Implementing a “Quality by Design” Approach to Assure the Safety and

Aug 31, 2012 - Integrated analytical assets aid botanical authenticity and adulteration management. Charlotte Simmler , James G. Graham , Shao-Nong Ch...
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Implementing a “Quality by Design” Approach to Assure the Safety and Integrity of Botanical Dietary Supplements Ikhlas A. Khan*,†,‡ and Troy Smillie† †

National Center for Natural Products Research and ‡Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ABSTRACT: Natural products have provided a basis for health care and medicine to humankind since the beginning of civilization. According to the World Health Organization (WHO), approximately 80% of the world population still relies on herbal medicines for healthrelated benefits. In the United States, over 42% of the population claimed to have used botanical dietary supplements to either augment their current diet or to “treat” or “prevent” a particular health-related issue. This has led to the development of a burgeoning industry in the U.S. ($4.8 billion per year in 2008) to supply dietary supplements to the consumer. However, many commercial botanical products are poorly defined scientifically, and the consumer must take it on faith that the supplement they are ingesting is an accurate representation of what is listed on the label, and that it contains the purportedly “active” constituents they seek. Many dietary supplement manufacturers, academic research groups, and governmental organizations are progressively attempting to construct a better scientific understanding of natural products, herbals, and botanical dietary supplements that have co-evolved with Western-style pharmaceutical medicines. However, a deficiency of knowledge is still evident, and this issue needs to be addressed in order to achieve a significant level of safety, efficacy, and quality for commercial natural products. The authors contend that a “quality by design” approach for botanical dietary supplements should be implemented in order to ensure the safety and integrity of these products. Initiating this approach with the authentication of the starting plant material is an essential first step, and in this review several techniques that can aid in this endeavor are outlined.

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INTRODUCTION The role of the natural product chemist is ever challenging, with the contemporary researcher in this field being often associated with drug discovery. In the model used commonly, selected biological activity is followed through various fractionation schemes for the purpose of lead compound discovery in order to target various therapeutic areas (e.g., infectious diseases, CNS, immune stimulation/suppression, cancer) in addition to the search for new compounds. However, “natural products” are now being extensively marketed in the form of dietary supplements, nutraceuticals, health products, or traditional medicines, or as prescriptions written from national pharmacopeias (e.g., Chinese and Indian). The use of dietary supplements in the U.S. has increased significantly over the past few decades.1 For example, since the passage of the Dietary Supplement Health and Education Act (DSHEA) in 1994, the botanical dietary supplement market in the U.S. has grown rapidly, from $2.9 billion in 19952 to $4.8 billion in 2008.3 This trend results in a considerable challenge to the natural products chemist to evaluate the quality of botanical ingredients, which is directly linked to the safety and efficacy of the final dietary supplement products. In comparison to pharmaceuticals, the quality and safety of botanical products are more complicated in their evaluation. In the case of pharmaceuticals, the physiochemical properties of the lead compound must be fully characterized before it is even considered for clinical development or marketing. After characterization, the drug is then developed under strict © 2012 American Chemical Society and American Society of Pharmacognosy

GLP/GMP guidelines. On the other hand, for botanicals, the raw materials are often grown in disparate conditions and geographic locations, harvested at various times, and processed differently, with often different species or varieties used interchangeably due to simple misidentification or as the result of the use of common names or synonyms. Therefore, the first step toward assuring the quality and safety of botanicals should be to inherently incorporate these attributes, through attaining a comprehensive knowledge of which phytochemical constituents make up a “quality” botanical product. The “quality by design” (QbD) concept was coined by Joseph M. Juran in 1992 to address the quality control issues involved in the manufacturing process.4 The premise behind the QbD philosophy is the idea that quality should be built into the product from the onset. Each step of the production process should be focused on avoiding potential risks if strict standards are not met. Immediate measures should be taken to correct any quality discrepancies in order to ensure that the end consumer is provided with a product that is safe and of superior quality. The U.S. FDA recently has adopted the QbD model for the process validation procedure of the pharmaceutical industry and has observed a significant improvement in product quality from companies following these guidelines. As an additional benefit, industries that undertake a QbD model typically experience an increase in efficiency, productivity, and cost Received: June 20, 2012 Published: August 31, 2012 1665

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particular analytical methods required in order to meet these stipulations. The preamble to regulation 21 CFR §111 does provide the reasoning behind why the FDA did not specify a particular “scientifically valid method”. This is because the selected method “... could become obsolete if we (FDA) based it on specific sources such as INA (Institute for Nutraceutical Advancement), AHP (American Herbal Pharmacopoeia), or USP (United States Pharmacopoeia)”.6 This “identity” criterion as stipulated by the FDA implies that each botanical component will require a specific, scientifically valid authentication method in order provide the necessary proof to comply with this regulation. A main purpose of this review is to outline some specific techniques and methodologies that, in combination with a quality by design approach, can aid investigators in the authentication of botanical ingredients and dietary supplement products.

savings because each step of the process provides a superior intermediate item. In this manner, production does not have to be impeded in order to correct flaws that occurred in the manufacturing process. It is important that a QbD model should be implemented for botanicals as well as pharmaceuticals, since plants have an inherent variation in consistency and possess a potential risk for adulteration. Such adulteration represents a major impediment in the control of product quality and potentially can adversely impact public health. For botanicals, quality by design should start with the knowledge of the seed/genetic source of the plant material; the identity of the specific species grown; information on the climate or environment of plant growth; the date of harvest; how the plant was stored; and details of the processing, extraction, and overall phytochemical composition for each sample in question. Unfortunately, the current U.S. market regulations do not yet stipulate a “quality standard” for commercial botanical products.

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AUTHENTICATION OF BOTANICALS One of the most critical issues involved in developing botanical products is the process for assessing the authenticity and quality of the individual raw ingredients. Since these ingredients are produced naturally, there is a possibility for the misidentification of the collected plant, potential adulteration with other related species, or contamination with extraneous ingredients during processing. From the perspective of a safety concern, these issues may range from simple misleading labeling to potential poisoning due to toxic contaminants.7 A considerable amount of research in this area has been applied to the use of a “standardized” botanical material, which usually implies a chemical standardization based on the quantification of one or more selected marker compound(s). However, while chemical standardization can provide a useful technique for authentication purposes, such standardization is of limited utility when the starting raw material is not authentically characterized botanically. For example, Tanacetum parthenium (L.) Schultz Bip. (feverfew) and Magnolia grandif lora L. are two distinctly dissimilar species botanically; however, both plants contain the purported “active” ingredient parthenolide.8 Therefore, if an analytical method that selectively detected this compound for authentication purposes is used, then there is the possibility of accidental misidentification for these two disparate species. Fortunately, there are several tools and approaches that have been developed and used for the authentication of botanical ingredients. Selection of the method is highly dependent on the species in question, the specific plant part(s) being studied, the sample processing method, and the inherent matrices of the specific formulation in question. The available techniques range from a straightforward botanical, organoleptic, or morphological identification of the plant to very elaborate genetic or phytochemical and analytical approaches. Many of the cutting-edge botanical authentication techniques used today stem from traditional pharmacognostic research methods.9−11 Each identification technique requires significant levels of prior information, infrastructure, and skill sets in order to achieve a full-spectrum approach for the authentication of botanical samples. Many botanical dietary supplement manufacturers currently utilize chemical “fingerprinting” techniques for identity purposes. However, there are several other methods that can be effectively employed to properly authenticate a botanical sample. Plant Sourcing and Classical Authentication Techniques. The initial criterion for the identification and authentication of botanical materials starts with the acquisition

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UNITED STATES REGULATORY ASPECTS While this review is not intended to summarize the entire U.S. regulatory procedures associated with dietary supplements, a synopsis of the current regulatory policies is outlined to provide a perspective of what is anticipated, from a legal standpoint, for dietary supplements marketed in the United States. New pharmaceutical drug candidates are required to have FDA approval before being marketed; however, DSHEA stipulates that dietary ingredients legally on the market prior to October 1994 are generally regarded as safe (GRAS). The assumption made is that if these ingredients were not safe, then the FDA would have already removed these ingredients from the market prior to that date. After 1994, the FDA required manufacturers to submit a “new dietary ingredient” (NDI) notification with complete safety information to the agency 75 days prior to the date of first marketing. The FDA has the authority to deny permission for the new ingredient to enter the marketplace based on safety concerns. Products in the marketplace that contain NDIs for which NDI applications were rejected by FDA, or for which no NDI notification was filed previously, are considered “adulterated” under the Food, Drug, and Cosmetic Act. In July of 2011, the FDA released a “Draft Guidance for Industry: Dietary Supplements: New Dietary Ingredient Notifications and Related Issues” document5 to aid dietary supplement manufacturers in determining whether or not a NDI notification is required for proposed dietary supplement ingredients. Even though there are many new dietary ingredients in the marketplace, to date the FDA has received very few NDI notifications and has approved only a few of these. Additionally, there is a concern from the botanical dietary supplement industry regarding what the FDA considers as “chemically altered” ingredients. Similar constituents or processing techniques have been deemed safe with regard to items in the current food supply chain, but these appear to be scrutinized more intensely when applied to botanical dietary supplements. Therefore, the FDA faces a daunting task in ensuring the safety and quality of an ever-increasing number of botanical products while not stifling activities of the current botanical dietary supplement industry. The current GMP (good manufacturing practice) regulations of the FDA stipulate that manufacturers have to provide full verification that “specifications are met for the identity, purity, strength, and composition of the dietary supplements”. On the other hand, the regulations do not instruct manufacturers about 1666

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For example, “chamomile” is the common name for many plants of the family Asteraceae, of which the flowers are commonly used in teas and infusions for their calming and antianxiolytic effects. The two main species of chamomile used in commerce are German chamomile (Matricaria recutita L.) and Roman chamomile [Chamaemelum nobile (L.) All.]. While both can be sold interchangeably on the market as “chamomile”, the phytochemical profile for these two species is significantly different, so the ability to utilize a macromorphological method to distinguish between these two species would be advantageous to those responsible for sourcing, distributing, or receiving bulk “chamomile” material. Fortunately, the most notable morphological difference between these two species occurs in the plant part (flowers) most commonly marketed. As shown in Figure 1, the flower

of authentic tissue samples from the representative species and selected target tissues. Initial tissue sourcing options for authenticated plant material can vary significantly from selected seed stock to germplasm propagation to field collection. The ability to grow the selected species in a controlled environment (e.g., greenhouse, shade house, or open plot) by trained agronomists or botanists can provide a certain level of consistency for given botanicals. However, many species thrive better living in the wild, and, under these circumstances, collections need to be carried out by either a trained herbalist or botanist to ensure that the correct species is collected and that proper harvesting techniques are followed. Regardless of the method by which the material is propagated and raised, the collected tissue needs to be characterized and authenticated by a taxonomist utilizing classical botanical methodologies. Vital collection and identification information needs to be captured and documented for each sample, and the information should include the proper Latin binomial nomenclature (vs common name); the name of the collector or collectors; the date and location of collection (GPS preferred); a geographical/habitat description; a unique collection number/code; plant part(s) collected; other habitat information; organoleptic characteristics (smell, taste, color, etc.); processing steps (drying method, time, and temperature); and a visual representative image (digital picture or sketch) of the plant in both its growing habitat and a postharvest mounted specimen. Additionally, a representative sample of the collected material in the form of a voucher specimen containing an accurate description of the plant needs to be stored in either a registered public herbarium, certified research institute, or, in the case of commercial materials, an on-site herbarium repository. Vouchering techniques have been thoroughly outlined in the scientific literature12,13 and require a properly dried sample of the aboveground portions of an “archival quality” plant specimen as a representative example of the collected material(s). This sample is then mounted on a herbarium sheet in a format used to conserve the identifying morphological characteristics of the specimen and thereby to assist in the identification and authentication of the overall population being collected. The herbarium voucher sample should include as many morphological identification “keys” as can be obtained at the time of collection. These taxonomic keys include fruits, flowers, and (less commonly) vegetative material and are often utilized as macrocharacteristics for the identification of most plants. Macroscopic and Microscopic Authentication. Both macroscopic and microscopic investigations constitute the mainstay of classical botanical authentication and characterization techniques for whole plants, plant parts, and, in some cases, the plant material that has been dried and processed. Macroscopic characteristics that may be examined to aid in this technique include traits such as woody/suffruticose/herbaceous; leaf shape, size, and morphology (e.g., leaf margins: entire, undulate, dentate, serrate, lobed, or pinnatifid); flower characters such as type of inflorescence (e.g., spike, raceme, panicle, cyme, corymb, helicoid cyme, head); floral morphology (e.g., epigynous, perigynous, hypogynous; stamen number and shape; number of carpels in ovary; number of seeds per carpel); and root characteristics including surface texture, type (corm, bulb, rhizome, etc.), and tissue layering (banding patterns). In many instances, macroscopic techniques can be used to discriminate between the desired plant species and potential adulterants that are morphologically similar, yet distinguishable by one or more of these “key” characteristics.12

Figure 1. German chamomile (Matricaria recutita) entire flower head (A) and vertically cut flower head (B) to show arrangement of florets and the hollow receptacle. Vertically cut Roman chamomile (Chamaemelum nobile) flower head (C) to show the solid receptacle and the arrangement of florets and presence of paleae, along with a cross section of the receptacle (D).

receptacle of M. recutita is hollow, whereas the receptacle of C. nobile is solid. Additional differentiation can be observed in the distribution of the petals as well as the overall shape of the flower head and inflorescence. Figure 2 also shows the

Figure 2. Images of (A) German chamomile (Matricaria recutita) and (B) Roman chamomile (Chamaemelum nobile), displaying differentiation characteristics of the stem and leaf morphology.

differentiation of the stems and leaves, with M. recutita having more erect and hairy stems with thin and feather-like leaves, while C. nobile has hairless stems that are more spread out and contain flatter and thicker leaves. Microscopic techniques are especially useful when attempting to establish authenticity or adulteration in a sample of ground plant material because most macroscopic characteristics are difficult to distinguish in such samples. Microscopic approaches involve techniques such as scanning electron microscopy14−16 or standard light microscopy14,17−19 to analyze characteristics such as the presence or absence of hairs (trichomes), oil glands, canals, particular cell types, seed morphology, pollen morphology, and vascular traces. Fluorescence microscopy20,21 can provide a selective colorful image 1667

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tives that can pose a serious detrimental health effects for consumers.22−30 While micro/macroscopic techniques can provide a classical botanical-/pharmacognostic-based authentication method for many herbal samples, these techniques lose their effectiveness when dealing with complicated multicomponent powdered samples, or when there is little to no cellular distinction between closely related genera, or where material is processed or formulated beyond the ability to provide distinct morphological characterization. When these circumstances occur, it is necessary to utilize alternative techniques in order to effectively identify and authenticate botanical samples. Genetic Fingerprinting Techniques. One burgeoning area of botanical authentication research is the field of genetic fingerprinting and profiling. Ideally, botanical genetic profiling methods should be cost-effective, adaptable to efficient highthroughput analysis, and utilize technology that can be easily transferred and validated. Additionally, it is important that one develops efficient and easily reproducible genetic extraction procedures that can yield sufficient quantities of high-quality DNA. Typically, the best quality DNA is obtained from freshly harvested, young, fast-growing tissues. Unfortunately, many botanical samples consist of dried powdered plant parts made up of highly differentiated tissues of various ages that contain DNA of relatively poor quality, so the extraction and manipulation steps are technically much more difficult. The harvesting, drying, storing, and processing of plants used in many dietary supplements tend to result in the degradation of the genetic material of the plant. In addition, inherent phytochemicals occur within the plant, and these can hinder DNA manipulation and analysis using polymerase chain reaction (PCR)-based technologies. Fortunately, there are commercially available kits, such as the DNAeasy genomic extraction kit from Qiagen, that can provide investigators with the ability to isolate quality DNA from various plant materials with very little need for modification. Once the genetic material is extracted, the considerable task of sequencing the isolated DNA must be addressed. The number of DNA regions available for potential sequencing is increasing rapidly as researchers search multiple genomic areas to obtain genetic markers. Many investigators target nuclear and/or chloroplast genomic regions. The advantage of studying chloroplast genomic regions is the fact that the chloroplast genome is small (between 120 and 200 kb) and that most of the genes are essentially a single copy. The chloroplast genome is considered to be conservative in its evolution and evolves slowly at the nucleotide level. Nonetheless, different portions of the chloroplast genome evolve at different rates. While focusing on one “select” nuclear or singlecopy chloroplast genomic region often cannot provide a means of developing a method that is suitable to differentiate all species, a perusal of the literature shows that one or more of the genomic regions described below (ITS, psbA-trnH, trnL-trnF, atpB-rbcL, and rpoC) can be used to provide good sequencing results for a variety of sample types. The nuclear ITS genomic region, between the 18S rRNA gene and the 25−28S rRNA gene, consists of an internal transcribed spacer 1 (ITS1), a 5.8S rRNA gene, and internal transcribed spacer 2 (ITS2).31−33 The psbA-trnH intergenic region is the noncoding region between the end of the gene for the D1 protein of photosystem II and the gene coding for the tRNA (tRNA) for histidine.34 This is one of the most variable regions in the angiosperm chloroplast genome.35−37 The trnL-

that can be diagnostic for a given species. For example, when examining various species of Ephedra from North America, South America, and China, it was possible to differentiate samples from these geographical regions based on the helical thickness of vessels and tracheids, the ratio of the vessel length as compared to tracheid length, and mean vessel diameter as well as distinct ray patterns.18 Figure 3 shows examples of these morphological differences.

Figure 3. Transverse sections of stems of various species of Ephedra showing secondary xylem features of (A) E. fedtschenkoae (mean vessel diameter 40−60 μm; China) and (B) E. coryi (mean vessel diameter 20−40 μm; North America) and ray patterns of (C) E. equisetina (China) and (D) E. nevadensis (North America).

Lastly, microscopic evaluation of herbal products can disclose unusual results. During a routine evaluation of an “herbal Viagra” product, it was observed that a crystalline substance was mixed within the ground plant tissue. These crystals were easily detectable at 10× magnification and appeared to be a crystallized synthetic chemical (Figure 4). The crystals were

Figure 4. Sildenafil citrate crystals in a purported “herbal Viagra” product.

collected manually and identified as sildenafil citrate (Viagra), indicating that either the supplier of the bulk material or the manufacturer of this product knowingly added a synthetic pharmaceutical ingredient into a purportedly “natural botanical”. While this not a common occurrence, there have been several reports of the adulteration of botanical products with pharmaceutical ingredients or closely related synthetic deriva1668

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trnF noncoding region is located between tRNAs for lysine and phenylalanine.38−40 The atpB-rbcL noncoding region is located between the 3′ ends of the ATP synthase subunits (atpB) and the large subunit of the ribulose 1,5-bisphosphate carboxylase gene RUBISCO (rbcL).41,35 The rpoC region for RNA polymerase consists of two genes, rpoC1 and rpoC2. The subunit C1 (rpoC1) gene is interrupted by a single intron in most, but not all, terrestrial plants.37,42 The fragments generated by PCR can then be cloned and sequenced for comparative analysis. If the sequence shows enough differences, then it is possible to design species-specific PCR primers that will yield a PCR product of a known size that can be used as a method to confirm the botanical content of a dietary supplement. One method is PCR restriction fragment length polymorphism (PCR-RFLP), where various gene regions are amplified by PCR then cut with restriction enzyme(s) and analyzed by gel electrophoresis. An example of results obtained by this technique is shown in Figure 5. Fragment length polymorphisms can then be identified and used to generate a specific DNA fingerprint.32

samples and identifying the presence of adulterants. Selection of the most appropriate method is highly dependent on the level of genetic relationship of the medicinal plant, and related genera, and other potential adulterants. Analytical Chemical Fingerprinting Techniques. Analytical separation techniques (e.g., HPTLC, HPLC, UPLC, CE, or GC with a suitable detection mode) currently provide the most reliable and applicable authentication methods for botanicals. By acquiring and analyzing a statistically significant representative collection of authentic plant specimens from multiple collection sources, a valid analytical “fingerprint” method for authentication purposes can be developed.47 However, in order to accomplish this, it is necessary to isolate and identify selected “marker” compounds that make up an analytical fingerprint that is distinct for the selected species. These makers should ideally be diagnostic for the selected species and preferably represent the health-relevant principal component(s) for the species in question. The more key identifying markers one utilizes in developing an analytical fingerprint, the higher the resultant level of confidence that can be achieved for the identification and authentication of “unknown” samples. Additionally, the use of multiple (and preferably highly diagnostic) marker compounds can dissuade dishonest bulk material distributors and manufacturers from “spiking” cheap botanical samples, since each additional constituent required to meet a stringent authentication level adds another layer of complexity and cost to the unsavory practice known as “economic adulteration”. However, in order for any analytical fingerprint method to be widely adopted and validated, requires that sufficient quantities of the selected markers are readily available. While some phytochemical markers can be synthesized, this is often not a cost-effective solution. Therefore, many of the constituents are only attainable utilizing classical pharmacognosy techniques (extraction, isolation, and spectroscopic identification). The lack of relevant marker compounds is the major limiting factor hindering the widespread adoption of quality control approaches for botanicals. The interest in finding suitable markers has waned significantly because this area of research has been relegated to being considered as “basic” in need and not seen as a valuable asset that can not only provide a quality control measure for botanicals but also be used as a cornerstone for the understanding of the phytochemical complexity of these widely used products. A longstanding research effort at the National Center for Natural Products Research (NCNPR) has included the investigation of hundreds of common botanicals and products, resulting in the isolation and characterization of thousands of both known and new compounds that have been utilized for the development of authentication methods as well as biological evaluation. Recent isolation work on plants such as Hoodia gordonii (Masson) Sweet ex Decne.,48−52 Scutellaria laterif lora L.,53 Labisia pumila (Blume) Fern.-Vill.,54 Casearia sylvestris Sw.,55−60 Terminalia arjuna (Roxb. ex DC.) Wight & Arn.,61−63 Centella asiatica (L.) Urb.,64 and Sutherlandia f rutescens (L.) R.Br. ex W.T. Aiton65,66 are prime examples of the type of effort that is required to facilitate a full understanding of the phytochemical complexity of botanical samples. It stands to reason that each botanical sample requires a tailored isolation scheme to address the specific phytochemical constituents that are inherent to that particular plant. Additionally, what is often overlooked is a full understanding of the intricacies involved in extracting, fractionating, isolating, and unambiguously identifying these constituents.

Figure 5. Nuclear ITS genomic region amplified from various Angelica species and then cut with a restriction endonuclease. M = molecular size standard.

An alternative is simple sequence repeat (SSR) polymorphism or microsatellites for authentication purposes. SSRs are short sequence motifs consisting of two or more nucleotides (e.g., CA or AGT), which repeat in tandem (e.g., CACACACA or AGTAGTAGT). The repeats vary in length and are ubiquitously and randomly distributed in the eukaryotic genome. Primers anchored at simple sequence repeats are used to amplify the DNA regions between the flanking SSR. The length polymorphisms of the generated amplicons can be detected by gel electrophoresis.43,44 The construction of SSR libraries provides an indispensable tool to search for molecular markers since complete genome sequences are still not available for the majority of species of interest. However, it is often necessary to selectively enrich genomic DNA libraries for repeat sequences in order to increase the number of cloned fragments that contain repeats as compared to the information that would be obtained from random-insert libraries. A highly efficient and simplified protocol for the successful generation of SSR-enriched genomic libraries has been developed and used on various organisms to demonstrate that this protocol works effectively across phyla.35,45,46 Genomic fingerprinting, with the methods mentioned above, can be useful for confirming the homogeneity of botanical 1669

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to compress existing fingerprinting profiles while simultaneously increasing resolution and sensitivity. Combining these advances with increased sensitivity for a variety of detection options (e.g., UV, ELS, MS, HRMS, MS/MS) has revolutionized the ability to quickly develop applicable analytical methods for authentication purposes. Fingerprinting methods and phytochemical identity techniques have been developed for a myriad of authenticated botanicals that aid significantly in the analysis of dietary supplement products as well as safety evaluations. For example, recently published methods for the following species have appeared: Hoodia gordonii (Masson) Sweet ex Decne.,75−77 Citrus aurantium L.,78−80 Actaea racemosa L.,81,82 Scutellaria laterif lora L.,83 Curcuma longa L.,84 Gardenia spp.,85 and Pausinystalia johimbe (K.Schum.) Pierre.14 Such work not only displays the utility of using advanced LC techniques but in many cases indicates how these methods can be combined with more classical authentication techniques to provide a more “holistic” designed approach toward establishing the identity of these species. Lastly, there are several other chromatographic techniques that can also be utilized for authentication purposes. Separation techniques such as GC15,86,87 and CE88−90 have been used extensively for qualitative and quantitative analysis of botanicals. Additionally, a re-emergence of supercritical fluid techniques is anticipated since this method has the potential to provide chiral separations as well as offering an affordable and safe mobile phase alternative.91−93 As mentioned earlier, a major limitation of analytical fingerprinting techniques is the requirement to have an established set of marker compounds that are unique to a given botanical specimen. Additionally, the reality is that one has to develop a method that often has to accommodate a potentially broad range of phytochemical analytes. Unfortunately, most developed analytical methods are quite often “myopically” restricted in their capability to recognize the broader chemical profile of the plant in question, and often researchers need to use multiple analytical methods in order to evaluate disparate sets of analytes. To address this issue, many researchers have turned to utilizing broad-range spectroscopicbased analysis techniques in combination with chemometric analysis. Chemometric Techniques (MS, NMR, UV, IR). An extension of phytochemical fingerprinting would be the utilization of statistical evaluation tools such as hierarchical cluster analysis (HCA) or principal component analysis (PCA) to evaluate either a broad-range spectroscopic scan of a given botanical or a representative chromatographic segment of a given sample as compared to a compiled population of authenticated reference samples.94,95 One of the advantages of this type of statistical approach is that it utilizes pattern recognition parameters to evaluate peaks/components within the data clusters in order to see if the “test” sample correlates to the population of authenticated samples. Since phytochemical chemometric techniques utilize a broadspectrum evaluation of the chemical profile of the botanical in question, the material analyzed can be a crude whole extract with very little prefractionation. The advantage of this type of methodology is that it is nonselective and can provide a more “holistic” phytochemical snapshot of the sample in question, thereby eliminating some of the bias or myopathy that can be inherent in classical fingerprinting techniques. One of the initial authentication techniques to use this type of statistical analysis was GC-MS, where the volatile

Once the indicative markers are obtained and fully characterized, then the task of method development must be undertaken. Selected experimental parameters, such as mobile phases, stationary phases, and operating conditions, need to be taken into account along with the particular chemical characteristics of the identified markers. Additionally, in order for an analytical method to be considered “validated”, it also needs to be evaluated for the following criteria: precision, accuracy, specificity, robustness, ruggedness, and repeatability. The proposed method will need to be selective and linear for the range in question and provide a reasonably low limit of detection and limit of quantitation.67,68 While development of an analytical fingerprint profile requires a significant investment in effort, the resultant methods provide an invaluable tool for the identification and authentication of botanical samples as well as providing the capability to utilize the developed method(s) for potential subsequent clinical sample evaluation. One of the more “classical” analytical techniques that is still quite practical in modern identification applications is highperformance thin-layer chromatography (HPTLC). HPTLC is an advance over standard TLC in utilizing smaller silica gel particles (5−7 μm vs 10−15 μm, respectively), thereby providing more theoretical plates for greater resolution of the constituent(s) of interest. Additionally, there are robotic applicator systems and development chambers along with digital imagery and densitometry capabilities that can be used for the development of qualitative and quantitative analysis of HPTLC data.69 HPTLC also allows the rapid evaluation of multiple samples side by side in identical conditions, thereby affording additional cost savings for comparative analyses. HPTLC has been used to both quantitatively and qualitatively evaluate several botanical species such as Caulophyllum thalictroides (L.) Michx.,70 Morus spp.,71 Lonicera japonica Thunb.,72 Lancea tibetica Hook. f. & Thomson,73 and Rhodiola spp.74 Figure 6 illustrates how HPTLC can provide a snapshot

Figure 6. HPTLC chromatogram of R. rosea extract (1); R. sachalinensis extract (2), commercial samples (3−5), a standard mixture (6), and extracts of authentic R. rosea samples from Nunavik, Canada (7−10).

profile of authentic samples of Rhodiola spp. as well as commercial products claiming to contain Rhodiola spp. While HPTLC provides a handy benchtop evaluation tool for rapid analysis of many types of botanicals, it does have its limitations with regard to sensitivity, resolution, and adaptability. Under circumstances where HPTLC cannot meet these criteria, it is imperative to utilize more robust techniques that can achieve the desired separations and quantitative evaluations. The evolution of LC technologies has resulted in significant advances in the design of column packing materials. Highperformance, high-pressure LC systems have made it possible 1670

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associated with this issue. Unfortunately, even the essential requirement of an authenticated reference plant sample that is properly vouchered and taxonomically identified by a qualified botanist is often overlooked. Additionally, one needs to understand the potential botanical product that is being analyzed such as the plant part utilized or the type of final preparation evaluated. Establishing a QbD model to evaluate as many of the key aspects that identify each botanical is imperative since there is no single method that can authenticate every plant sample or characterize each dietary supplement. For each botanical, there needs to be a full understanding of the constituents being considered and the capabilities of the techniques specifically suited for authentication purposes. While a significant amount of research has been published to aid in the authentication of botanicals, it is abundantly clear that more work needs to be done in order to fill in the substantial gaps that still exist, such as evaluating possible adulterants that can be associated with the botanical of interest including spiked pharmaceutical additives, economic adulterants, and accidental substitution by commonly misidentified or improperly characterized material. Additionally, safety concerns surrounding botanicals do not end with the “authentication” of the plant material but must also include analyses for potential microbe, pesticide, and heavy metal contaminations. It is clear that only a systematic designed approach can provide the required solution for complete botanical characterization (genetic, morphological, phytochemical, etc.), authentication, and safety evaluation. In order to fully undertake this approach, continued research effort in the related fields of authentication is required. This, in turn, demands that more individuals will need to be trained in the performance of these techniques as well as the provision of a committed level of support for the basic sciences that promote this type of research.

components of a complex plant extract are injected and the resultant peaks are identified by their specific mass, which then provides the basis for statistical analyses and clusters the “like” samples into grouped populations.96−99 Similar statistical methods (PCA, HCA, LDA, etc.) have since evolved and been coupled with other chromatographic systems such as HPLC-UV and HPLC-MS to provide new tool sets for population analysis.100−108 Recently, the inclusion of 1H NMR chemometric profiling has emerged as yet another methodology for the evaluation of botanical extracts.109−111 Broadspectrum 1H NMR spectroscopic evaluation of crude botanical extracts provides researchers with the capability to understand the full chemical profile and offers additional information regarding potential phytochemical structural characteristics that give rise to the profile in question. Lastly, infrared (IR) and ultraviolet (UV), visible spectroscopy, in combination with other detection techniques, have also been utilized to ascertain the authenticity of plant samples.112−118 Many of these techniques use either near-IR (NIR ≈ 4000−12500 cm−1), mid-IR (MIR)/Fourier transform IR (FT-IR ≈ 400−4000 cm−1), or FT-Raman (∼400−4000 cm−1) spectra for botanical analysis. While NIR provides ease of use for a rapid evaluation of raw materials, this method often falls short of providing a reliable technique for authentication purposes due to its inability to provide a high-resolution profile required to identify peaks of interest, and this technique is susceptible to interference from residual water signals. FT-IR analysis provides greater spectroscopic resolution than NIR; however, the FT-IR spectrum can also be significantly impacted by residual water signals. Unlike NIR and FT-IR, which rely on absorbance of IR radiation, FT-Raman spectroscopy measures the resultant scattering at a specific wavelength. This allows for the removal of errant water peaks and also provides the ability to perform measurements through some packaging materials. Both the FT-IR and FT-Raman IR techniques combined with statistical analyses can provide analysts with a valuable resource for identification purposes. All of the aforementioned chemometric methods require that investigators have a significant population of authenticated specimens for spectroscopic measurement in order to construct a spectroscopic library. Post analysis, the compiled library typically consists of alignment, regional exclusion, binning, normalization, and scaling criteria. Then through the use of statistical analysis packages such as HCA, PCA, or LDA, investigators can then begin to cluster like populations of samples based on recognized similarities within the populations. While much of the analysis can be undertaken automatically with the requisite software packages, it should be stressed that the analyst must have an in-depth understanding as to what each set of signals in the given spectra represents, pytochemically, so that the identification of key constituents within a material can be assigned logically. It is also important to note that these methods are better suited for single-constituent (one plant sample) analysis and do not lend themselves to multicomponent “finished” products.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (662)-915-1090. Fax: (662)-915-7989. E-mail: ikhan@ olemiss.edu. Author Contributions

Adapted from the Varro E. (Tip) Tyler Award for Botanical Research Address by Ikhlas A. Khan at the 52nd Annual Meeting of the American Society of Pharmacognosy, July 30−August 3, 2011, San Diego, CA, USA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01 FD004246-01. Many of the examples described were investigated by the following scientists: E. Abourashed, Z. Ali, B. Avula, E. Bedir, M. Ganzera, A. Jadhav, V. Joshi, S. Khan, R. Pawar, V. Raman, C. Rumalla, B. Schaneberg, Y. Shukla, N. Techen, A. Weerasooriya, W. Wang, Y.-H. Wang, and J. Zhao, who, along with many other collaborators throughout the years, have diligently worked on this research initiative.

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SUMMARY AND CONCLUSIONS While admittedly difficult to undertake, a quality by design approach can provide the best suitable solution for the longstanding problems associated with botanical authenticity. Taking this type of methodical approach to understanding the inherent attributes of a given botanical from seed to shelf is probably the most logical way to approach the problems

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REFERENCES

(1) Barnes, P. M.; Powell-Griner, E.; McFann, K.; Nahin, R. L. Adv. Data 2004, 343, 1−19. 1671

dx.doi.org/10.1021/np300434j | J. Nat. Prod. 2012, 75, 1665−1673

Journal of Natural Products

Review

(2) Nutrition Business Journal; New Hope Natural Media, Penton Media Inc.: Boulder, CO, June, 1998. (3) Nutrition Business Journal. New Hope Natural Media, Penton Media Inc.: Boulder, CO, September, 2009. (4) Juran, J. M. Juran on Quality by Design: The New Steps for Planning Quality into Goods and Services; The Free Press: New York, 1992. (5) http://www.fda.gov/food/ guidancecomplianceregulatoryinformation/guidancedocuments/ dietarysupplements/ucm257563.htm#app-a. (6) Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding Operations for Dietary Supplements. Fed. Regist. 2007, 72(121), 34805. (7) Betz, J. M., Garland, T., Page, S. W. In Functional FoodsBiochemical and Processing Aspects; Shi, J., Mazza, G., Maguer, M. L., Eds.; CRC Press: Boca Raton, FL, 2002; Vol. 2, pp 367−394. (8) Ganzera, M.; Mair, M.; Stuppner, H.; Fischer, N. H.; Khan, I. A. Chromatographia 2001, 54, 665−668. (9) Evans, W. C. Trease and Evans’ Pharmacognosy, 16th ed.; Saunders Elsevier Limited: Oxford, UK, 2009. (10) Bruneton, J. Pharmacognosy, Phytochemistry, Medicinal Plants, 2nd ed.; Intercept: London, 1999. (11) Hänsel, R.; Sticher, O.; Alban, S.; Franz, G.; Spieß, E. Pharmakognosie-Phytopharmazie, 7th ed.; Springer-Verlag: Heidelberg, 2004. (12) Applequist, W.; Blumenthal, M.; Foster, S. The Identification of Medicinal Plants. A Handbook of the Morphology of Botanicals in Commerce; Missouri Botanical Garden Press: St. Louis, MO, 2006. (13) Hildreth, J.; Hrabeta-Robinson, E.; Applequist, W.; Betz, J.; Miller, J. Anal. Bioanal. Chem. 2007, 389, 13−17. (14) Raman, V.; Avula, B.; Galal, A. M.; Wang, Y.-H.; Khan, I. A. J. Nat. Med. Online 2012, DOI: 10.1007/s11418-012-0642-2. (15) Joshi, V. C.; Pullela, S. V.; Khan, I. A. J. AOAC Int. 2005, 88, 703−706. (16) Joshi, V. C.; Khan, I. A.; Sharaf, M. H. M. Pharm. Forum 2008, 34, 1075−1078. (17) Joshi, V. C.; Khan, I. A. J. AOAC Int. 2005, 88, 707−713. (18) Joshi, V. C.; Navarrete, A.; Khan, I. A. J. AOAC Int. 2005, 88, 1621−1625. (19) Joshi, V. C.; Avula, B.; Khan, I. A. J. Nat. Med. 2008, 62, 117− 121. (20) Avula, B.; Navarrete, A.; Joshi, V. C.; Khan, I. A. Pharmazie 2006, 61, 590−594. (21) Joshi, V. C.; Khan, I. A. Acta Hortic. 2006, 720, 73−79. (22) Israelsen, L. Nutraceutical World 2009, 57−58. (23) Huang, W. F.; Wen, K.-C.; Hsiao, M.-L. J. Clin. Pharmacol. 1997, 37, 344−350. (24) Singer, N. FDA Finds ‘Natural’ Diet Pills Laced with Drugs. New York Times, 2009, February 9, B1. (25) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm253416.htm. (26) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm301621.htm. (27) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm291577.htm. (28) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm290518.htm. (29) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm274746.htm. (30) http://www.fda.gov/Safety/MedWatch/SafetyInformation/ SafetyAlertsforHumanMedicalProducts/ucm248293.htm. (31) Baldwin, B. G.; Sanderson, M. J.; Wojciechowski, M. F.; Campbell, C. S.; Donoghue, M. J. Ann. Mo. Bot. Gard. 1995, 82, 247− 277. (32) Techen, N.; Pan, Z.; Scheffler, B. E.; Khan, I. A. Planta Med. 2009, 75, 392−395. (33) Law, S. K.; Simmons, M. P.; Techen, N.; Khan, I. A.; He, M. F.; Shaw, P. C.; But, P. H. Phytochemistry 2011, 72, 21−26. (34) Sang, T.; Crawford, D. J.; Stuessy, T. F. Am. J. Bot. 1997, 84, 1120−1136.

(35) Techen, N.; Arias, R. S.; Glynn, N. C.; Pan, Z.; Khan, I. A.; Scheffler, B. E. Mol. Ecol. Resour. 2010, 10, 508−515. (36) Li, M.; Ling, K. H.; Lam, H.; Shaw, P. C.; Ling, C.; Techen, N.; Khan, I. A.; Chang, Y. S.; But, P. H. J. Ethnopharmacol. 2010, 130, 429−432. (37) Techen, N.; Joshi, V.; Rumalla, C. S.; Khan, I. A. Manuscript in preparation. (38) Taberlet., P.; Gielly, L.; Pautou, G.; Bouvet, J. Plant Mol. Biol. 1991, 17, 1105−1109. (39) Gielly, L.; Taberlet, P. Mol. Biol. Evol. 1994, 11, 769−777. (40) Gielly, L.; Taberlet, P. Bot. J. Linnean Soc. 1996, 120, 57−75. (41) Chiang, T. Y.; Schaal, B. A.; Peng, C. I. Bot. Bull. Acad. Sin. 1998, 39, 245−250. (42) Downie, S. R.; Llanas, E.; Katz-Downie, D. S. Syst. Bot. 1996, 21, 135−151. (43) Zietkiewicz, E.; Rafalski, A.; Labuda, D. Genomics 1994, 20, 176−83. (44) Lata, H.; Chandra, S.; Techen, N.; Khan, I. A.; ElSohly, M. A. Planta Med. 2010, 76, 97−100. (45) Arias, R. S.; Techen, N.; Rinehart, T. A.; Olsen, R. T.; Kirkbride, J. H.; Scheffler, B. E. Hortic. Sci. 2011, 46, 23−29. (46) Chandra, S.; Lata, H.; Techen, N.; Khan, I. A.; ElSohly, M. A. Manuscript in preparation. (47) Betz, J. M.; Fisher, K. D.; Saldahna, L. G.; Coates, P. M. Anal. Bioanal. Chem. 2007, 389, 19−25. (48) Pawar, R. S.; Shukla, Y. J.; Khan, I. A. Steroids 2007, 72, 881− 891. (49) Pawar, R. S.; Shukla, Y. J.; Khan, S. I.; Avula, B.; Khan, I. A. Steroids 2007, 72, 524−534. (50) Avula, B.; Wang, Y.-H.; Pawar, R. S.; Shukla, Y. J.; Smillie, T. J.; Khan, I. A. Rapid Commun. Mass Spectrom. 2008, 22, 2587−2596. (51) Shukla, Y. J.; Fronczek, F. R.; Pawar, R. S.; Khan, I. A. Acta Crystallogr., Sect. E: Struct. Rep. Online 2008, E64, o1643−o1644, o1643/1−o1643/10. (52) Shukla, Y. J.; Pawar, R. S.; Ding, Y.; Li, X.-C.; Ferreira, D.; Khan, I. A. Phytochemistry 2009, 70, 675−683. (53) Li, J.; Ding, Y.; Li, X.-C.; Ferreira, D.; Khan, S.; Smillie, T.; Khan, I. A. J. Nat. Prod. 2009, 72, 983−987. (54) Ali, Z.; Khan, I. A. Phytochemistry 2011, 72, 2075−2080. (55) Wang, W.; Ali, Z.; Shen, Y.; Li, X.-C.; Khan, I. A. Helv. Chim. Acta 2010, 93, 139−146. (56) Wang, W.; Ali, Z.; Li, X.-C.; Khan, I. A. Nat. Prod. Commun. 2010, 5, 771−774. (57) Wang, W.; Zhao, J.; Wang, Y.-H.; Smillie, T. J.; Li, X.-C.; Khan, I. A. Planta Med. 2009, 75, 1436−1441. (58) Wang, W.; Li, X.-C.; Ali, Z.; Khan, I. A. Chem. Pharm. Bull. 2009, 57, 636−638. (59) Wang, W.; Ali, Z.; Li, X.-C.; Smillie, T. J.; Guo, D.-A.; Khan, I. A. Fitoterapia 2009, 80, 404−407. (60) Wang, W.; Ali, Z.; Li, X.-C.; Khan, I. A. Helv. Chim. Acta 2009, 92, 1829−1839. (61) Wang, W.; Ali, Z.; Shen, Y.; Li, X.-C.; Khan, I. A. Planta Med. 2010, 76, 903−908. (62) Wang, W.; Ali, Z.; Shen, Y.; Li, X.-C.; Khan, I. A. Planta Med. 2010, 76, 1751−1754. (63) Wang, W.; Ali, Z.; Shen, Y.; Li, X.-C.; Khan, I. A. Fitoterapia 2010, 81, 480−484. (64) Rumalla, C. S.; Ali, Z.; Weerasooriya, A. D.; Smillie, T. J.; Khan, I. A. Planta Med. 2010, 76, 1018−1021. (65) Fu, X.; Li, X.-C; Wang, Y.-H.; Avula, B.; Smillie, T. J.; Mabusela, W.; Syce, J.; Johnson, Q.; Folk, W.; Khan, I. A. Planta Med. 2010, 76, 178−181. (66) Fu, X.; Li, X.-C.; Smillie, T. J.; Carvalho, P.; Mabusela, W.; Syce, J.; Johnson, Q.; Folk, W.; Avery, M. A.; Khan, I. A. J. Nat. Prod. 2008, 71, 1749−1753. (67) Betz, J. M.; Brown, P. N.; Roman, M. C. Fitoterapia 2011, 82, 44−52. (68) Rambla-Alegre, M.; Esteve-Romero, J.; Carda-Broch, S. J. Chromatogr., A 2012, 1232, 101−109. 1672

dx.doi.org/10.1021/np300434j | J. Nat. Prod. 2012, 75, 1665−1673

Journal of Natural Products

Review

(69) Reich, E.; Schibli, A. High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants; Thieme Medical Publishers, Inc.: New York, 2007. (70) Avula, B.; Wang, Y.-H.; Rumalla, C. S.; Ali, Z.; Smillie, T. J.; Khan, I. A. J. Pharm. Biomed. Anal. 2011, 56, 895−903. (71) Ayinampudi, S.; Wang, Y.-H.; Avula, B.; Smillie, T.; Khan, I. JPC - J Plan. Chromatogr. - Modern TLC 2011, 24, 125−129. (72) Rumalla, C. S.; Avula, B.; Zhao, J.; Smillie, T. J.; Khan, I. A. J. Liq. Chromatogr. Relat. Technol. 2011, 34, 38−47. (73) Song, Z.-H.; Qian, Z.-Z.; Rumalla, C. S.; Smillie, T. J.; Khan, I. A. J. Planar Chromatogr.−Mod. TLC 2011, 24, 312−315. (74) Rumalla, C.; Avula, B.; Ali, Z.; Smillie, T.; Filion, V.; Cuerrier, A.; Arnason, J.; Khan, I. J. Planar Chromatogr.−Mod. TLC 2011, 24, 116−120. (75) Avula, B.; Wang, Y.-H.; Pawar, R. S.; Shukla, Y. J.; Schaneberg, B.; Khan, I. A. J. AOAC Int. 2006, 89, 606−611. (76) Avula, B.; Wang, Y.-H.; Pawar, R. S.; Shukla, Y. J.; Khan, I. A. J. AOAC Int. 2007, 90, 1526−1531. (77) Avula, B.; Wang, Y.-H.; Pawar, R. S.; Shukla, Y. J.; Smillie, T. J.; Khan, I. A. J. Pharm. Biomed. Anal. 2008, 48, 722−731. (78) Avula, B.; Upparapalli, S. K.; Khan, I. A. Chromatographia 2005, 62, 151−157. (79) Avula, B.; Upparapalli, S. K.; Navarrete, A.; Khan, I. A. J. AOAC Int. 2005, 88, 1593−1606. (80) Avula, B.; Upparapalli, S. K.; Khan, I. A. J. AOAC Int. 2007, 90, 633−640. (81) Avula, B.; Ali, Z.; Khan, I. A. Chromatographia 2007, 66, 757− 762. (82) Avula, B.; Wang, Y.-H.; Smillie, T. J.; Khan, I. A. Planta Med. 2009, 75, 381−386. (83) Li, J.; Wang, Y.-H.; Smillie, T. J.; Khan, I. A. J. Pharm. Biomed. Anal. 2012, 63, 120−127. (84) Avula, B.; Wang, Y.-H.; Khan, I. A. J. Chromatogr. Sep. Tech. 2012, 3, 120−125. (85) Lakavath, S.; Avula, B.; Wang, Y.-H.; Rumalla, C. S.; Gandhe, S.; Venkatrao, A. R. B.; Satishchandra, P. A.; Bobbala, R. K.; Khan, I. A.; Narasimha, A. R. A. V. J. AOAC Int. 2012, 95, 67−73. (86) Tabanca, N.; Demirci, B.; Ozek, T.; Kirimer, N.; Baser, K. H. C.; Bedir, E.; Khan, I. A.; Wedge, D. E. J. Chromatogr., A 2006, 1117, 194− 205. (87) Tabanca, N.; Douglas, A. W.; Bedir, E.; Dayan, F. E.; Kirimer, N.; Baser, K. H. C.; Aytac, Z.; Khan, I. A.; Scheffler, B. E. Plant Genet. Resour. 2005, 3, 149−169. (88) Avula, B.; Begum, S.; Ahmed, S.; Choudhary, M. I.; Khan, I. A. Pharmazie 2008, 63, 20−22. (89) Avula, B.; Upparapalli, S. K.; Khan, I. A. Chromatographia 2005, 62, 151−157. (90) Avula, B.; Khan, I. A. Chromatographia 2004, 59, 71−77. (91) Mich, A.; Matthes, B.; Chen, R.; Buehler, S. LC-GC Eur. 2010, March, 12−13. (92) Kohler, M.; Haerdi, W.; Christen, P.; Veuthey, J.-L. Phytochem. Anal. 1997, 8, 223−227. (93) Taylor, S. L.; King, J. W.; Snyder, J. M. J. Microcolumn Sep. 1994, 6, 467−74. (94) Soares, P. K.; Scarminio, I. S. Phytochem. Anal. 2008, 19, 78−85. (95) Zhao, Y. Y.; Zhang, Y.; Lin, R. C.; Sun, W. J. Fitoterapia 2009, 80, 333−338. (96) Rocha, S.; Maeztu, L.; Barros, A.; Cid, C.; Coimbra, M. A. J. Sci. Food Agric. 2004, 84, 43−51. (97) Rubiolo, P.; Belliardo, F.; Cordero, C.; Liberto, E.; Sgorbini, B.; Bicchi, C. Phytochem. Anal. 2006, 17, 217−25. (98) Rocha, S.; Maeztu, L.; Barros, A.; Cid, C.; Coimbra, M. A. J. Sci. Food Agric. 2004, 84, 43−51. (99) Sim, C. O.; Ahmad, M. N.; Ismail, Z.; Othman, A. R.; Noor, N. A. M.; Zaihidee, E. M. Sensors 2003, 3, 458−471. (100) Wang, L.; Wang, X.; Kong, L. Biochem. Syst. Ecol. 2012, 40, 138−145. (101) Zhao, Y.; Chen, P.; Lin, L.; Harnly, J. M.; Yu, L.; Li, Z. Food Chem. 2011, 126, 1269−1277.

(102) Sun, J.; Chen, P.; Lin, L.-Z.; Harnly, J. M. J. AOAC Int. 2011, 94, 487−497. (103) Chen, P.; Lin, L.-Z.; Harnly, J. M. J. AOAC Int. 2010, 93, 1148−1154. (104) Sun, X.; Chen, P.; Cook, S. L.; Jackson, G. P.; Harnly, J. M.; Harrington, P. B. Anal. Chem. 2012, 84, 3628−3634. (105) Dong, W.-W.; Au, D.; Cao, X.-W.; Li, X.-B.; Yang, D.-J. J. Food Drug Anal. 2011, 19, 495−501. (106) Cheng, X.-M.; Zhao, T.; Yang, T.; Wang, C.-H.; Bligh, S. W. A.; Wang, Z.-T. Phytochem. Anal. 2010, 21, 279−289. (107) Garza-Juarez, A.; Waksman-De-Torres, N.; Ramirez-Duron, R.; Cavazos, M. L. S. Acta Chromatogr. 2009, 21, 217−235. (108) Ma, H.-L.; Qin, M.-J.; Qi, L.-W.; Wu, G.; Shu, P. Biomed. Chromatogr. 2007, 21, 931−939. (109) Zhao, J.; Avula, B.; Chan, M.; Clement, C.; Kreuzer, M.; Khan, I. A. Planta Med. 2012, 78, 90−101. (110) Zhao, J.; Avula, B.; Joshi, V. C.; Techen, N.; Wang, Y.-H.; Smillie, T. J.; Khan, I. A. Planta Med. 2011, 77 (8), 851−7. (111) Frédérich, M.; Jansen, C.; de Tullio, P.; Tits, M.; Demoulin, V.; Angenot, L. Phytochem. Anal. 2010, 21, 61−65. (112) Sandasi, M.; Kamatou, G. P. P.; Gavaghan, C.; Baranska, M.; Viljoen, A. M. Vib. Spectrosc. 2011, 57, 242−247. (113) Luthria, D. L.; Mukhopadhyay, S.; Lin, L.-Z.; Harnly, J. M. Appl. Spectrosc. 2011, 65, 250−259. (114) Chen, P.; Luthria, D.; Harrington, P. D. B.; Harnly, J. M. J. AOAC Int. 2011, 94, 1411−1421. (115) Wu, Y.; Zheng, Y.; Li, Q.; Iqbal, J.; Zhang, L.; Zhang, W.; Du, Y. Vib. Spectrosc. 2011, 55, 201−206. (116) Maree, J. E.; Viljoen, A. M. Vib. Spectrosc. 2011, 55, 146−152. (117) Kokalj, M.; Kolar, J.; Trafela, T.; Kreft, S. Phytochem. Anal. 2011, 22, 541−546. (118) Vermaak, I.; Hamman, J. H.; Viljoen, A. M. Food Chem. 2010, 120, 940−944.

1673

dx.doi.org/10.1021/np300434j | J. Nat. Prod. 2012, 75, 1665−1673