Curcuma comosa loaded transfersomal gel for transdermal application: formulation, in vitro and in vivo evaluation.

RATIONAL: Diarylheptanoids, extracted from the Curcuma comosa (CC) rhizome, have been reported to exhibit estrogenic activity. However, oral administration of the extract showed a short half-life. OBJECTIVES: This study aimed to formulate and to investigate the potential of transfersomal gels for the transport of phytoestrogenic diarylheptanoids across the skin into the blood circulation. MATERIALS AND METHODS: The transfersomes were developed and optimized for their compositions including sources of phospholipid (egg yolk and soybean), types of edge activators (polysorbate 80, sorbitan oleate 80, and sodium cholate), and concentrations of CC extract (10-60 mg). The optimal formulation was further incorporated into Carbopol® Ultrez 21 gel and evaluated for in vitro release, permeation, and in vivo absorption. RESULTS: The optimal transfersomes containing 10% of polysorbate 80 were selected due to high drug entrapment efficiency and a small diameter. The release kinetic of transfersomal gels followed a zero model. The maximum permeation flux through porcine ear skin was 1.38 ± 0.25 µg/cm2/h for (4E, 6E)-1, 7-diphenylhepta-4, 6-dien-3-ol, and 0.40 ± 0.11 µg/cm2/h for (6E)-1, 7-diphenylhept-6-en-3-ol. Results of the in vivo pharmacokinetics study in rats showed that transfersomal gel provided a maximum concentration of 219.71 ± 4.05 ng/ml and prolonged plasma concentration of diarylheptanoids for over 12 h. There was no significant variation found in the physical characteristics including viscosity, pH, and size after six months of storage at room temperature (30 ± 1 °C) and high temperature (40 ± 1 °C). CONCLUSIONS: The obtained data suggested that the developed transfersomal gel of CC extract should be beneficial for improving the delivery of phytoestrogenic diarylheptanoids.

In vivo effect of Kaempferia parviflora extract on pharmacokinetics of acetaminophen.

Kaempferia parviflora is widely used as a food supplement and a herbal medicine for vitalization. Previous study has shown that K. parviflora had CYP2E1 inducer activity. It is likely to affect the metabolism of CYP2E1 substrates such as acetaminophen which is a common household pain relief medicine. This study investigated the possible pharmacokinetic interaction between K. parviflora and acetaminophen in rats. Acetaminophen (100 mg/kg, p.o) was administered to rats for nine consecutive days. On days 4-9, K. parviflora extract (250 mg/kg, p.o) was given to the acetaminophen-treated rats. After co-administration with K. parviflora, the concentrations of acetaminophen during day 5-8 markedly decreased compared with acetaminophen-only group. At day 9, the pharmacokinetic parameters of acetaminophen in the presence of K. parviflora extract also decreased, including area under the concentration-time curve (from 1.68 ± 0.16 to 0.34 ± 0.04 mg.min/mL), the maximum concentration (from 19.10 ± 1.90 to 4.48 ± 0.56 µg/mL), and half-life (from 21.29 ± 1.36 to 10.81 ± 1.24 min). In addition, clearance and the elimination rate constant of acetaminophen were significantly increased (from 0.003 ± 0.000 to 0.006 ± 0.001 L/min and 0.03 ± 0.00 to 0.07 ± 0.01 min-1, respectively) in the presence of K. parviflora extract. These findings provide the data for in vivo herb-drug interaction between K. parviflora extract and acetaminophen. Therefore, the concomitant use of K. parviflora as a food supplement and acetaminophen should occasion therapeutic and safety concerns.

Antitumor Efficacy of the Herbal Recipe Benja Amarit against Highly Invasive Cholangiocarcinoma by Inducing Apoptosis both In Vitro and In Vivo.

Thailand is the country with highest incidence and prevalence of cholangiocarcinoma (CCA) in the world. Due to the frequently late diagnosis that is associated with this disease, most CCA patients are prescribed chemotherapy as a form of treatment. However, CCA is able to resist the presently available chemotherapy, so to the prognosis of this disease is still very poor. In this study, we investigated the anticancer potential of a Thai herbal recipe, Benja Amarit (BJA) against CCA and the relevant mechanisms of action that are involved. We found that BJA inhibited CCA cell viability in a dose-dependent manner, especially in highly invasive KKU-213 cells. The extract induced mitochondrial- and caspase-dependent apoptosis in CCA cells by regulating the nuclear factor-κB (NF-κB) signaling pathway. BJA also triggered autophagy in CCA cells. Nonetheless, the inhibition of autophagy enhanced BJA-induced CCA cell death via apoptosis. An in vivo xenograft model revealed the growth-inhibiting and death-inducing effects of BJA against CCA by targeting apoptosis. However, general toxicity to blood cells, kidneys and the liver, as well as changes in body weight, did not appear. Our findings suggest that the herbal recipe BJA might be used as a potentially new and effective treatment for cholangiocarcinoma patients.

Potential of Thai Herbal Extracts on Lung Cancer Treatment by Inducing Apoptosis and Synergizing Chemotherapy.

The incidence of lung cancer has increased while the mortality rate has continued to remain high. Effective treatment of this disease is the key to survival. Therefore, this study is a necessity in continuing research into new effective treatments. In this study we determined the effects of three different Thai herbs on lung cancer. Bridelia ovata, Croton oblongifolius, and Erythrophleum succirubrum were extracted by ethyl acetate and 50% ethanol. The cytotoxicity was tested with A549 lung cancer cell line. We found four effective extracts that exhibited toxic effects on A549 cells. These extracts included ethyl acetate extracts of B. ovata (BEA), C. oblongifolius (CEA), and E. succirubrum (EEA), and an ethanolic extract of E. succirubrum (EE). Moreover, these effective extracts were tested in combination with chemotherapeutic drugs. An effective synergism of these treatments was found specifically through a combination of BEA with methotrexate, EE with methotrexate, and EE with etoposide. Apoptotic cell death was induced in A549 cells by these effective extracts via the mitochondria-mediated pathway. Additionally, we established primary lung cancer and normal epithelial cells from lung tissue of lung cancer patients. The cytotoxicity results showed that EE had significant potential to be used for lung cancer treatment. In conclusion, the four effective extracts possessed anticancer effects on lung cancer. The most effective extract was found to be E. succirubrum (EE).

Kaempferia parviflora Nanosuspension Formulation for Scalability and Improvement of Dissolution Profiles and Intestinal Absorption.

Kaempferia parviflora (KP) is an herbal medicine for enhancement of physical fitness and male sexual function improvement with low oral absorption of the main active compounds, methoxyflavones. The purpose of this study is to optimize the preparation of nanosuspensions of KP extract for enhancing intestinal absorption using antisolvent precipitation technique which is an accessible nanomanufacturing methodology in the small industrial factory. Nanosuspensions were prepared using various types and concentrations of stabilizers. Then, the dry powder of KP nanosuspension was produced by spray drying. Its dissolution rate was determined using USP dissolution apparatus II. The rat everted intestinal sac was tested to confirm the improvement of intestinal absorption of KP nanosuspension. The result showed that 3% sodium lauryl sulfate (SLS) was the optimal condition for covering the nano-size of KP nanosuspension. KP nanosuspensions had particle sizes ranging from 100 to 300 nm with narrow size distribution (PDI < 0.60) and zeta potential at - 58 to - 70 mV. These characteristics were stable at 4°C and 25°C/60%RH for 1-month storage. Its methoxyflavones content also unchanged at 4°C and 25°C/60%RH for 1-month storage. KP nanosuspension released > 80% of the methoxyflavones within 30 min both in 0.1 N HCl and 0.01 M phosphate buffer solution (pH 6.8). Moreover, the developed nanosuspension dramatically improved the rat intestinal absorption about 10-fold. Therefore, the KP nanosuspension was successfully prepared. It has relatively high stability, fast dissolution rate, and high intestinal absorption.

Development and evaluation of topical films containing phytoestrogenic diaryheptanoids from Curcuma comosa extract.

RATIONAL: Phytoestrogens have been found to delay signs of skin aging in post-menopausal women, in a way similar to the effects of estrogens. Diarylheptanoids from a rhizome of traditional Thai herb named Curcuma comosa is considered to be a novel class of phytoestrogens. OBJECTIVES: The aims of this study were to prepare effective topical films using mixed types and vary ratios of hydrophobic (Eudragit RL, Eudragit RS, and Eudragit NE) and hydrophilic polymer (hydroxylpropyl methycellulose, HPMC) with Transcutol as a permeation enhancer for delivery of diarylheptanoids to improve signs of skin aging in post-menopausal women. MATERIAL AND METHODS: Topical films were characterized for their physical and mechanical properties. In vitro release, skin permeation and accumulation were evaluated using Franz diffusion cell and the concentrations of diarylheptanoids were determined using high-performance liquid chromatography. RESULTS: The combined formulations between HPMC and Eudragit NE showed the satisfactory physical and mechanical properties, and also provided the highest amount of drug released compared to Eudragit RL and Eudragit RS. When the proportion of HPMC amount in the polymer matrix increased, the cumulative drug release also increased (HPMC: Eudragit NE 6:4 > 5:5 > 4:6). Moreover, they provided a high accumulation of diarylheptanoids within skin when using transcutol as a permeation enhancer. CONCLUSION: The obtained data provided the skin permeation and accumulation behavior of diarylheptanoids, indicating the feasibility of a skin delivery of the C. comosa extract. The developed films might be topically used as an alternative therapy for protection of skin aging in peri and post-menopausal women.

Effects of Vehicles and Enhancers on the Skin Permeation of Phytoestrogenic Diarylheptanoids from Curcuma comosa.

Curcuma comosa (C. comosa) is widely used in traditional medicine as a dietary supplement for health promotion in postmenopausal women in Thailand. It contains several diarylheptanoids, which are considered to be a novel class of phytoestrogens. However, the diarylheptanoids isolated from the plant rhizome are shown to have low oral bioavailability and faster elimination characteristics. The aim of this study was to investigate the permeation behavior of the active compounds of diarylheptanoids. The effects of binary vehicle systems and permeation enhancers on diarylheptanoids permeation and accumulation within the skin were studied using side-by-side diffusion cells through the porcine ear skin. Among the tested binary vehicle systems, the ethanol/water vehicle appeared to be the most effective system for diarylheptanoids permeation with the highest flux and shortest lag time. The presence of transcutol in the vehicle system significantly increased diarylheptanoid's permeation and accumulation within the skin in a concentration-dependent manner. Although the presence of terpenes in formulation decreased the flux of diarylheptanoids, it raised the amount of diarylheptanoids retained within the skin substantially. Based on the feasibility of diarylheptanoid permeation, C. comosa extract should be further developed into an effective transdermal product for health benefits and hormone replacement therapy.

Antioxidant effect of Phyllanthus amarus after moderate-intensity exercise in sedentary males: a randomized crossover (double-blind) study.

[Purpose] We aimed to evaluate the effects of Phyllanthus amarus (PA) on oxidative stress and damage, inflammation, and soreness in muscle after a single session of moderate-intensity exercise. [Subjects and Methods] Twelve men randomly participated in 2, three-day phases with a one-week washout period. On the first day, participants consumed two capsules of PA or placebo control (CTL) before 20 min of cycling. They then consumed four capsules on the same day after exercise and six capsules/day for the next two days. Blood samples were obtained before, immediately after exercise and 24 h and 48 h after exercise. The pain tolerance was measured at both legs. [Results] Plasma vitamin C levels in the PA group were higher than those in the CTL group after exercise. At 48 h after exercise, vitamin C levels were higher in the PA group, but those in the CTL group were lower than the pre-exercise levels. However, plasma levels of creatine kinase were increased in both groups after exercise compared with the pre-exercise levels. The neutrophil count was higher immediately after exercise than the pre-exercise levels in the CTL group. [Conclusion] Acute supplementation with PA improves antioxidant status after a single session of moderate-intensity exercise.

Hair growth-promoting effect of Carthamus tinctorius floret extract.

The florets of Carthamus tinctorius L. have traditionally been used for hair growth promotion. This study aimed to examine the potential of hydroxysafflor yellow A-rich C. tinctorius extract (CTE) on hair growth both in vitro and in vivo. The effect of CTE on cell proliferation and hair growth-associated gene expression in dermal papilla cells and keratinocytes (HaCaT) was determined. In addition, hair follicles from mouse neonates were isolated and cultured in media supplemented with CTE. Moreover, CTE was applied topically on the hair-shaved skin of female C57BL/6 mice, and the histological profile of the skin was investigated. C. tinctorius floret ethanolic extract promoted the proliferation of both dermal papilla cells and HaCaT and significantly stimulated hair growth-promoting genes, including vascular endothelial growth factor and keratinocyte growth factor. In contrast, CTE suppressed the expression of transforming growth factor-ß1 that is the hair loss-related gene. Furthermore, CTE treatment resulted in a significant increase in the length of cultured hair follicles and stimulated the growth of hair with local effects in mice. The results provided the preclinical data to support the potential use of CTE as a hair growth-promoting agent.

Development of a nanoemulsion of Phyllanthus emblica L. branch extract.

For potential topical administration, we formulated a nanoemulsion containing phenolic constituents of Phyllanthus emblica branch extract. The nanoemulsion has high entrapment efficiency, small particle size, is stable, and can release its main chemical components. Branches of P. emblica were extracted with 50% ethanol (EPE) with 5.4% yield. HPLC analysis indicated several phenolic compounds, including gallic acid, vanillic acid, epigallocatechin (EGC), epigallocatechin gallate (EGCG) and ellagic acid. These were selected as chemical markers of EPE in the nanoemulsion development. The nanoemulsion was prepared by microemulsion techniques with hot high pressure homogenization. A ternary phase diagram was constructed to obtain the optimized nanoemulsion. The obtained transparent EPE nanoemulsion is composed of isopropyl myristate (0.6% w/w), Brij® 78 (0.35% w/w), and 0.15% (w/w) EPE. The optimized EPE nanoemulsion had a median particle size of 191.63 ± 4.07 nm with a narrow particle size distribution, a zeta potential of -10.19 ± 0.54 mV, high entrapment efficiency at 67.99 ± 0.87% and good stability at 4 °C after 90 d of storage. The release of active ingredients from the EPE nanoemulsion was slower than that of the EPE aqueous formulation. The loading ratios of the five phenolic compounds were high, with relative order of EGC > EGCG > vanillic acid > gallic acid > ellagic acid, resulting in slow release profiles of EGC and EGCG in the EPE nanoemulsion. In conclusion, the obtained EPE nanoemulsion has good characteristics for future clinical trials.

Transdermal permeation of Kaempferia parviflora methoxyflavones from isopropyl myristate-based vehicles.

Kaempferia parviflora (K. parviflora) rhizomes have long been used in traditional folk medicines and as general health-promoting agents. Several biological activities of K. parviflora, especially its anti-inflammatory effect, are due to its major constituents, methoxyflavones. However, the oral bioavailability of these methoxyflavones has been shown to be low. The aim of this study was to investigate the permeation behaviors of K. parviflora methoxyflavones from isopropyl myristate (IPM)-based vehicles. We studied the effects of ethanol and propylene glycol (PG) as the hydrophilic, solvent-type vehicles as well as fatty acids as the permeation enhancers. A permeation experiment was performed in vitro, using side-by-side diffusion cells through the full thickness of pig ear skin. The solubility and permeation of methoxyflavones were able to be modified by choice and ratio of vehicles. The ethanol/IPM vehicle was shown to be more effective in enhancing the solubility and permeation of methoxyflavones when compared to the PG/IPM vehicle. Regarding an optimal balance between solubility or affinity to vehicle and skin to vehicle partition coefficient, the ethanol/IPM vehicle in the ratio of 1:9 maximized the flux. Among the investigated fatty acids, oleic acid showed the greatest enhancing effect on the permeation of methoxyflavones, indicating that saturated fatty acids are less effective than unsaturated fatty acids. Long chain fatty acids increased diffusion coefficient parameter and shortened the lag time. The number of carbon atoms and double bonds of fatty acids did not show direct relation to the profile of permeation of methoxyflavones.

Thai traditional medicines reduce CD147 levels in lung cells: Potential therapeutic candidates for cancers, inflammations, and COVID-19.

ETHNOPHARMACOLOGICAL RELEVANCE: The cluster of differentiation 147 (CD147) is identified as the signaling protein relevant importantly in various cancers, inflammations, and coronavirus disease 2019 (COVID-19) via interacting with extracellular cyclophilin A (CypA). The reduction of CD147 levels inhibits the progression of CD147-associated diseases. Thai traditional medicines (TTMs): Keaw-hom (KH), Um-ma-ruek-ka-wa-tee (UM), Chan-ta-lee-la (CT), and Ha-rak (HR) have been used as anti-pyretic and anti-respiratory syndromes caused from various conditions including cancers, inflammations, and infections. Thus, these medicines would play a crucial role in the reduction of CD147 levels. AIM OF THE STUDY: This article aimed to investigate the effects of KH, UM, CT, and HR for reducing the CD147 levels through in vitro study. Additionally, in silico study was employed to screen the active compounds reflexing the reduction of CD147 levels. MATERIALS AND METHODS: The immunofluorescent technique was used to evaluate the reduction of CD147 levels in human lung epithelial cells (BEAS-2B) stimulated with CypA for eight extracts of KH, UM, CT, and HR obtained from water decoction (D) and 70% ethanol maceration (M) including, KHD, UMD, CTD, HRD, KHM, UMM, CTM, and HRM. RESULTS: UM extracts showed the most efficiency for reduction of CD147 levels in the cytoplasm and perinuclear of BEAS-2B cells stimulated with CypA. Phenolic compounds composing polyphenols, polyphenol sugars, and flavonoids were identified as the major chemical components of UMD and UMM. Further, molecular docking calculations identified polyphenol sugars as CypA inhibitors. CONCLUSIONS: UMD and UMM are potential for reduction of CD147 levels which provide a useful information for further development of UM as potential therapeutic candidates for CD147-associated diseases such as cancers, inflammations, and COVID-19.

Increased in situ intestinal absorption of phytoestrogenic diarylheptanoids from Curcuma comosa in nanoemulsions.

Curcuma comosa has long been used as a gynecological medicine. Several diarylheptanoids have been purified from this plant, and their pharmacological effects were proven. However, there is no information about the absorption of C. comosa components to support the formulation usage. In the present study, C. comosa hexane extract and the mixture of its two major compounds, (4E,6E)-1,7-diphenylhepta-4,6-dien-3-ol (DA1) and (6E)-1,7-diphenylhept-6-en-3-ol (DA2), were formulated into nanoemulsions. The physical properties of the nanoemulsions and the in situ intestinal absorptions of DA1 and DA2 were evaluated. The results demonstrated the mean particle sizes at 0.207 ± 0.001 and 0.408 ± 0.014 µm, and the zeta potential at -14.57 ± 0.85 and -10.47 ± 0.32 mV for C. comosa nanoemulsion (C.c-Nano) and mixture of diarlylheptanoid nanoemulsions (DA-Nano), respectively. The entrapments of DA1 and DA2 were 76.61% and 75.41%, and 71.91% and 71.63% for C.c-Nano and DA-Nano, respectively. The drug loading ratios of DA1 and DA2 were 351.47 and 614.53 µg/mg, and 59.48 and 126.72 µg/mg for C.c-Nano and DA-Nano. The intestinal absorption rates of DA1 and DA2 were 0.329 ± 0.015 and 0.519 ± 0.026 µg/min/cm2 in C.c-Nano, and 0.380 ± 0.006 and 0.428 ± 0.036 µg/min/cm2 in DA-Nano, which were five to ten times faster than those in oil. In conclusion, the formulation in nanoemulsion forms obviously increased the intestinal absorption rate of diarylheptanoids.

Formulation and In Vitro Evaluation of Mucoadhesive Sustained Release Gels of Phytoestrogen Diarylheptanoids from Curcuma comosa for Vaginal Delivery.

Diarylheptanoids (DAs) characterized by a 1,7-diphenylheptane structural skeleton are considered a novel class of phytoestrogens. The DAs available in Curcuma comosa Roxb. (C. comosa) extract demonstrated significant estrogenic activities both in vitro and in vivo. This study aimed to develop and comprehensively evaluate a mucoadhesive vaginal gel for the sustained release of DAs. Different mucoadhesive polymers as gelling agents were investigated. C. comosa ethanolic crude extract was used as a source of DAs. All C. comosa gels were light brown homogeneous with pH within 4.4-4.6. Their flow behaviors were pseudoplastic with a flow behavior index of 0.18-0.38. The viscosity at a low shear rate varied from 6.2 to 335.4 Pa·s. Their mechanical and extrudability properties were associated well with rheological properties. Polycarbophil (PCP):hydroxypropyl methylcellulose (HPMC) blends had a higher mucoadhesiveness to porcine vaginal mucosa than those of PCP-based or HPMC-based gels. All C. comosa gels exhibited a sustained, zero-order DA release pattern over 72 h. Korsmeyer and Peppas equation fitting indicated a non-Fickian, case II transport release mechanism. C. comosa gels had good physical and chemical stability under low-temperature storage for up to 12 months. PCP:HPMC-based mucoadhesive gels could be a proper delivery system for vaginal administration of DAs.

Pharmacokinetics, bioavailability, tissue distribution, excretion, and metabolite identification of methoxyflavones in Kaempferia parviflora extract in rats.

Kaempferia parviflora (KP) is an herbal plant in the family of Zingiberaceae. KP mainly contains methoxyflavones, especially 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF). The present study was designed to characterize the pharmacokinetics, including bioavailability, distribution, excretion, and identification of metabolites after administration of a KP ethanolic extract. Male rats were orally or intravenously administered a 250 mg/kg concentration of a KP extract, and blood samples were obtained at selected times to determine pharmacokinetic parameters of PMF, TMF, and DMF. For distribution and excretion studies, the organs, urine, and feces samples were collected at various times after oral administration of a larger (750 mg/kg) dose of KP extract. Methoxyflavones in the biological samples were quantified by high-performance liquid chromatography-UV, and the metabolites in urine and feces were further identified by using liquid chromatography-tandem mass spectrometry. After oral administration, concentrations of the three methoxyflavones quickly approached their maximal concentration, ranging from 0.55 to 0.88 µg/ml within 1 to 2 h after administration, and then were gradually excreted with half-lives of 3 to 6 h. The methoxyflavones showed low oral bioavailability of 1 to 4%. Three methoxyflavones were detected at their highest levels in liver followed by kidney. They were also found in lung, testes, and brain. After absorption, organ distribution, and metabolism, the components of KP were mainly eliminated through urine in the forms of demethylated, sulfated, and glucuronidated products and as demethylated metabolites in the feces. The parent compounds were found to have 0.79, 1.76, and 3.10% dose recovery in urine and 1.06, 1.77, and 0.96% dose recovery in feces for PMF, TMF, and DMF, respectively. These studies are the first to describe the pharmacokinetics of KP extract to provide the information on blood and tissue levels.

Positive Modulation Effect of 8-Week Consumption of Kaempferia parviflora on Health-Related Physical Fitness and Oxidative Status in Healthy Elderly Volunteers.

Health-related physical fitness declines as the age advances. Oxidative stress is reported to contribute the crucial role on this phenomenon. This condition is also enhanced by antioxidant. Therefore, we aimed to determine the effect of Kaempferia parviflora, a plant reputed for antifatigue, longevity promotion, and antioxidant effects, on health-related quality physical fitness and oxidative status of the healthy elderly volunteers. Total 45 subjects had been randomized to receive placebo or K. parviflora extract at doses of 25 or 90 mg once daily for 8 weeks. They were determined baseline data of physical performance using 30 sec chair stand test, hand grip test, 6 min walk test, and tandem test. Serum oxidative stress markers including malondialdehde (MDA) level and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were also assayed. All assessments were performed every 4 weeks throughout the 8-week study period. The results showed that K. parviflora increased performance in 30-second chair stand test and 6 min walk test together with the increased all scavenger enzymes activities and the decreased MDA level. Therefore, K. parviflora can enhance physical fitness partly via the decreased oxidative stress. In conclusion, K. parviflora is the potential health supplement for elderly. However, further study is required.

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea).

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

The effect of ovariectomy on learning and memory and relationship to changes in brain volume and neuronal density.

The loss of sex hormones in postmenopausal women has been suggested to be involved in cognitive degenerative diseases, such as Alzheimer's disease. In the present study, ovariectomized (OVX) and control rats were tested for 4 months in a Morris water maze (MWM) task to track their memory status. Thereafter, postmortem frozen brain sections were analyzed to determine if changes in brain area volumes and neuronal density were related to changes in cognitive ability. A modified artificial-land-mark-based method was used to assure the fidelity of the three dimensions (3D) reconstructed structures. Volumetric areas of the hippocampus, cortex, caudate putamen (cpu), and cerebellum were estimated from the reconstructions, and neuron densities of CA1 and CA3 subregions of the hippocampus were measured in an adjacent second series of Nissl-stained sections. Compared to the control rats, OVX rats displayed memory impairments, beginning in the second month after the ovariectomy (p < .05). Assessments at the end of the study demonstrated that OVX (compared to control) rats displayed reduced brain volume in the hippocampus and neocortex and in the brain as a whole. In contrast, when compared to controls, the volumes of cpu and cerebellum of OVX rats increased slightly. CA3 neuron density of OVX (compared to controls) rats was significantly lower, but the CA1 neuron density was significantly higher. In conclusion, ovariectomy impaired spatial memory and led to morphological changes in cognitive centers of rat brain. The results demonstrate that the 3D reconstructed method is useful for the study of brain morphological abnormality in rats.

Supplementation of Kaempferia parviflora Extract Enhances Physical Fitness and Modulates Parameters of Heart Rate Variability in Adolescent Student-Athletes: A Randomized, Double-Blind, Placebo-Controlled Clinical Study.

This randomized double-blind controlled study aimed to investigate the effects of a standardized Kaempferia parviflora (KP) extract on the physical fitness and heart rate variability (HRV) parameters in adolescent sport school students. 194 male students were recruited and randomized into two groups (n = 97), matched by age and sports. The KP-treated group received KP extract capsules at a dose of 360 mg/day and the control group received placebo capsules, continuously for 12 weeks. Physical fitness performance and HRV parameters were monitored with blood biochemical analysis for product safety. KP extract significantly increased the right-hand grip strength, the back-leg strength and maximal oxygen consumption (VO2 max) and decreased the time used for 50-meter sprint test without changing the sit-and-reach test and the 40 yard technical test. For HRV parameters, KP extract significantly increased standard deviation of normal to normal intervals (SDNN), square root of the mean of square of successive normal to normal interval differences (RMSSD) and high frequency (HF) norm, without changing low frequency (LF) norm and LF/HF ratio. The increase in stress resistance and decrease in stress index were found in the KP-treated group, without changing the autonomic nervous system (ANS) activity and balance. Blood biochemical analysis showed normal values of all participants. This data indicates the safety and positive effects of KP on muscle strength, endurance and speed, but not on the flexibility and agility. The modulatory effects of KP extract on HRV parameters suggest its anti-stress effects and would encourage the application in a sport training and exercise.

Centella asiatica Improves Physical Performance and Health-Related Quality of Life in Healthy Elderly Volunteer.

Recently, oxidative stress has been reported to contribute an important role in the decline of physical function as age advances. Numerous antioxidants can improve both physical and psychological performances resulting in the increase of health-related quality of life (HQOL). Therefore, we hypothesized that Centella asiatica, a medicinal plant reputed for nerve tonic, strength improvement and antioxidant activity, could improve the physical performance and HQOL especially in the physical satisfaction aspect, of the healthy elderly volunteer. To test this hypothesis, a double-blind, placebo-controlled, randomized trial was performed. Eighty healthy elderly were randomly assigned to receive placebo or standardized extract of C. asiatica at doses of 250, 500 and 750 mg once daily for 90 days. The subjects were evaluated to establish baseline data of physical performance using 30-s chair stand test, hand grip test and 6-min walk test. The health-related quality of life was assessed using SF-36. These assessments were repeated every month throughout the 3-month experimental period using the aforementioned parameters. Moreover, 1 month after the cessation of C. asiatica treatment, all subjects were also evaluated using these parameters again. The results showed that after 2 months of treatment, C. asiatica at doses of 500 and 750 mg per day increased lower extremity strength assessed via the 30-s chair stand test. In addition, the higher doses of C. asiatica could improve the life satisfaction subscale within the physical function subscale. Therefore, the results from this study appear to support the traditional reputation of C. asiatica on strength improvement, especially in the lower extremities of the elderly. C. asiatica also possesses the potential to be a natural resource for vigor and strength increase, in healthy elderly persons. However, further research is essential.