A validated method for detection of cannflavins in hemp extracts.

A selective and sensitive liquid chromatography mass spectrometry assay was developed for the detection of cannflavin A, B, and C in hemp extract specimens. A deuterated analog cannabidiol-D3 was used as the internal standard and the isocratic method used a mobile phase consisting of acetonitrile and water with 0.1 % formic acid [83:17]. Detection was carried out by electrospray positive ionization in single-ion monitoring mode through a C-18 analytical column. The assay (total run time <20 min) had excellent linearity and a lower limit of quantification of 0.5 µg/mL and a limit of detection of 0.25 µg/mL with a 10 µL injection. The method possessed suitable measures of stability, sensitivity, and selectivity for detecting cannflavins in several specimen types. The method was successfully applied to the analysis of samples of cannflavin release from prototype topical formulations.

Therapeutic Potential and Predictive Pharmaceutical Modeling of Stilbenes in Cannabis sativa.

Cannabis sativa is a plant used for recreational and therapeutic purposes; however, many of the secondary metabolites in the plant have not been thoroughly investigated. Stilbenes are a class of compounds with demonstrated anti-inflammatory and antioxidant properties and are present in cannabis. Many stilbenes present in cannabis have been investigated for their therapeutic effects. Fourteen stilbenes have been identified to be present in cannabis, all of which are structurally dihydrostilbenoids, with half possessing a prenylated moiety. The stilbenes summarized in this analysis show varying degrees of therapeutic benefits ranging from anti-inflammatory, antiviral, and anti-cancer to antioxidant effects. Many of the identified stilbenes have been researched to a limited extent for potential health benefits. In addition, predictive in silico modeling was performed on the fourteen identified cannabis-derived stilbenes. This modeling provides prospective activity, pharmacokinetic, metabolism, and permeability data, setting the groundwork for further investigation into these poorly characterized compounds.

Investigations of the antipyretic effect and safety of Prasachandaeng, a traditional remedy from Thailand national list of essential medicines.

Prasachandaeng (PSD) remedy from the Thailand National List of Essential Medicines (NLEM) has been used as an antipyretic for chronic fever in both adults and children for centuries. Its therapeutic effect in treating fever and its safety have not been studied in animal models. We evaluated its antipyretic activity on lipopolysaccharide (LPS)-induced fever and safety in the liver in comparison with acetaminophen (ACP). Correlation between biochemistry of liver function and the level of cytochrome P450 (CYP2E1) was also evaluated using an ELISA kit. All doses of PSD powder (PSDP) and a 95% ethanol extract of PSD (PSDE) (50, 200, and 400 mg/kg) showed a significant antipyretic effect (* p < 0.05) as compared to ACP. We investigated clinical biochemistry of liver and kidney functions, histopathology, and concentrations of CYP2E1. All treatment groups demonstrated a normal range of clinical biochemistry of liver and kidney functions in comparison with ACP on days 1, 3, 7, and 10. Serum AST, ALP, and LDH levels of PSDE and PSDP showed mean values less than that of ACP on the corresponding days (* p < 0.05). None of the treatment groups showed evidence of hepatocellular damage, nor did they affect CYPE21. The results of histopathology on liver tissue correlated with the biochemistry of liver functions which indicated no hepatotoxicity effect in liver tissue during the seven day treatment. These findings suggest that both forms of PSD remedy possessed marked antipyretic activity and were not hepatotoxic during the seven days of administration in rats.

Traditional Chinese Medicine "Pill", an Ancient Dosage Form with Surprising Modern Pharmaceutical Characteristics.

Currently, the use of Traditional Chinese Medicine (TCM) for healthy living in daily practice is widely accepted across the world. However, not much attention has been paid to the particular characteristics of TCM "pills", one of the classic dosage forms in TCM. For a better understanding, this review was undertaken to provide a modern pharmaceutical overview of pills. Over many centuries, pills have been developed in different types (honeyed pill, water-honeyed pill, watered pill, pasted pill, waxed pill, concentrated pill, and dripping pill) to achieve varying intended TCM release patterns. It suggests that knowledge relating to the impact of binders and excipients on drug release from TCM pills can be traced back to before dissolution testing was invented. Therefore, although Pills may be considered as an ancient and outdated dosage form compared to current drug delivery systems, they have surprisingly modern pharmaceutical properties that is highlighted in this article. In addition, this review found that the quality control standards for TCM pill are globally substantially different. Hence, greater effort should be taken to establish an internationally harmonized and proper standard to safeguard the quality of this dosage form and to ensure the alignment with TCM use.

Are the release characteristics of Erzhi pills in line with traditional Chinese medicine theory? A quantitative study.

OBJECTIVE: Traditional Chinese medicine (TCM) has been widely used throughout China to prevent and cure diseases for thousands of years, and now it is a part of the integrative medicine field that is available in Western societies. To ensure the safety and quality of the herbal medicines that are a major part of the TCM tradition, they must be held to modern pharmaceutical standards. Erzhi pill (EZP) is a Chinese Pharmacopeia-listed herbal preparation that is used in the long-term clinical management of post-menopausal symptoms, osteoporosis and menstrual disorders. Until now, whether the drug release mechanism of EZP is in line with its intended TCM usage has not been studied. METHODS: The release of specnuezhenide from three EZPs (self-made, Leiyunshang and Renhe) in simulated gastric fluid (SGF), acetate buffer (pH 4.5 buffer) and simulated intestinal fluid (SIF) was investigated in a dissolution test. The water uptake capacity and erosion extent of the three EZPs were investigated using swelling and erosion studies. The drug release mechanism was further assessed through statistical model fitting, using DDSolver software. RESULTS: The release of specnuezhenide from all three EZPs in SGF was less than 50% within a 4 h period. However, over 70% of the specnuezhenide was released from each EZP in both pH 4.5 buffer and SIF in the same time. Analysis of the swelling and erosion behaviors and the drug release mechanism of the three EZPs confirmed that the release rate from EZP followed a sustained release profile, which was an interactive combination of swelling and erosion. CONCLUSION: This study showed that the release pattern from the pills was in line with the intended TCM use of EZP. TCM had not only theoretically considered sustained release from the pills, but also formulated them to achieve this release pattern. When establishing quality control standards for pills, the theoretical TCM usage and the actual release patterns need to be considered.

A new medium-throughput screening design approach for the development of hydroxymethylnitrofurazone (NFOH) nanostructured lipid carrier for treating leishmaniasis.

Hydroxymethilnitrofurazone (NFOH) is a nitrofurazone derivative and has potential use in treating leishmaniasis. However, due to low water solubility and bioavailability, NFOH has failed in in vivo tests. Nanostructured lipid carrier (NLC) is an alternative to overcome these limitations by improving pharmacokinetics and modifying drug delivery. This work is focused on developing a novel NFOH-loaded NLC (NLC-NFOH) using a D-optimal mixture statistical design and high-pressure homogenization, for oral administration to treat leishmaniasis. The optimized NLC-NFOH consisted of Mygliol® 840, Gelucire® 50/13, and Precirol® ATO 5 as lipids. These lipids were selected using a rapid methodology Technobis Crystal 16 T M, microscopy, and DSC. Different tools for selecting lipids provided relevant scientific knowledge for the development of the NLC. NLC-NFOH presented a z-average of 198.6 ±â€¯5.4 nm, PDI of 0.11 ±â€¯0.01, and zeta potential of -13.7 ±â€¯0.7 mV. A preliminary in vivo assay was performed by oral administration of NLC-NFOH (2.8 mg/kg) in one healthy male Wistar rat (341 g) by gavage. Blood from the carotid vein was collected, and the sample was analyzed by HPLC. The plasma concentration of NFOH after 5 h of oral administration was 0.22 µg/mL. This same concentration was previously found using free NFOH in the DMSO solution (200 mg/kg), which is an almost 100-fold higher dose. This study allowed a design space development approach of the first NLC-NFOH with the potential to treat leishmaniasis orally.

Esculetin as bioactive marker: towards a rational scientific approach for the treatment of hyperuricemia using Traditional Chinese Medicine

Traditional Chinese Herbal Medicine has been used to prevent and cure disease in China for thousands of years and has gained global interest in recent decades. The Erding formula is a Chinese Pharmacopeia (ChP)-listed herbal preparation used for treating sore throat, carbuncles and boils. Esculetin is a ChP quality control (QC) marker for these indications. A previous study found that a new indication, hyperuricemia, can be added to the Erding formula. Therefore, this study aimed to evaluate whether the traditionally used marker, esculetin, still has bioactivity for hyperuricemia, which is substantially different from the original indications. The study analyzed the quantity of esculetin by high-performance liquid chromatography, assessed the therapeutic effect of esculetin using animal model, and then characterized esculetin and its metabolites in serum via ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The results showed that the esculetin content in the aqueous Erding extract was 0.26±0.05% (w/w). Both the Erding extract and esculetin significantly reduced uric acid levels. Six metabolites of esculetin were identified in mice serum. This study revealed a rational scientific approach to prove esculetin is a reliable bioactive and QC marker for Erding formula in hyperuricemia treatment which contributed to ensure product quality and therapeutic efficacy.

Erding Formula in hyperuricaemia treatment: unfolding traditional Chinese herbal compatibility using modern pharmaceutical approaches.

OBJECTIVES: Traditional Chinese herbal formulas are difficult to be understood because of complex compositions and specific therapeutic principles. To better understand herbal compatibility in Traditional Chinese medicine (TCM), this study was conducted to investigate the effects of a Chinese pharmacopoeia-listed formula, Erding Formula (EF) and its constituent herbs for a new indication, hyperuricaemia. METHODS: A hypoxanthine and potassium oxonate-induced hyperuricemic mouse model, a xylene-induced inflammatory mouse model and an acetic acid-induced pain model were used to test the effects of EF and its constituent herbs. In addition, we investigated whether EF and/or its relevant herbs had an impact on the expression of URAT1 and OAT3 mRNA. KEY FINDINGS: The results showed EF and individual herbs had pharmacological effects on selected targets. Only Viola yedoensis Makino (Viola) lowered uric acid levels, while all four herbs had anti-inflammatory and analgesic effects. The EF may lower the uric acid level through inhibiting the expression of URAT1 mRNA and enhancing the expression of OAT3 mRNA. CONCLUSIONS: These findings provide pharmacological insights into the effects of EF and individual herbs on UA excretion. This study suggests that Viola is the main herb in EF. This study facilitates better understanding of TCM principles and theories using modern pharmaceutical approaches.

Combinational siRNA delivery using hyaluronic acid modified amphiphilic polyplexes against cell cycle and phosphatase proteins to inhibit growth and migration of triple-negative breast cancer cells.

Triple-negative breast cancer is an aggressive form of breast cancer with few therapeutic options if it recurs after adjuvant chemotherapy. RNA interference could be an alternative therapy for metastatic breast cancer, where small interfering RNA (siRNA) can silence the expression of aberrant genes critical for growth and migration of malignant cells. Here, we formulated a siRNA delivery system using lipid-substituted polyethylenimine (PEI) and hyaluronic acid (HA), and characterized the size, ζ-potential and cellular uptake of the nanoparticulate delivery system. Higher cellular uptake of siRNA by the tailored PEI/HA formulation suggested better interaction of complexes with breast cancer cells due to improved physicochemical characteristics of carrier and HA-binding CD44 receptors. The siRNAs against specific phosphatases that inhibited migration of MDA-MB-231 cells were then identified using library screen against 267 protein-tyrosine phosphatases, and siRNAs to inhibit cell migration were further validated. We then assessed the combinational delivery of a siRNA against CDC20 to decrease cell growth and a siRNA against several phosphatases shown to decrease migration of breast cancer cells. Combinational siRNA therapy against CDC20 and identified phosphatases PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 successfully inhibited cell growth and migration, respectively, without interfering the functional effect of the co-delivered siRNA. The identified phosphatases could serve as potential targets to inhibit migration of highly aggressive metastatic breast cancer cells. Combinational siRNA delivery against cell cycle and phosphatases could be a promising strategy to inhibit both growth and migration of metastatic breast cancer cells, and potentially other types of metastatic cancer. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of a tailored polymeric siRNA delivery system formulation as well as combinational siRNA therapy in metastatic breast cancer cells to inhibit malignant cell growth and migration. The siRNA delivery was undertaken by non-viral means with PEI/HA. We identified six phosphatases that could be critical targets to inhibit migration of highly aggressive metastatic breast cancer cells. We further report on specifically targeting cell cycle and phosphatase proteins to decrease both malignant cell growth and migration simultaneously. Clinical gene therapy against metastatic breast cancer with effective and safe delivery systems is urgently needed to realize the potential of molecular medicine in this deadly disease and our studies in this manuscript is intended to facilitate this endeavor.

Multiple siRNA delivery against cell cycle and anti-apoptosis proteins using lipid-substituted polyethylenimine in triple-negative breast cancer and nonmalignant cells.

Conventional breast cancer therapies have significant limitations that warrant a search for alternative therapies. Short-interfering RNA (siRNA), delivered by polymeric biomaterials and capable of silencing specific genes critical for growth of cancer cells, holds great promise as an effective, and more specific therapy. Here, we employed amphiphilic polymers and silenced the expression of two cell cycle proteins, TTK and CDC20, and the anti-apoptosis protein survivin to determine the efficacy of polymer-mediated siRNA treatment in breast cancer cells as well as side effects in nonmalignant cells in vitro. We first identified effective siRNA carriers by screening a library of lipid-substituted polyethylenimines (PEI), and PEI substituted with linoleic acid (LA) emerged as the most effective carrier for selected siRNAs. Combinations of TTK/CDC20 and CDC20/Survivin siRNAs decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth. The effects of combinational siRNA therapy was higher when complexes were formulated at lower siRNA:polymer ratio (1:2) compared to higher ratio (1:8) in nonmalignant cells. The lead polymer (1.2PEI-LA6) showed differential transfection efficiency based on the cell-type transfected. We conclude that the lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets in breast cancer therapy. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3031-3044, 2016.

Antiulcerogenic potential activity of free and nanoencapsulated Passiflora serratodigitata L. extracts.

This paper provides evidence that the leaves and stem of Passiflora serratodigitata L. dry crude extract (DCE), ethylacetate fraction (EAF), and residual water fraction show potential antiulcerogenic activity. Interestingly, the polymeric nanocapsule loaded with EAF had 10-fold more activity than the free EAF. Furthermore, the polymer nanoparticles provided homogeneous colloidal drug delivery systems and allowed overcoming challenges such as poor aqueous solubility as well as the physical-chemical instability of the organic extract, which presented 90% (w/w) of the flavonoid content. The entrapment efficiency of the total flavonoid was 90.6 ± 2.5% (w/v) for the DCE and 79.9 ± 2.7% (w/v) for the EAF. This study shows that nanoencapsulation improves both the physicochemical properties and the efficacy of the herbal formulations. Therefore, free and encapsulated extracts have the potential to be suitable drug design candidates for the therapeutic management of ulcer.

Establishing the pharmaceutical quality of Chinese herbal medicine: a provisional BCS classification.

The Biopharmaceutical Classification System (BCS), which is a scientific approach to categorize active drug ingredient based on its solubility and intestinal permeability into one of the four classes, has been used to set the pharmaceutical quality standards for drug products in western society. However, it has received little attention in the area of Chinese herbal medicine (CHM). This is likely, in part, due to the presence of multiple active components as well as lack of standardization of CHM. In this report, we apply BCS classification to CHMs provisionally as a basis for establishing improved in vitro quality standards. Based on a top-200 drugs selling list in China, a total of 31 CHM products comprising 50 official active marker compounds (AMCs) were provisionally classified according to BCS. Information on AMC content and doses of these CHM products were retrieved from the Chinese Pharmacopoeia. BCS parameters including solubility and permeability of the AMCs were predicted in silico (ACD/Laboratories). A BCS classification of CHMs according to biopharmaceutical properties of their AMCs is demonstrated to be feasible in the current study and can be used to provide a minimum set of quality standards. Our provisional results showed that 44% of the included AMCs were classified as Class III (high solubility, low permeability), followed by Class II (26%), Class I (18%), and Class IV (12%). A similar trend was observed when CHMs were classified in accordance with the BCS class of AMCs. Most (45%) of the included CHMs were classified as Class III, followed by Class II (16%), Class I (10%), and Class IV (6%); whereas 23% of the CHMs were of mixed class due to the presence of multiple individual AMCs with different BCS classifications. Moreover, about 60% of the AMCs were classified as high-solubility compounds (Class I and Class III), suggesting an important role for an in vitro dissolution test in setting quality control standards ensuring consistent biopharmaceutical quality for the commercially available CHM products. That is, provisionally, more than half of the AMCs of the top-selling CHMs included in this study would be candidates for a bioequivalence (BE) biowaiver, based on WHO recommendations and EMEA guidelines. Thus a dissolution requirement on these AMCs would represent a significant advance in the pharmaceutical quality of CHM today.

Provisional biopharmaceutical classification of some common herbs used in Western medicine.

The aim of this study was to classify some markers of common herbs used in Western medicine according to the Biopharmaceutical Classification System (BCS). The BCS is a scientific approach to classify drug substances based upon their intestinal permeability and their solubility, at the highest single dose used, within the physiologically relevant pH ranges. Known marker components of twelve herbs were chosen from the USP Dietary Supplement Compendium Monographs. Different BCS parameters such as intestinal permeability (P(eff)) and solubility (C(s)) were predicted using the ADMET Predictor, which is a software program to estimate biopharmaceutical relevant molecular descriptors. The dose number (D0) was calculated when information from the literature was available to identify an upper dose for individual markers. In these cases the herbs were classified according to the traditional BCS parameters using P(eff) and D0. When no upper dose could be determined, then the amount of a marker that is just soluble in 250 mL of water was calculated. This value, M(x), defines when a marker is changing from highly soluble to poorly soluble according to BCS criteria. This biopharmaceutically relevant value can be a useful tool for marker selection. The present study showed that a provisional BCS classification of herbs is possible but some special considerations need to be included into the classification strategy. The BCS classification can be used to choose appropriate quality control tests for products containing these markers. A provisional BCS classification of twelve common herbs and their 35 marker compounds is presented.

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit.

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 µg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.

Investigation of the performance of the disintegration test for dietary supplements.

The aim of this study was to investigate how beaker size, basket assembly, use of disk, and immersion medium impact the disintegration time of dietary supplements. The disintegration times were determined for five tablet and two capsule products. A two-station disintegration tester was used with Apparatus A or Apparatus B as described in the United States Pharmacopeia (USP) chapters, <701> and <2040>. Two beakers complying with the harmonized specifications were used, one with a volume of 1,000 mL and one with a 1,500-mL volume. The disintegration data were analyzed using ANOVA for the following factors: beaker size, equipment (App A and B) and condition (with/without disk). Two tablet products were not sensitive to any changes in the test conditions or equipment configurations. One product was only partially sensitive to the test conditions. The other products showed impact on the disintegration time for all test conditions. The results revealed that these tablet products might pass or fail current USP disintegration requirements depending on the equipment configuration. Similar results were obtained for the two investigated capsule formulations. One product might fail current USP disintegration requirements if the large beaker was used, but might pass the disintegration requirements when the small beaker was used. Hydroxy propyl methyl cellulose capsules were mostly influenced if sodium instead of a potassium buffer was used as the immersion medium. The results demonstrate that the current harmonized ICH specifications for the disintegration test are insufficient to make the disintegration test into reliable test for dietary supplements.

Investigation of vitamin and mineral tablets and capsules on the Canadian market.

PURPOSE: The goal of this study was to investigate the disintegrating properties of tablets and capsules containing minerals and vitamins commercially available on the Canadian market and to review their label information. METHODS: The labels were examined for product-related information. The first disintegration test stage was performed using Simulated Intestinal Fluid (SIF) pH 6.8 for 20 minutes. Products which did not disintegrate were further investigated using USP disintegration conditions for dietary supplements. RESULTS: The provided label information is difficult to understand and in some cases pseudo-scientific. Thirty out of thirty-nine tablets and six out of ten capsules had a Drug Identification Number (DIN). Twenty-one of thirty-nine tablets and four out of the ten capsules did not disintegrate within 20 minutes. Using the USP disintegration conditions for dietary supplements nine tablet products did not fully disintegrate but all capsules passed the test. None of the three "time-released" products disintegrated under the applied conditions. CONCLUSIONS: Industry should follow already existing label recommendations more closely to allow the consumers to make an informed decision on their products by providing only essential information rather than using pseudo-scientific terms. The results of the disintegration study indicated that disintegration, one of the most basic quality control parameters, is still a concern for dietary supplements.

A method for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides using high-performance liquid chromatography with ultraviolet and positive mode electrospray ionization mass spectrometric detection.

A high-performance liquid chromatographic separation coupled to diode array absorbance and positive mode electrospray mass spectrometric detection has been developed for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides in extracts of Asian ginseng (Panax ginseng) and American ginseng (P. quinquefolius). The method is capable of separating, identifying, and quantifying the predominant ginsenosides found in heated alcoholic extracts of Asian and American ginseng roots routinely sold as nutraceuticals. It also separates and identifies the malonyl ginsenosides often found in cold alcoholic extracts of ginseng root and has the potential to quantify these compounds if pure standards are available. Furthermore, it can separate and identify ginsenoside hydrolysis products such as those readily produced in situations mimicking gastric situations, including those used for dissolution studies (i.e., 0.1 N HCl, 37 degrees C).