o.). Anthropometric parameters, serum lipid profiles, lipase activity, and liver and renal functional markers were measured. Histopathological examination of the liver and any steatosis was carried out at the end of this study. Consecutive treatment with NawaTab (125 mg/kg/day) in HFD-induced hyperlipidemic rats caused a significant reduction in serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, and very low-density lipoprotein cholesterol levels. However, at the tested dose, NawaTab failed to prevent the onset of hepatic steatosis and adipose tissue accumulation. No adverse events due to the consumption of NawaTab on liver and kidney function markers were noted. These findings are the first suggestive evidence for the lipid-lowering capability of NawaTab. However, to promote the use of this formulation in the management of borderline hyperlipidemic patients, elucidation of the underlying mechanisms of action, quantification of biological markers, and clinical trials of NawaTab are urgently needed.

Clinical Evaluation of a Novel Tablet Formulation of Traditional Thai Polyherbal Medicine Named Nawametho in Comparison with Its Decoction in the Treatment of Hyperlipidemia.

In the traditional medical system in Thailand, medicinal plants and polyherbal medicines have been prescribed as lipid-lowering agents, including Nawametho decoction. This polyherbal formulation is described in the Worayokasan scripture. It consists of nine medicinal plants (Aegle marmelos (L.), Carthamus tinctorius L., Hibiscus sabdariffa Linn., Phyllanthus emblica L., Piper longum L., Piper nigrum L., Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula Retz., and Zingiber officinale Roscoe). Apart from its utilization in Thai traditional medicine, there is a lack of evidence supporting its use. This research work thereby aims to formulate and evaluate the tablet containing Nawametho decoction. The feasibility of Nawametho decoction and NawaTab for patients with borderline hyperlipidemia was additionally examined using a prospective, open-label, randomized, parallel-group design. The dry granulation technique was employed to formulate the polyherbal tablets. The tablets were developed using the spray-dried Nawametho decoction as the active ingredient in addition to other excipients. The chosen formulation, the F B (NawaTab), consisted of 385 milligrams of the extract, 12% w/w of a diluent (lactose), 8% w/w of a lubricant (magnesium stearate), 5% w/w of a disintegrant (microcrystalline cellulose), and 5% w/w of an anti-adherent (talcum). Their hardness, friability, and disintegration time were 4.4 ± 0.32 kg, 0.05 ± 0.02%, and 4.60 ± 0.05 min, respectively. Accelerated stability study results revealed that NawaTab was stable for six months at 40°C/75% RH and 25°C/60% RH. Even though taking NawaTabs (500 mg twice daily) for eight consecutive weeks was unable to improve the lipid profile of the patients, the administration of Nawametho decoction (30 mL twice daily) was associated with a significant decrease in serum triglycerides of the patients. The results show that the dry granulation technique is suitable for the formulation of NawaTab based on the tablet evaluation. Furthermore, the triglyceride-lowering effect of Nawametho decoction was reported for the first time.

Evaluation of Microwave-Assisted Extraction Method for Preparation and Assessment of Thai Herbal Medicine Oral Tablets With Enriched Phytochemical Compounds.

In developing countries, populations have employed herbal medicines for primary health care because they are believed to be more appropriate to the human body and have less side effects than chemically synthesized drugs. The present study aimed to develop and evaluate herbal tablets incorporated with a Thai traditional medicinal extract, U-pa-ri-waat (URW), using microwave-assisted extraction (MAE). The extraction efficiency for URW using MAE and traditional solvent extraction was compared based on the percent yield after spray drying. URW tablets were prepared using the dry granulation method. The optimized products were assessed using standard characterization methods based on the United States and British Pharmacopeias. DPPH and ABTS radical scavenging assays were performed to analyze the antioxidant capacity of the microwave-assisted extracts. The results revealed that the flowability of the dry granule with added maltodextrin was improved compared to a granule without additives, as indicated by an angle of repose of 33.69 ± 2.0°, a compressibility index of 15.38 ± 0.66, and a Hausner's ratio of 1.18 ± 0.06. The resulting formulation produced flat tablets with uniform weight variation, hardness, thickness, friability, and optimum disintegration time. The URW extracts showed antioxidant activity and MAE with maltodextrin carrier displayed the strongest DPPH and ABTS radical activities with IC50 values of 1.60 ± 0.02 µg/mL and 4.02 ± 0.24 µg/mL, respectively. The URW tablet formulation passed the quality control tests. Storage of the formulation tablets for 90 days under accelerated conditions had minimal effects on tablet characteristics.

Antioxidant and antiaging effect of traditional Thai rejuvenation medicines in Caenorhabditis elegans.

OBJECTIVE: This study explored the rejuvenation mechanisms of Thai polyherbal medicines using different approaches, including in vitro methods, as well as a well-defined nematode model, Caenorhabditis elegans. METHODS: THP-R-SR012 decoction was selected from 23 polyherbal medicines, based on metal-chelating and chain-breaking antioxidant capacities. The influences of this extract on the survival and some stress biomarkers of C. elegans under paraquat-induced oxidative stress were evaluated. Furthermore, lifespan analysis and levels of lipofuscin accumulation were examined in senescent nematodes. The phytochemical profile of THP-R-SR012 was analyzed. RESULTS: Supplementation with THP-R-SR012 decoction significantly increased the mean lifespan and reduced the oxidative damage to C. elegans under oxidative stress conditions. Further, THP-R-SR012 supplementation slightly influenced the lifespan and the level of lipofuscin accumulation during adulthood. Antioxidant-related phytochemical constituents of THP-R-SR012 decoction were rutin, naringenin, 3,4-dihydroxybenzoic acid, gallic acid, glycyrrhizic acid, demethoxycurcumin and 18α-glycyrrhetinic acid. CONCLUSION: The antioxidant potential of THP-R-SR012 was due to its scavenging properties, its enhancement of antioxidant-related enzyme activities, and the presence of the antioxidant-related compound. These results support the traditional use of THP-R-SR012 decoction as a tonic for nourishing and strengthening the whole body.

Enhanced Oral Bioavailability and Improved Biological Activities of a Quercetin/Resveratrol Combination Using a Liquid Self-Microemulsifying Drug Delivery System.

Both quercetin and resveratrol are promising plant-derived compounds with various well-described biological activities; however, they are categorized as having low aqueous solubility and labile natural compounds. The purpose of the present study was to propose a drug delivery system to enhance the oral bioavailability of combined quercetin and resveratrol. The suitable self-microemulsifying formulation containing quercetin together with resveratrol comprised 100 mg Capryol 90, 700 mg Cremophor EL, 200 mg Labrasol, 20 mg quercetin, and 20 mg resveratrol, which gave a particle size of 16.91 ± 0.08 nm and was stable under both intermediate and accelerated storage conditions for 12 months. The percentages of release for quercetin and resveratrol in the self-microemulsifying formulation were 75.88 ± 1.44 and 86.32 ± 2.32%, respectively, at 30 min. In rats, an in vivo pharmacokinetics study revealed that the area under the curve of the self-microemulsifying formulation containing quercetin and resveratrol increased approximately ninefold for quercetin and threefold for resveratrol compared with the unformulated compounds. Moreover, the self-microemulsifying formulation containing quercetin and resveratrol slightly enhanced the in vitro antioxidant and cytotoxic effects on AGS, Caco-2, and HT-29 cells. These findings demonstrate that the self-microemulsifying formulation containing quercetin and resveratrol could successfully enhance the oral bioavailability of the combination of quercetin and resveratrol without interfering with their biological activities. These results provide valuable information for more in-depth research into the utilization of combined quercetin and resveratrol.

Thai herbal formulation 'Ya-Pit-Samut-Noi': Its antibacterial activities, effects on bacterial virulence factors and in vivo acute toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: A Thai herbal formulation 'Ya-Pit-Samut-Noi' containing Nigella sativa (seed), Piper retrofractum (fruit), Punica granatum (pericarp), and Quercus infectoria (nutgall) has long been traditionally used to treat diarrhea or bloody mucous diarrhea. Scientific information is very important to support its therapeutic effects and traditional drug development. AIM OF THE STUDY: This study aimed to evaluate the antibacterial activities of Ya-Pit-Samut-Noi against diarrhea-causing bacteria and determine its effects on bacterial virulence factors and in vivo acute toxicity. MATERIALS AND METHODS: Ethanol and water extracts of Ya-Pit-Samut-Noi and its plant components were prepared. The agar diffusion method was used for preliminary screening of antibacterial activity of the extracts against diarrhea-causing bacteria including Staphylococcus aureus, Vibrio cholerae, and Vibrio parahaemolyticus. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were assessed using broth microdilution method. The effects on bactericidal activity, bacterial cell wall, and cell membrane were examined by time-kill, lysis, and leakage assays, respectively. The effects on bacterial virulence factors including quorum-sensing system, biofilm production, and swarming motility were determined. Phytochemical screening was carried out to identify the group of chemical compounds present in the formulation extracts. Acute toxicity study was conducted by a single oral dose of 2000 mg/kg body weight in Wistar albino rats. RESULTS: Ethanol and water extracts of Ya-Pit-Samut-Noi and Quercus infectoria demonstrated antibacterial efficacy against all bacterial strains as revealed by zones of inhibition ranging from 7.0 to 24.5 mm. The ethanol and water extracts of Ya-Pit-Samut-Noi and Quercus infectoria produced strong bacteriostatic activity against V. parahaemolyticus (n = 11) with an MIC range of 7.81-250 µg/ml. Only the ethanol extract of Ya-Pit-Samut-Noi produced MBC values less than or equal to 1000 µg/ml against all V. parahaemolyticus. Based on time-kill study, no surviving V. parahaemolyticus (ATCC 17802 and 5268) cells were detected within 6-12 h after treatment with the ethanol extract of Ya-Pit-Samut-Noi at MBC-4MBC concentrations. Vibrioparahaemolyticus ATCC 17802 cells treated with the ethanol extract of Ya-Pit-Samut-Noi demonstrated no lysis or leakage through the bacterial membrane was not observed. At low concentrations (0.125-0.25 µg/ml) the ethanol extract of Ya-Pit-Samut-Noi inhibited violacein production by Chromobacterium violaceum DMST 21761 without affecting the bacterial growth. The ethanol (31.25-62.5 µg/ml) and water (31.25-250 µg/ml) extracts of Ya-Pit-Samut-Noi inhibited biofilm production by S. aureus. The ethanol and water extracts of Ya-Pit-Samut-Noi at 1000 µg/ml reduced the swarming motility of Escherichia coli O157: H7 by 74.98% and 52.65%, respectively. Tannins and terpenoids were detected in both the ethanol and water extracts. Flavonoids were present only in the ethanol extract. Alkaloids and antraquinones were not noticed in either extract. In the acute toxicity study, there were no significant changes in hematological and biochemical parameters nor were adverse effects on mortality, general behaviors, body weight, or organ weights detected. CONCLUSIONS: The scientific evidence from this study supported the therapeutic effects and safety of the traditional Thai herbal formulation 'Ya-Pit-Samut-Noi' which has been used as an alternative treatment for gastrointestinal infections in Thailand.

Traditional tonifying polyherbal infusion, Jatu-Phala-Tiga, exerts antioxidant activities and extends lifespan of Caenorhabditis elegans.

BACKGROUND: The imbalance between the generation of free radicals and natural cellular antioxidant defenses, known as oxidative stress, can cause oxidation of biomolecules and further contribute to aging-associated diseases. The purpose of this study was to evaluate the antioxidant capacities of Thai traditional tonifying preparation, Jatu-Phala-Tiga (JPT) and its herbal ingredients consisting of Phyllanthus emblica, Terminalia arjuna, Terminalia chebula, and Terminalia bellirica and further assess its effect on longevity. METHOD: Antioxidant activities of various extracts obtained from JPT and its herbal components were carried out using well-established methods including metal chelating, free radical scavenging, and ferric reducing antioxidant power assays. Qualitative analysis of the chemical composition from JPT water extract was done by high-performance liquid chromatography tandem with electrospray ionisation mass spectrometry. The effect of JPT water extract on the lifespan of Caenorhabditis elegans were additionally described. RESULTS: Among the extracts, JPT water extract exerted remarkable antioxidant activities as compared to the extracts from other solvents and individual constituting plant extract. JPT water extract was found to possess the highest metal chelating activity, with an IC50 value of 1.75 ± 0.05 mg/mL. Moreover, it exhibited remarkable scavenging activities towards DPPH, ABTS, and superoxide anion radicals, with IC50 values of 0.31 ± 0.02, 0.308 ± 0.004, and 0.055 ± 0.002 mg/mL, respectively. The ORAC and FRAP values of JPT water extract were 40.338 ± 2.273 µM of Trolox/µg of extract and 23.07 ± 1.84 mM FeSO4/mg sample, respectively. Several well-known antioxidant-related compounds including amaronols, quinic acid, gallic acid, fertaric acid, kurigalin, amlaic acid, isoterchebin, chebulagic acid, ginkgolide C, chebulinic acid, ellagic acid, and rutin were found in this extract. Treatment with JPT water extract at 1 and 5 mg/mL increased C. elegans lifespan under normal growth condition (7.26 ± 0.65 vs. 10.4 0± 0.75 (p < 0.01) and 10.00 ± 0.73 (p < 0.01) days, respectively). CONCLUSIONS: The results indicated that JPT and its herbal ingredients exhibited strong antioxidant activities, in particular the water extract of the polyherbal tonic. These findings rationalize further investigation in JPT infusion as a promising agent for anti-aging and oxidative stress prevention.

Effects of a traditional Thai polyherbal medicine 'Ya-Samarn-Phlae' as a natural anti-biofilm agent against Pseudomonas aeruginosa.

Ya-Samarn-Phlae (YaSP) is a traditional Thai polyherbal formula for the treatment of chronic wounds. Although its ethanol extract has been proven to possess several wound-related biological activities, there is no scientific information available for the infused oil of YaSP, which is its traditionally prepared form. This present study therefore aimed to evaluate the efficacy of different infused oils obtained from either fresh or oven-dried herbal parts of YaSP (F-YaSP and D-YaSP) against biofilms of Pseudomonas aeruginosa, which reside in chronic wounds. Its main active herbal component, Garcinia mangostana (F-GM and D-GM), as well as α-mangostin were also tested in this study. All infused oils significantly inhibited the biofilm formation of P. aeruginosa with a percentage of reduction ranging from 50 to 90%. Visualization of the inhibition of biofilm development was confirmed using scanning electron and atomic force microscopes. All tested agents resulted in a reduction in the mean average roughness of the biofilm, whereas only treating with D-YaSP, D-GM, and α-mangostin led to a decrease in both peak height and peak-valley height. MTT reduction assays revealed that the metabolic activity of P. aeruginosa mature biofilms decreased considerably up to 50% after only 3 h of incubation and after only 9 h of exposure to D-YaSP. Confocal laser scanning micrographs illustrated that a maximum biofilm eradication was found when treated with the extracts for 3 h, whereas the biomass, the average thickness, maximum thickness, and the surface to volume ratio of the treated biofilm was reduced after up to 18 h of contact time. It can be concluded that D-YaSP can effectively inhibit biofilm formation and eradicate mature biofilms of P. aeruginosa. It should be noted that G. mangostana and α-mangostin contribute in YaSP as principle active agents for anti-biofilm efficacy.

New alkylamide from the stems of Zanthoxylum nitidum.

A new alkylamide, named (2E,6E,8E)-N-(2-methylpropyl)-10-oxo-2,6,8-decatrienamide (1), together with 22 known compounds (2-23), were isolated from the stems of Zanthoxylum nitidum. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR spectroscopy. The isolated compounds exhibited slightly antioxidant activities through DPPH and ABTS radical scavenging assays but showed no antibacterial activity against Streptococcus mutans ATCC2517, a dental caries causing bacteria.

Antioxidant capacities and total phenolic contents of 20 polyherbal remedies used as tonics by folk healers in Phatthalung and Songkhla provinces, Thailand.

BACKGROUND: Uses of polyherbal formulations have played a major role in traditional medicine. The present study is focused on the formulations used in traditional Thai folkloric medicine as tonics or bracers. Twenty documented polyherbal mixtures, used as nourishing tonics by the folk healers in Phatthalung and Songkhla provinces in southern Thailand, are targeted. Despite traditional health claims, there is no scientific evidence to support the utilization of polyherbal formulations. METHODS: The phenolic and flavonoid contents of the polyherbal formulations and a series of antioxidant tests were applied to measure their capability as preventive or chain-breaking antioxidants. In addition, the cytotoxic activity of effective formulations was assayed in Vero cells. RESULTS: Ninety-eight plant species belonging to 45 families were used to prepare the tested formulation. The preliminary results revealed that water extracts of THP-R016 and THP-R019 contain a high level of total phenolic and flavonoid contents and exhibit remarkable antioxidant activities, as tested by DPPH, ABTS, and FRAP assays. The extract of THP-R019 also showed the strongest metal chelating activities, whereas THP-R016 extract possessed notable superoxide anion and peroxyl radical scavenging abilities. CONCLUSIONS: The data provide evidence that the water extracts of folkloric polyherbal formulations, particularly THP-R016, are a potential source of natural antioxidants, which will be valuable in the pharmaceutical and nutraceutical industries. The free radical scavenging of THP-R016 may be due to the contribution of phenolic and flavonoid contents. Useful characteristics for the consumer, such as the phytochemical profiles of active ingredients, cellular based antioxidant properties and beneficial effects in vivo, are under further investigation.

Lupinifolin from Albizia myriophylla wood: A study on its antibacterial mechanisms against cariogenic Streptococcus mutans.

OBJECTIVE: To determine the anti-Streptococcus mutans mechanisms of action of lupinifolin from Albizia myriophylla Benth. (Fabaceae) wood and provide scientific evidence to support the traditional use of the plant against dental caries. METHODS: The minimum inhibitory concentration (MIC) was evaluated using the broth micro-dilution method. The effects of lupinifolin on bactericidal activity, bacterial cell walls, and membranes were investigated by time-kill, lysis, and leakage assays, respectively. Electron microscopy was utilized to observe any cell morphological changes caused by the compound. Localization of lupinifolin in S. mutans was detected using the thin layer chromatography technique. RESULTS: The MIC range of lupinifolin against S. mutans (n=6) was 2-4 µg/ml. This compound displayed bactericidal effects on S. mutans ATCC 25175 by 90-99.9% killing at 4MIC-16MIC after 8-24 hours. Lupinifolin-treated cells demonstrated no lysis. However, significant cytoplasmic leakage through the bacterial membrane was observed after treatment with lupinifolin at 4MIC-16MIC. As revealed by ultrastructural analysis, lupinifolin produced some changes in bacterial cell walls and membranes. Moreover, the compound was observed in the cytoplasmic fraction of the lupinifolin-treated cells. These results suggest that lupinifolin can enter the cell of bacteria but does not accumulate in the cell envelope and subsequently disrupts the integrity of the cytoplasmic membrane, leading to cell death. CONCLUSION: The scientific evidence from this study offers valuable insights into the potential role of lupinifolin in pharmaceutical and antibiotic applications and supports the therapeutic effects of A. myriophylla, which has traditionally been used as an alternative treatment for dental caries.

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.

Potential wound healing activity of Quercus infectoria formulation in diabetic rats.

BACKGROUND: Quercus infectoria G. Olivier (Fagaceae) nutgalls have been widely employed in traditional Asian medicine for several treatments, especially wounds and skin disorders. However, the effects of this plant on wound healing have not yet been clearly elucidated. This present work was focused on utilization of Quercus infectoria (Qi) as a topical agent for chronic wound treatment. METHODS: Twenty Qi formulations (QiFs) were pharmaceutically formulated and antibacterial activity of all formulations was performed. The best formulation based on an antibacterial activity was selected for evaluation of wound healing property. Total phenolics, total flavonoids, and an anti-oxidant activity of the selected formulation were also investigated. Wound healing activity was assessed in streptozotocin-induced diabetic rats and control rats. Streptozotocin injection (50 mg/kg) was found to induce marked hyperglycaemia, compared with citrate-injected controls. Two wounds were created on the upper back of each animal. QiF was topically applied three days after wounding to one of the duplicate wounds on each animal and physiological saline (control) was applied to the other. All wounds were cleaned once a day until wound closure. RESULTS: QiF10, which exhibited antibacterial and anti-oxidant activities, had the ability to enhance the wound healing process in diabetic rats with abundant cellular infiltration, collagen deposition, and re-epithelialization when compared with the control. DISCUSSION: This study suggested that QiF10 could be a novel alternative treatment for diabetic wounds.

Improved Lipid Profile Associated with Daily Consumption of Tri-Sura-Phon in Healthy Overweight Volunteers: An Open-Label, Randomized Controlled Trial.

Tri-Sura-Phon (TSP), a traditional Thai polyherbal formula renowned for its rejuvenating properties, is commonly used as a blood tonic. It comprises Cinnamomum bejolghota, Cinnamomum parthenoxylon, and Aquilaria crassna. The aim of this study is to evaluate the beneficial properties of TSP tea consumption on blood glucose regulation and serum lipid profiles of healthy overweight volunteers. This open-label, randomized controlled trial was conducted in 70 healthy overweight adults. Two groups of 35 subjects took a TSP infusion or a placebo (cornstarch) twice daily for 8 weeks. The blood glucose regulation, serum lipid profiles, BMI, and liver function tests of the subjects were determined at the baseline, 4th week, and endpoint (8th week). Significant decreases in the average fasting levels of total cholesterol (p = 0.013), triglyceride (p = 0.001), and low-density lipoprotein (LDL, p = 0.017) were observed in the TSP group at the 8th week compared to those at the baseline. The average HDL level in the TSP group at the beginning of the study was 65.2 mg/dL, and it increased significantly (p = 0.005) to 72.4 mg/dL after 8 weeks of TSP intake. This study showed that the intake of TSP tea as an antioxidant-rich beverage might be safe and improve lipid profiles in overweight adults.

In vitro and in vivo assessments of Rhodomyrtus tomentosa leaf extract as an alternative anti-streptococcal agent in Nile tilapia (Oreochromis niloticus L.).

PURPOSE: Rhodomyrtustomentosa is a Thai medicinal plant that has been attracting attention for its remarkable antibacterial properties against Gram-positive pathogenic bacteria. The purpose of this study was to evaluate the antibacterial properties of R. tomentosa leaf extract against Streptococcus agalactiae and Streptococcus iniae isolated from infected tilapia. METHODOLOGY: The anti-streptococcal activity of R. tomentosa was determined using broth microdilution assays. RESULTS: The extract demonstrated strong antibacterial activity against the fish pathogens, with minimum inhibitory concentrations (MICs) ranging from 7.8‒62.5 µg ml-1. It was found to possess a dose-dependent bacteriostatic effect on this organism. Scanning electron microscopy revealed irregular and long chains of swollen cells, as well as corkscrew shapes andincomplete separation of cell division of S. agalactiae cells following the treatment at sub-MIC. Moreover, S. agalactiae cells pre-treated with the extract became more sensitive to oxidative stress induced by H2O2 than the untreated cells. Based on the mortality of Nile tilapia after intraperitoneal infection of S. agalactiae at median lethal dose (LD50), the pre-treated cells caused a significant (P<0.01) reduction in mortality of S. agalactiae-infected Nile tilapia. CONCLUSION: The results suggested that R. tomentosa could be further developed as a simple and effective agent for the treatment of streptococcosis in Nile tilapia.

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.

Evaluation of antibacterial activity, phytochemical constituents, and cytotoxicity effects of Thai household ancient remedies.

AIM: Household ancient remedies reported here are described in the National List of Essential Medicines and have traditionally been used in Thailand to treat infection-related ailments. However, the safety and effectiveness of these remedies have been poorly evaluated. The aim of this study was to evaluate the antibacterial properties of these remedies against seven gram-positive and gram-negative multidrug-resistant bacteria species. Phytochemical constituents and cytotoxicity of these remedies were also determined. METHODS: Seven remedies, consisting of Um-Ma-Luk-Ka-Wa-Tee, Chan-Ta-Lee-La, Kheaw-Hom, Learng-Pid-Sa-Mud, Pra-Sa-Chan-Dang, Dhart-Ban-Chob, and Tree-Hom, were prepared by a licensed traditional medical doctor using a mixture of medicinal plants. Antibacterial activity of ethanol extracts of the remedies was determined by using a broth microdilution method. Qualitative phytochemical screening analysis was carried out to identify the presence of major components. Cytotoxicity activities of the extracts against Vero cells were assessed by green fluorescent protein-based assay. RESULTS: With the exception of Dhart-Ban-Chob extract, significant minimum inhibitory concentrations (MICs) of <16 to 32 µg/mL were observed for the remedy extracts depending on the bacterial strains. The Um-Ma-Luk-Ka-Wa-Tee extract was noncytotoxic against Vero cells and possessed the highest activity, with MICs of <16 to 31 µg/mL against all methicillin-resistant Staphylococcus aureus isolates. CONCLUSIONS: Remarkable antibacterial activities against multidrug-resistant pathogens, as well as low toxicity on Vero cells, of Um-Ma-Luk-Ka-Wa-Tee support the use of this remedy in traditional medicine. Further investigation on other biological activities related to traditional applications, appropriate biomarkers, and treatment mechanisms of the household remedy are required.

Inhibition of microbial adhesion to plastic surface and human buccal epithelial cells by Rhodomyrtus tomentosa leaf extract.

OBJECTIVE: The adherence of oral pathogenic microorganisms to host tissues is the initial step for successful process of oral diseases. This study aimed to determine the effect of the Rhodomyrtus tomentosa leaf extract and rhodomyrtone, an antibacterial compound from R. tomentosa leaf, on adhesion of some oral pathogens to polystyrene plastic surface and human buccal epithelial cells. METHODS: The minimum inhibitory concentration (MIC) was evaluated using broth microdilution method. The microbial adhesion to the plastic surface and buccal cells was determined using microtiter plate method and microscopy technique. RESULTS: The ethanol extract of leaf demonstrated antibacterial activity against oral microorganisms including Staphylococcus aureus ATCC 25923, Streptococcus mutans (clinical isolate), and Candida albicans ATCC 90028 with the MIC values of 31.25, 15.62, and 1000µg/ml, respectively. Rhodomyrtone displayed activity with the MIC values of 0.78 and 0.39µg/ml against S. aureus ATCC 25923 and S. mutans, respectively. The MIC value of the compound against C. albicans ATCC 90028 was more than 100µg/ml which was the highest test concentration. All pathogenic microorganisms treated with the extract and rhodomyrtone at their subinhibitory concentrations resulted in a decrease in their adherence ability to both plastic surface and buccal cells. CONCLUSION: It is suggested that R. tomentosa extract and rhodomyrtone may be useful in therapy or as prophylaxis in infections involving oral pathogens.