Hair growth is promoted by BeauTop via expression of EGF and FGF­7.

Minoxidil and finasteride have been approved to treat hair loss by the Food and Drug Administration. However, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. BeauTop (BT) is a health food supplement which contains Ginseng radix, Astragali radix, Radix Angelicae sinensis, Ligustri fructus, Rehmannia glutinosa and Eclipta prostrata (Linn). Susbsequent to oral administration of BT at 0.6 g/kg/day to wax/rosin­induced alopecia in C57BL/6 mice, BT significantly induced hair growth at day 8 compared with control treatment (P<0.05). The expression levels of epidermal growth factor (EGF), and fibroblast growth factor (FGF)­7 were increased compared with control animals on day 8. In contrast, levels of FGF­5 of the BT group were reduced compared with the control on day 12. There were no effects on the expression of insulin­like growth factor 1. The results demonstrated that the mechanism of BT improving alopecia is potentially associated with modulation of EGF and FGF­7 levels. Taken together, it is suggested that BT may have a potential effect of the promotion of hair growth.

Hair growth effect of traditional Chinese medicine BeauTop on androgenetic alopecia patients: A randomized double-blind placebo-controlled clinical trial.

The aim of the present study was to evaluate the treatment effects of BeauTop in alopecia by observing its effectiveness in improving androgenetic alopecia. Hair growth was observed using a dermatoscope and clinical photos, and was scored by three dermatologists. Dermatologists evaluated and selected suitable participants for this study using the Norwood scale or Ludwig scale. A total of 40 participants with androgenetic alopecia were recruited in this study, and 32 participants completed the 6-month trial. The results revealed that in the BeauTop treatment group, 9/17 participants (52.9%) showed increased hair growth. Changes in hair growth were as follows: No change, 47.1% patients; minimally improved, 5.9% patients; moderately improved, 29.4% patients; and significantly improved, 17.6% patients. In the placebo group, 2/15 participants (13%) showed increased hair growth. A Chi-square test was performed and attained a value of 0.01

Neuroprotective Effects of the Herbal Formula B401 in Both Cell and Mouse Models of Alzheimer's Disease.

In this study, we have reported the herbal formula B401 that has neuroprotective effects via multifunction, multitarget characteristics. It is possible that the herbal formula B401 may also provide new insights for AD. Here, we studied protective effects in the Tet-On Aß42-GFP SH-SY5Y cell model and the APP/PS1/Tau triple transgenic mouse model by the herbal formula B401. In in vitro experiments, we showed that the herbal formula B401 treatment effectively reduces glutamate-induced excitotoxicity and acetylcholinesterase activity in Tet-On Aß42-GFP SH-SY5Y cells. In in vivo experiments, we found that oral B401 treatment effectively ameliorates neurocognitive dysfunctions of 3× Tg-AD mice via motor and cognitive behavior tests. By using magnetic resonance imaging, moorFLPI instruments, and chemiluminescence methods, we reported that oral B401 treatment effectively alleviates brain atrophy, improves subcutaneous blood flow, and reduces blood ROS in 3× Tg-AD mice. As observed from results of immunohistochemistry staining and western blotting, we found that oral B401 treatment significantly enhances expressions of neuroprotective proteins, while reducing expressions of AD derived proteins such as amyloid beta, phosphorylated Tau, neurofibrillary tangles, and 3-nitrotyrosine in the brain of 3× Tg-AD mice. Thus, the herbal formula B401 may have the potential to be developed into optimum TCM for AD patients.

Oral treatment with the herbal formula B401 protects against aging-dependent neurodegeneration by attenuating oxidative stress and apoptosis in the brain of R6/2 mice.

BACKGROUND: Neurodegeneration is characterized by progressive neurological deficits due to selective neuronal loss in the nervous system. Huntington's disease (HD) is an incurable neurodegenerative disorder. Neurodegeneration in HD patients shows aging-dependent pattern. Our previous study has suggested that a herbal formula B401 may have neuroprotective effects in the brains of R6/2 mice. OBJECTIVE: To clarify possible mechanisms for neurodegeneration, which improves the understanding the aging process. This study focuses on clarifying neurodegenerative mechanisms and searching potential therapeutic targets in HD patients. METHODS: The oxidative stress and apoptosis were compared in the brain tissue between R6/2 HD mice with and without oral B401 treatment. Expressions of proteins for oxidative stress and apoptosis in the brain tissue of R6/2 HD mice were examined by using immunostaining and Western blotting techniques. RESULTS: R6/2 HD mice with oral B401 treatment significantly reduced reactive oxygen species levels in the blood, but markedly increased expressions of superoxide dismutase 2 in the brain tissue. Furthermore, R6/2 HD mice with oral B401 treatment significantly increased expressions of B-cell lymphoma 2 (Bcl-2), but significantly reduced expressions of Bcl-2-associated X protein (Bax), calpain, and caspase-3 in the brain tissue. CONCLUSION: Our findings provide evidence that the herbal formula B401 can remedy for aging-dependent neurodegeneration of R6/2 mice via suppressing oxidative stress and apoptosis in the brain. We suggest that the herbal formula B401 can be developed as a potential health supplement for ameliorating aging-dependent neurodegeneration.

Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington's disease via suppressing oxidative stress, inflammation, and apoptosis.

Cardiac failure is often observed in aging patients with Huntington's disease (HD). However, conventional pharmacological treatments for cardiac failure in HD patients have rarely been studied. Chinese herbal medicines, especially combined herbal formulas, have been widely used to treat cardiac dysfunctions over the centuries. Thus, we assess whether oral treatment with herbal formula B307 can alleviate cardiac failure in transgenic mice with HD. After oral B307 or vehicle treatment for 2 weeks, cardiac function and cardiomyocytes in 12-week-old male R6/2 HD mice and their wild-type littermate controls (WT) were examined and then compared via echocardiography, immunohistochemistry, and Western blotting. We found that cardiac performance in aging R6/2 HD mice had significantly deteriorated in comparison with their WT (P<0.01). Cardiac expressions of superoxide dismutase 2 (SOD2) and B-cell lymphoma 2 (Bcl-2) in aging R6/2 HD mice were significantly lower than their WT (P<0.01), but cardiac expressions of tumor necrosis factor alpha (TNF-α), neurotrophin-3 (3-NT), 4-hydroxynonenal (4-HNE), Bcl-2-associated X protein (Bax), calpain, caspase 12, caspase 9, and caspase 3 of aging R6/2 HD mice were significantly higher than their WT (P<0.05). Furthermore, we found that cardiac performance in aging R6/2 HD mice had significantly improved under oral B307 treatment (P<0.05). Cardiac expressions of SOD2 and Bcl-2 of aging R6/2 HD mice were significantly higher under oral B307 treatment (P<0.01), but cardiac expressions of TNF-α, 3-NT, 4-HNE, Bax, calpain, caspase 12, caspase 9, and caspase 3 of aging R6/2 HD mice were significantly reduced under oral B307 treatment (P<0.05). Oral B307 treatment may briefly alleviate cardiac failure in aging HD R6/2 mice via suppressing cardiac oxidative stress, inflammation, and apoptosis. We suggested that the herbal formula B307 may be further developed as a potential health supplement for ameliorating cardiac failure associated with aging.

Oral treatment with herbal formula B401 alleviates penile toxicity in aging mice with manganism.

The present study aims to elucidate the roles of nitric oxide synthase activity, oxidative stress, inflammation, and apoptosis in penile toxicity of aging mice associated with excess manganese (Mn) treatment and to investigate the effect of oral treatment with the herbal formula B401 in this respect. ICR strain mice were divided into two groups: the vehicle (sham group) and the B401 (50 mg/kg) group. The mice were orally treated for 5 days; then a high single dose of MnCl2 (100 mg/kg) was given by intraperitoneal injection to the mice. One day after MnCl2 treatment, corpora cavernosal tissues of both Mn-treated mice and their controls were simultaneously sampled to examine their immunohistochemical staining and Western blot analysis. Nitric oxide (NO) production, levels of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), expression levels of factors governing angiogenesis (vascular endothelial growth factor), oxidative stress (catalase, superoxide dismutase 2,4-hydroxynonenal), inflammation (tumor necrosis factor alpha), apoptosis (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein [Bax], cleaved poly(adenosine diphosphate-ribose) polymerase [c-PARP], cytochrome C, caspase-12, and caspase-3) were evaluated in penile corpus cavernosum of the mice. We found that penile toxicity in the mice was enhanced under excess Mn treatment through reduction of NOS activity and increase in oxidative stress, inflammation, and apoptosis in the penile cavernous tissue. Furthermore, the penile toxicity in mice with manganism was alleviated by oral B401 treatment through enhancement of both nitric oxide synthesis and angiogenesis, with simultaneous reduction of oxidative stress, inflammation, and apoptosis in penile corpus cavernosum. We suggest that the herbal formula B401 may serve as a potential dietotherapeutic supplement for penile toxicity or dysfunction in aging males.

Oral treatment with the herbal formula B307 alleviates cardiac toxicity in doxorubicin-treated mice via suppressing oxidative stress, inflammation, and apoptosis.

OBJECTIVE: This study aimed to investigate whether the herbal formula B307 could alleviate doxorubicin (DOX)-induced acute cardiotoxicity. If so, we further unraveled possible molecular mechanisms of cardiac protection under treatment with the herbal formula B307. METHODS: Before the animal experiment, we examined relative viabilities of Huh7 cancer cells under treatment with the herbal formula B307. To test whether oral treatment with the herbal formula B307 could alleviate cardiotoxicity, equal volumes of B307 (50 mg/kg) or saline (sham treatment) were administered to 20-week-old male mice once daily for 14 consecutive days. Then, DOX (10 mg/kg; ip) was administered to male mice under B307 and sham treatments at 22-23 weeks of age. Cardiac functions in these mice were assessed via echocardiography at 23-24 weeks of age. Then, expressions of oxidative stress, inflammation, and apoptosis-related proteins were examined in the heart tissue by immunohistochemistry and Western blotting at 24-25 weeks of age. Apart from this, mortality rate and body weight were measured during the experiment. RESULTS: In vitro, the relative viabilities of Huh7 cancer cells under treatment with the herbal formula B307 had shown no obvious change at doses of 10-160 ng/mL. Furthermore, the relative viabilities of Huh7 cancer cells were significantly reduced under DOX treatment but showed no significant change under DOX only and DOX plus B307 treatment. In vivo, the mortality rate, body weight, and cardiac function of DOX-treated mice were obviously improved under oral treatment with the herbal formula B307. Furthermore, cardiac expressions of endothelial nitric oxide synthase, superoxide dismutase 2, and B-cell lymphoma 2 were significantly enhanced, but tumor necrosis factor alpha, NFKB1 (p50 and its precursor, p105), neurotrophin-3, Bcl-2-associated X protein, calpain, caspase 12, caspase 9, and caspase 3 were significantly suppressed in DOX-treated mice under oral treatment with the herbal formula B307. CONCLUSION: Our results revealed that oral treatment with the herbal formula B307 may provide cardioprotection in DOX-treated mice via suppressing oxidative stress, inflammation, and apoptosis in heart tissue. We believe that the herbal formula B307 may be developed as a potential alternative treatment for cancer patients under DOX treatment.

Treatment with a herbal formula B401 enhances neuroprotection and angiogenesis in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disease characterized by motor dysfunction and early death. Despite years of research, the mechanisms responsible for chronic neurodegeneration of HD remain elusive. Chinese traditional medicines might provide new insights or new therapy for HD. The Chinese herbal formula B401 is a well-known Taiwan-US patent formula and a health supplement for promoting blood circulation and enhancing brain function. This study aimed to elucidate the neuroprotective effects of the Chinese herbal formula B401 on the syndrome of HD. Then, we compared the life span and body weight of R6/2 HD mice with and without oral B401 treatment. The ameliorative effects of B401 on the symptom of HD mice were investigated through behavior tests. Expressions of proteins for neuroprotection, angiogenesis, and inflammation in the brain tissue of R6/2 HD mice were compared by using immunostaining and Western blotting techniques. Our study in vitro showed that viabilities of glutamate-treated SH-SY5Y cells were significantly increased under B401 treatment. Our results in vivo showed that duration of survival was increased, body weight loss was reduced, and motor ability was improved in R6/2 HD mice under oral B401 treatment. Subcutaneous microcirculation was enhanced in 3-month R6/2 HD mice under intraperitoneal B401 injections as observed by using moorFLPI laser Doppler imager. Atrophy of cerebrum, midbrain, and cerebellum in 3-month R6/2 HD mice under oral B401 treatment was alleviated as observed by utilizing magnetic resonance imaging. Evidence from immunostaining and Western blotting analysis showed that expressions of mutant huntingtin and tumor necrosis factor-alpha were reduced, while expressions of brain-derived neurotrophic factor and vascular endothelial growth factor were enhanced in the brain tissue of 2-month R6/2 HD mice under oral B401 treatment. We suggest that the herbal formula B401 can be developed as a medical supplement for ameliorating neurodegenerative diseases of HD via reducing mutant huntingtin aggregation and excitotoxicity, enhancing neuroprotection and angiogenesis, and alleviating inflammation in the brain.

Lignans and anthraquinones from the fruits of Morinda citrifolia.

Two new anthraquinones, 1,6-dihydroxy-5-methoxy-2-methoxymethylanthraquinone (1) and 1,5,7-trihydroxy-6-methoxy-2-methoxymethylanthraquinone (2), and one new lignan isoamericanoic acid A (3) were isolated from the fruits of Morinda citrifolia along with 11 known compounds scopoletin, luteolin, americanin A, americanin D, 3,3'-bisdemethylpinoresinol, p-cresol, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde, 4-hydroxy-3-methoxycinnamaldehyde, and 2,5-dihydroxy-4-methoxybenzaldehyde. The structures of all compounds were elucidated by spectroscopic analysis.

Origin identification on the commercial samples of Aurantii Fructus.

A total of 50 commercial samples of Aurantii Fructus, originating from Poncitrus trifoliata for Aurantii Fructus Immaturus, Citrus aurantium, and C. wilsonii for Aurantii Fructus Maturus, respectively, were collected from Taiwan and China herbal markets. Contents of the constituents in the samples were determined within 60 min using a developed HPLC method, which had been validated in terms of precision, repeatability, and accuracy. The results of the analyses showed that the constituents were closely related to the species and also the degree of maturity of the fruits, especially in the case of hesperidin (HE), naringin (NG), neohesperidin (NE), naringenin-7-glucoside (NGC), narirutin (NR), and quercetin (QU). The mature fruits (C. aurantium and C. wilsonii) contained chiefly NG, HE, and NE and the immature ones (P. trifoliata) had NG, NR, and QU majorly. Within the mature samples, the ratios of HE/NE and NGC/NE values were higher than 2.94 and 0.21 in C. aurantium and lower than 1.31 and 0.02 in C. wilsonii, respectively. A flowchart that is useful for identifying Aurantii Fractus was devised, based on the various chemical identification methods.

Phenolic derivatives from Aster indicus.

Investigation of the constituents of whole herbs of Aster indicus L. led to isolation of 4-hydroxy-3-[1-(methoxycarbonyl)vinyloxy]benzoic acid (1), 5-(1-carboxylvinyloxy)-2-hydroxybenzoic acid (2), 4-allyl-2,6-dimethoxyphenyl 3-methylbutanoate (3), and 4-allyl-2-methoxyphenyl 2-methylbutanoate (4), together with 27 previously known compounds. The structures of compounds 1-4 were established by application of spectroscopic (NMR and MS) analyses.

Classification of Aurantii Fructus samples by multivariate analysis.

Twenty commercial samples of Aurantii Fructus Immaturus (Poncitrus trifoliata) and 30 of Aurantii Fructus Maturus (Citrus aurantium and C. wilsonii) were collected from the Taiwan and China herbal markets. The contents of 12 constituents in these samples were determined by HPLC and were used to assess the potential relationships with their plant origins. Multivariate analysis including principal component analysis (PCA), cluster analysis (CA), and linear discriminant analysis (LDA) were used as classification procedures. Natural groupings of the samples divided into three sets successfully, 20 P. trifoliata, 15 C. aurantium, and 15 C. wilsonii, were observed by using PCA and CA. The application of LDA gave correct assignation percentages of 100.0% for all three groups.

Determination of 19 rhubarb constituents by high-performance liquid chromatography-ultraviolet-mass spectrometry.

Rhubarb (Rhei rhizoma), a commonly used Chinese herb, contains anthraquinones, anthrones, galloylglucoses, stilbenes, and flavan-3-ols compounds, etc. as major constituents. Using 19 of these compounds as markers, an HPLC-UV-MS method was developed to estimate the quality of rhubarb samples within a period of 70 min. Extracts were analyzed with a Cosmosil 5C18-MS column and eluted with a gradient comprising an aqueous solution of acetic acid and methanol at a flow rate of 0.9 mL/min. Peaks were detected by absorbance measurements at 254 nm (6 and 8-19) and 280 nm (1-5 and 7), and the peaks of the marker substances were identified from their UV spectra and MS fragmentation patterns. The proposed method yielded a peak-area ratio RSD value with an intraday SD falling within 0.71-1.78% and an interday SD within 0.78-1.98% at a detection limit of 0.2-3.2 microg/mL. The ESI negative ion mode was used to collect data (molecular weight, CID fragments from MS and MS/MS spectra) for 19 compounds from four types of structure categories: anthraquinones, dianthrone glycosides, stilbenes, and galloylglucosides. The information gathered can be used to identify the structures of various peaks appearing in the LC chromatograms of rhubarb samples.

Separation and identification of the organic acids in Angelicae Radix and Ligustici Rhizoma by HPLC and CE.

Angelicae Radix (AR) and Ligustici Rhizoma (LR) are both derived from the Umbelliferae plants and contain similar organic acids as their bioactive compounds. Nine of these organic acids, including nicotinic acid, protocatechuic acid, phthalic acid, folinic acid, p-hydroxybenzoic acid, folic acid, vanillic acid, caffeic acid, and ferulic acid were separated by HPLC and CE. Detection at 210 nm with a linear gradient containing 20 mM KH2PO4 (pH 3.5) and H2O-CH3CN in HPLC and with a buffer solution containing 10 mM LTAC, 2 mM Na2HPO4, 9 mM Na2B4O7(pH 9.56), and CH3CN in CE were found to be the most efficient eluents for this separation. The contents of the nine components in crude extracts of either AR or LR could easily be determined within 60 min by LC and within 20 min by CE. The structures of the individual peaks in the LC chromatogram were identified by LC-MS. The effects of buffers on the separation and validation of the two methods were examined.

Determination of 12 pueraria components by high-performance liquid chromatography-mass spectrometry.

Puerariae radix, a commonly used Chinese herb drug derived from the dried root of legume plant, contains a series of isoflavones as its chief pharmacologically active constituents. Using 12 pueraria components as markers, an LC-UV-MS method requiring less than 60 min, was developed for estimating the quality of pueraria samples within 60 min. Extracts were analyzed using a Cosmosil 5C18-MS column, by gradient elution with an aqueous solution of acetic acid and methanol-ACN at a flow-rate of 1.0 mL/min. Peaks were detected at 254 nm and each peak was identified by LC/MS. The reproducibilities (RSD) of this proposed method, on the basis of peak-area ratios from six replicate injections, were 0.93-1.42% (intraday) and 1.05-1.63% (interday) at a detection limit of 0.12-0.78 microg/mL. Most of the markers used in this study can be classified, respectively, into three major categories, namely, isoflavones, O-glycosidic isoflavones, and C-glycosidic isoflavones. The structures of the compounds were determined from LC-MS fragmentation data and data from the literature.

Chemical identification of the sources of commercial Fructus Chebulae.

Twenty-eight commercial samples of Fructus Chebulae were collected from local herbal markets in Taiwan and were determined to have been derived from Terminalia chebula Retz. and Terminalia chebula Retz. var. parviflora Thwaites, which differ markedly in external appearance. Ten tannin-related constituents [gallic acid (1), chebulic acid (2), punicalagin (4), chebulanin (7), corilagin (8), neochebulinic acid (9), ellagic acid (11), chebulagic acid (12), chebulinic acid (13) and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (14)] were identified and quantified by HPLC. Samples derived from T. chebula. var. parviflora, which are typically round-shaped, generally contained higher concentrations of 13 and 14 but lower levels of 12 compared with those from T. chebula, which are largely oval-shaped. The ratio of the concentration of 14 to that of 4 may serve as a potential parameter for differentiating samples from the two origins (T. chebula, ratio 0.6 +/- 0.3; T. chebula. var. parviflora, ratio 3.4 +/- 2.2). Levels of the three major bioactive constituents 12-14 were found to provide good references for the quality assessment of Fructus Chebulae. The ratio of the concentration of 12 to that of 14 may offer a guideline for determining quality as well as origin of the drug (lower-grade T. chebula, ratio 12.4 +/- 6.0; medium-grade T. chebula, ratio 8.8 +/- 7.9; higher-grade T. chebula, ratio 3.2 +/- 0.8; T. chebula var. parviflora, ratio 1.6 +/- 0.7).

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis.

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.

Analysis of ephedra-alkaloids using sweeping and cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography methods.

Two stacking methods of capillary electrophoresis (CE) were developed for the separation of very dilute solutions of ephedra-alkaloids, namely ephedrine, pseudoephedrine, methylephedrine, methylpseudoephedrine, norephedrine, and norpseudoephedrine. A sweeping method which uses a carrier comprised of phosphoric acid, sodium dodecyl sulfate (SDS), diethylamine and acetonitrile permits the detection of the alkaloids down to the 10(-1) microg/mL level, and the cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) method using phosphoric acid, SDS, and acetronitrile as electrolytes can detect down to the 10(-3) microg/mL level. The former requires the conductance of the sample solution to be adjusted beforehand, and only five peaks were observed, two of which were overlapped. The latter is capable of separating the six alkaloids but has a somewhat poorer reproducibility. Using an optimized injection time, it was found that the more diluted a solution is, the greater the sweeping effect will be. The CSEI-Sweep-MEKC method with a 600 s injection time and a 10(-1) microg/mL solution concentration provides an amplification effect of approximately 10(4). The method is suitable for analyses of dilute herb drug extracts and mouse sera. The effect of buffers on the separation and validation of the methods in this study are also discussed.