Validation of standardized high-performance thin-layer chromatographic methods for quality control and stability testing of herbals

In herbal medicinal products the entire herbal drug or an herbal drug preparation is regarded as the active pharmaceutical ingredient, regardless of whether constituents with defined therapeutic activity are known. In quality control and stability testing of herbal medicinal products, fingerprint chromatograms are used as powerful tools to evaluate and compare the composition of compounds in such products. To fulfill the International Conference on Harmonization and Good Manufacturing Practice-based regulatory requirements in pharmaceutical quality control, chromatographic fingerprint analysis needs to be validated. Based on a standardized methodology, this paper provides a comprehensive concept for evaluating validation parameters for planar chromatographic fingerprinting by considering the stationary phase, sample application, developing solvent, chromatogram development, plate labeling, derivatization, documentation, and chromatographic equipment. Validation parameters addressed include stability of the analyte, selectivity, robustness testing, and method reproducibility.     

Établissement du profil chromatographique liquide non aqueux des métabolites phytochimiques apolaires des phytomédicaments

Chromatographic profiling of plant metabolites is therefore a good tool for quality control of such herbal medicinal products. Our objective was to propose a protocol for sample preparation and liquid chromatographic profiling of non-polar metabolites for quality assessment of African herbal medicinal products. The methodology is based on the chemometric assessment of liquid chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber. Metabolic profiling is carried out by non-aqueous liquid chromatography on porous carbon graphite, coupled with mass spectrometry, after extraction with dichloromethane and removal of chlorophyll. Our method using liquid chromatography, coupled to mass spectrometry can detect non-polar metabolites already identified in the two herbal drugs. Chemometric data analysis of chromatographic profiles using the PLS-discriminant analysis with or without orthogonal signal correction, allowed a distinction between the two herbal drugs.     

The quality of commercially available herbal products

Herbal products are believed to be safe as they are naturally occurring, however, this is a misconception. Currently, there are many herbal products which are not manufactured under the Good Manufacturing Practice (GMP) guidelines and hence the quality of the product is not controlled. Quality can be assessed by evaluation of data on levels of either active constituent(s) or chemical markers indicative of origin of plant material. Assessment of published data on levels of quality of herbal medicinal products revealed that only a percentage of the total number of products investigated were found to contain the levels of markers as stated on the label, and others had widely varying levels of constituents. Thus few products complied with acceptable quality control standards. Herbal products are increasingly self-prescribed for the treatment of minor ailments. Patients relying on herbal products for therapeutic effects may expose themselves to either low doses of active constituents causing insufficient effects or alternatively take higher levels than expected, with the increased risk of toxicity or adverse effects, or be affected by the inadvertent inclusion of unexpected components with associated potential health risks.     

Orthogonal projection (OP) technique applied to pattern recognition of fingerprints of the herbal medicine houttuynia cordata Thunb. and its final injection products

It is a crucial issue to determine the origins of herbal medicinal materials and identify the quality grades and fakes of their final products collected from different pharmaceutical corporations. Pattern recognition technique may assist the manufacturers to achieve this purpose and effectively control the quality of their products. In this work, a widely used method in chemometrics, orthogonal projection (OP) technique, was applied to discrimination analysis and identification of fingerprints of the herbal medicine houttuynia cordata Thunb. (HCT) and its final injection products. The advantages of the OP technique are clearly shown after comparing with the conventional methods such as principal component analysis (PCA), Mahalanobis distance (MD), and similarity comparison method (SCM). Three different sources of medicinal material HCT and its final injection products from six different manufacturers were studied under 'sixfold', 'threefold' and 'threefold-bis' cross-validation procedures. The good performance of the proposed method in determination and identification of unknown samples shows it could be a powerful tool for quality control in herbal medicine production and other related research fields.     

Recent applications of capillary electrophoresis in the analysis of traditional Chinese medicine

Traditional Chinese medicines (TCMs) are generally mixtures of herbal plants or extracts which comprise hundreds of different constituents with widely difference in the content and physiochemical properties. In order to analyze bioactive compounds in TCMs and control the quality, a large number of analytical tools have been developed, among which capillary electrophoresis (CE) has become a powerful technique with increasing importance. Some formats of CE, including capillary zone electrophoresis, micellar electrokinetic chromatography, non-aqueous CE and capillary electrochromatography have been widely employed for the analysis of TCMs. The general characteristics of these formats are briefly described, and their applications to the analysis of TCMs during the past five years are summarized.     

Thermal characterization of dried extract of medicinal plant by DSC and analytical techniques

The increased search for herbal products has generated an increasing interest in improving the quality control of dried extracts by pharmaceutical industry since these are raw materials of great importance by their quality and versatility. This work aimed at the application of various analytical techniques (thermal analysis, X-ray diffraction, scanning electron microscopy, and infrared Fourier transform spectroscopy) in the characterization of dried extracts of two plants from the Brazilian semiarid region with medicinal properties. The DSC curves for the dried extracts of Ximenia americana L. and Schinopsis brasiliensis Engl. showed that thermal processes occur between 33.50 and 118.58 °C and between 39.17 and 126.14 °C. The X-ray powder diffraction revealed high degree of amorphization, but the dried extract of X. americana L. showed some diffraction peaks of high intensity. The IR spectra showed high variety of metabolites in the extracts dried. Through this study it was possible to verify the feasibility of applying these techniques in the characterization of raw materials from medicinal plants for use in the herbal medicines production.     

Diagnosis of public programs focused on herbal medicines in Brazil

The present study is aimed to diagnose the current public programs focused on herbal medicines in Brazil by means of in loco visits to 10 programs selected by means of questionnaires sent to 124 municipalities that count on herbal medicine services. The main purpose of the implementation of program programs is related to the development of medicinal herbs. 70% of them are intended for the production of herbal medicines and 50% are aimed to ensure the access of the population to medicinal plants and or herbal medicines. The initiative of the implementation of these programs was related to the managers (60%). The difficulties in this implementation were due to the lack of funding (100%) of the programs. In 60% of the programs, the physicians did not adhere to herbal medicine services due to the lack of knowledge of the subject. Training courses were proposed (80%) to increase the adhesion of prescribers to the system. Some municipalities use information obtained from patients to assess the therapeutic efficiency of medicinal plants and herbal medicines. Of the programs underway, cultivation of medicinal plants was observed in 90% and 78% of them adopt quality control. In most programs, this control is not performed in accordance with the legal requirements. The programs focused on medicinal plants and herbal medicines implemented in Brazil face some chronic problems of infrastructure, management, operational capacity and self-sustainability, which can be directly related to the absence of a national policy on medicinal plants and herbal medicines.     

Chromatographic separation techniques and data handling methods for herbal fingerprints: a review

As herbal medicines have an important position in health care systems worldwide, their current assessment and quality control are a major bottleneck. Over the past decade, major steps were taken not only to improve the quality of the herbal products but also to develop analytical methods ensuring their quality. Nowadays, chromatographic fingerprinting is the generally accepted technique for the assessment and quality control of herbal products. This paper briefly considers the evolution of the regulations and guidelines on the quality control of herbal medicines, and reviews the established analytical techniques for herbal fingerprinting with an emphasis on the most recent developments, such as miniaturized techniques, new stationary phases, analysis at high temperatures and multi-dimensional chromatography. Accessory to the new analytical techniques, the chemometric data handling techniques applied are discussed. Chemometrics provide scientists with useful tools in understanding the huge amounts of data generated by the analytical advances and prove to be valuable for quality control, classification and modelling of, and discrimination between herbal fingerprints.     

Chionanthus virginicus L.: phytochemical analysis and quality control of herbal drug and herbal preparations

Root barks of Chionanthus virginicus L. are used in homeopathic medicines in the treatment of icterus and hepatitis. The objective of this study is to identify novel secoiridoids and lignans and to develop a simple and reliable HPLC method for the determination of oleuropein, phillyrin, total secoiridoids and total lignans for quality control and stability studies of C. virginicus herbal drug and preparations. Secoiridoids and lignans were purified by preparative HPLC. Compounds previously described were identified by HPLC according to their retention times and UV spectra. Structures of new compounds were determined by NMR. Two compounds namely excelside B and acetoxypinoresinol-4"-O-beta-D-glucoside are described for the first time in the drug. HPLC separation was performed on Symmetry C18 (Waters) by gradient elution using acetonitrile and 0.2% aqueous phosphoric acid. The method was validated for specificity, linearity, precision, accuracy, limits of detection and quantification for simultaneous determination of secoiridoids and lignans in herbal drug and herbal preparations as mother tinctures. The proposed HPLC method is linear in the range studied (r2 > or = 0.9989) for all the analytes. The method is precise with intra- and inter-day variations of less than 4%. The mean recoveries of the analytes range from 99.65 to 102.81%. The method is successfully applied to the quantification of nine compounds belonging to secoiridoids and lignans and for the stability studies of these compounds. The study allowed completing the phytochemical knowledge of C. virginicus. This simple developed assay could be used as tools for routine quality control of C. virginicus herbal drug and herbal medicinal products.     

A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants

Sample preparation is the crucial first step in the analysis of herbs. In recent years there has been increasing interest worldwide in the use of alternative/herbal medicine for the prevention and treatment of various illnesses. Currently, however, quality-related problems (lack of consistency, safety, and efficacy) seem to be overshadowing the potential genuine health benefits of various herbal products. Thus, the development of "modern" sample-preparation techniques with significant advantages over conventional methods for the extraction and analysis of medicinal plants is likely to play an important role in the overall effort of ensuring and providing high-quality herbal products to consumers worldwide. In this article, recent developments and applications of modern sample-preparation techniques for the extraction, clean-up, and concentration of analytes from medicinal plants or herbal materials are reviewed. These modern techniques include solid-phase microextraction, supercritical-fluid extraction, pressurized-liquid extraction, microwave-assisted extraction, solid-phase extraction, and surfactant-mediated extraction.     

Evaluation of thin-layer chromatography methods for quality control of commercial products containing Aesculus hippocastanum, Turnera diffusa, Matricaria recutita, Passiflora incarnata, and Tilia occidentalis

In Mexico, plant-derived products with health claims are sold as herbal dietary supplements, and there are no rules for their legal quality control. Aesculus hippocastanum, Turnera diffusa, Matricaria recutita, Passiflora incarnata, and Tilia occidentalis are some of the major commercial products obtained from plants used in this region. In this paper, we describe the effectiveness of thin-layer chromatography methods to provide for the quality control of several commercial products containing these plants. Standardized extracts were used. Of the 49 commercial products analyzed, only 32.65% matched the chromatographic characteristic of standardized extracts. A significant number of commercial products did not match their label, indicating a problem resulting from the lack of regulation for these products. The proposed methods are simple, sensitive, and specific and can be used for routine quality control of raw herbals and formulations of the tested plants. The results obtained show the need to develop simple and reliable analytical methods that can be performed in any laboratory for the purpose of quality control of dietary supplements or commercial herbal products sold in Mexico.     

Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb.

Bulbus Fritillariae derived from plants of various Fritillaria species is the most commonly used antitussive traditional Chinese medicinal herb and is called Beimu. Herbs derived from similar and/or different species of Fritillaria are also used in Japan and Turkey as traditional or folk medicines. Isosteroidal alkaloids are the main bioactive ingredients in Fritillaria species. As the contents and structure types of these bioactive alkaloids vary in different Fritillaria species, quality control of these active principles in herbal Beimu is very important to ensure its safe and effective clinical use. This review describes the development of chromatographic analyses for the simultaneous qualitative and quantitative determination of the main bioactive Fritillaria isosteroidal alkaloids in herbal and biological samples. The recently developed direct HPLC-evaporative light scattering detection method is the most simple, selective and sensitive assay, and is readily used as a suitable quality control method for the analysis of the active principles of herbal Beimu.     

Recent developments in chromatographic fingerprints from herbal products: set-up and data analysis

The use of chromatographic fingerprints from herbal products where the whole chromatographic profile is applied to evaluate the quality of the investigated product. In this paper, recent developments in the set-up and data analysis of chromatographic fingerprints for herbal products are discussed. First different set-ups for fingerprint development are reviewed. Prior to fingerprint development, a suitable sample preparation, e.g. extraction, should be considered. In a second instance, this review focuses on the data analysis with regards to the different applications of fingerprints. Usually, chemometric data pretreatment is necessary. This is discussed first, followed by a short overview of the data handling techniques used in the two main application areas of herbal fingerprints, i.e. quality assurance and classification or calibration. The quality assurance, which involves the identification and quality control of the herbal products, is reviewed, followed by the use of fingerprints in classification or modelling. The different application areas are illustrated and discussed with several case studies.     

Evaluation of Chinese medicinal herbs fingerprinting by HPLC-DAD for the detection of toxic aristolochic acids

Aristolochic acids are known to contribute to various renal disorders; therefore, expanding the availability of analytical methodology to detect these compounds is important in order to assess the quality of Chinese herbal medicines in which they can be found. Twelve medicinal herbal samples were procured from various sources and extracted in duplicate prior to a "fingerprint" analysis using conventional HPLC-DAD. Multivariate analysis was performed on the entire chromatographed fingerprints. The resulting output was a partial least-square discriminant analysis model, which was able to evaluate the potential presence of aristolochic acids I and II as well as providing an individual herbal "fingerprint". The results of this study provide evidence that the presence of aristolochic acids contained within certain herbal extractions could be detected using a simple method, although some limitations apply to this method for quality control, since newly detected samples for aristolochic acid (positives) will need further confirmation with purity checks or MS hyphenation.     

RP‐HPLC‐DAD method for the estimation of embelin as marker in Embelia ribes and its polyherbal formulations

Evidence‐based herbal products with assured quality are assuming importance for complementary and alternative medicine. Traditional medicines by and large are not standardized and validated to meet the new requirements. In the present study, marker (embelin)‐based standardization of a major medicinal plant, Embelia ribes and its polyherbal formulations was attempted. Conditions for the quantitative extraction of the marker compound embelin from E. ribes fruits and herbal formulations were also optimized. Reversed‐phase high‐performance liquid chromatography, coupled with diode array detection (RP‐HPLC–DAD) for the quantification of embelin was developed and validated. Satisfactory results were obtained with respect to linearity (15–250 µg/mL), LOD (3.97 µg/mL), LOQ (13.2 µg/mL), recovery (99.4–103.8%) and precision (1.43–2.87%). The applicability of the method was demonstrated with selected phytopharmaceuticals. The present method was sensitive, accurate, simple and reproducible and therefore can be recommended for marker‐based standardization, and quality assurance of E. ribes herbal formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Promising therapeutics with natural bioactive compounds for improving learning and memory - a review of randomized trials

Cognitive disorders can be associated with brain trauma, neurodegenerative disease or as a part of physiological aging. Aging in humans is generally associated with deterioration of cognitive performance and, in particular, learning and memory. Different therapeutic approaches are available to treat cognitive impairment during physiological aging and neurodegenerative or psychiatric disorders. Traditional herbal medicine and numerous plants, either directly as supplements or indirectly in the form of food, improve brain functions including memory and attention. More than a hundred herbal medicinal plants have been traditionally used for learning and memory improvement, but only a few have been tested in randomized clinical trials. Here, we will enumerate those medicinal plants that show positive effects on various cognitive functions in learning and memory clinical trials. Moreover, besides natural products that show promising effects in clinical trials, we briefly discuss medicinal plants that have promising experimental data or initial clinical data and might have potential to reach a clinical trial in the near future.     

Chemical fingerprint analysis for quality assessment and control of Bansha herbal tea using paper spray mass spectrometry

Chemical fingerprinting methodology is an approach for quality assessment and control of herbal medicines and related products based on the holistic chemical profile obtained by various analytical techniques. This study demonstrates the first application of paper spray mass spectrometry (PS-MS) as a chemical fingerprinting methodology for tracing the origins, establishing the authenticity, and assessing the overall quality of a famous herbal product, Bansha herbal tea (BHT). A negative ion PS-MS spectrum yielded the best chemical profiling information and was most appropriate for fingerprint analysis of BHT. In addition to the identification of active ingredients, various compounds present in BHT were simultaneously detected without any sample pretreatment and chromatographic separation, providing valuable information for the quality assessment and control of this herbal product. According to the principal component analysis of the PS-MS fingerprints, two sources of commercially available BHT products made by different manufacturers were easily differentiated. Qualified and expired products from the two manufacturers were also successfully distinguished, and the consistency of the quality between the manufacturers was assessed. Our experimental data demonstrated that the PS-MS chemical fingerprinting is a simple, rapid, and robust methodology for pharmaceutical analysis, with promising prospects for quality assessment and control of herbal medicines and related products with high-throughput.     

Quality control of medicinal herbs Fructus gardeniae, Common Andrographis Herb and their preparations for their active constituents by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

Dehydroandrographolide, andrographolide and geniposide are the main active constituents of many herbal medicines, e.g., Fructus gardeniae, Common Andrographis Herb. They are used as the markers to control the quality of such herbal medicines and their herbal preparations. In this paper, a simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) were developed to determine the three compounds simultaneously in extracts of medicinal herbs and herbal preparations produced by different companies. The extracts were separated on a C₁₈ reversed phase HPLC column, with a gradient solvent system, the time for the separation of the three target analytes was 10 min. The abundance ions were recorded using selected ion monitoring (SIM) mode with m/z 297.3, 297.3 and 411.1 for dehydroandrographolide, andrographolide and geniposide, respectively. The limit of detection for dehydroandrographolide, andrographolide and geniposide were 20, 30 and 150 ng mL⁻¹, respectively. The proposed method was successfully applied to the determination of the contents of the compounds in related to medicinal herbs and preparations.     

A rapid site‐specific PCR based one‐tube authentication method for saffron in crude drugs and processed herbal medicines

Saffron is one of the world's most precious medicinal herbs and often found to be adulterated with other cheaper materials. The chemical compounds in Crocus sativus L. such as crocin, picrocrocin also exist in other plants, which makes the chemotype‐driven analysis not ideal for the quality control of saffron. Herein, we developed a rapid authentication method for saffron in crude drugs by the site‐specific PCR. In order to realize fast high‐throughput analysis, a one‐tube identification approach was further established by using a universal fluorescent dye to detect the PCR products. In addition, this method was also applied to the authentication of saffron in a processed herbal medicine “Er shi wu wei shan hu wan” which consists of twenty‐five kinds of medicinal materials including plants, minerals and animals. Additionally, this method was also proved to be with high specificity and repeatability. The flexibility of choosing different primers also made this method versatile for other medicinal materials.     

Similarity analyses of chromatographic herbal fingerprints: A review

Herbal medicines are becoming again more popular in the developed countries because being “natural” and people thus often assume that they are inherently safe. Herbs have also been used worldwide for many centuries in the traditional medicines. The concern of their safety and efficacy has grown since increasing western interest. Herbal materials and their extracts are very complex, often including hundreds of compounds. A thorough understanding of their chemical composition is essential for conducting a safety risk assessment. However, herbal material can show considerable variability. The chemical constituents and their amounts in a herb can be different, due to growing conditions, such as climate and soil, the drying process, the harvest season, etc. Among the analytical methods, chromatographic fingerprinting has been recommended as a potential and reliable methodology for the identification and quality control of herbal medicines. Identification is needed to avoid fraud and adulteration. Currently, analyzing chromatographic herbal fingerprint data sets has become one of the most applied tools in quality assessment of herbal materials. Mostly, the entire chromatographic profiles are used to identify or to evaluate the quality of the herbs investigated. Occasionally only a limited number of compounds are considered. One approach to the safety risk assessment is to determine whether the herbal material is substantially equivalent to that which is either readily consumed in the diet, has a history of application or has earlier been commercialized i.e. to what is considered as reference material. In order to help determining substantial equivalence using fingerprint approaches, a quantitative measurement of similarity is required. In this paper, different (dis)similarity approaches, such as (dis)similarity metrics or exploratory analysis approaches applied on herbal medicinal fingerprints, are discussed and illustrated with several case studies.