Alginate-Based Carriers Loaded with Mulberry (Morus alba L.) Leaf Extract: A Promising Strategy for Prolonging 1-Deoxynojirimicyn (DNJ) Systemic Activity for the Nutraceutical Management of Hyperglycemic Conditions.

The iminosugar 1-deoxynojirimicyn (DNJ) contained in mulberry leaves has displayed systemic beneficial effects against disorders of carbohydrate metabolism. Nevertheless, its effect is impaired by the short half-life. Alginate-based carriers were developed to encapsulate a DNJ-rich mulberry extract: Ca-alginate beads, obtained by external gelation, and spray-dried alginate microparticles (SDMs). Mean size and distribution, morphology, drug loading, encapsulation efficiency, experimental yield, and release characteristics were determined for the two formulations. Ca-alginate beads and SDMs exhibited an encapsulation efficiency of about 54% and 98%, respectively, and a DNJ loading in the range of 0.43-0.63 µg/mg. The in vitro release study demonstrated the carriers' capability in controlling the DNJ release in acid and basic conditions (<50% in 5 h), due to electrostatic interactions, which were demonstrated by 1H-NMR relaxometry studies. Thus, alginate-based particles proved to be promising strategies for producing food supplements containing mulberry leaf extracts for the management of hyperglycemic state.

In vitro bioactivity evaluation of mulberry leaf extracts as nutraceuticals for the management of diabetes mellitus.

There is an increasing need for new options to treat diabetes mellitus at its early stage and natural remedies have been recently reassessed as potential candidates owing to their low-cost and effectiveness. Genus Morus plants contain many active compounds with hypoglycaemic, hypolipidemic, and antioxidant effects. Current research on mulberry chemical composition and bioactivity has been generally carried out only on Asian cultivation, where this plant has been traditionally used in the form of leaf infusion for decades. In this work, twelve Italian mulberry cultivars were fully characterised to fill this gap of knowledge, since a strong correlation among composition, genetics and growing area was proven. Antiglycative and hypoglycaemic effects of leaf extracts were evaluated using different in vitro models. The results indicate that the inhibitory effect on carbohydrate digestive enzymes was likely mediated by 1-deoxynojirimycin, kaempferol, quercetin, and chlorogenic acid, acting in a synergistic way. Besides, the combined antiglycative and carbonyl trapping capacities, tested here for the first time, may help in preventing long-term complications related to AGEs in diabetic patients.

Characterization and Valorization of the Agricultural Waste Obtained from Lavandula Steam Distillation for Its Reuse in the Food and Pharmaceutical Fields.

The main focus of the current research was the characterization of the by-products from the steam distillation of Lavandula angustifolia Mill. (LA) and Lavandula x intermedia Emeric ex Loisel (LI) aerial parts, as they are important sources of bioactive compounds suitable for several applications in the food, cosmetic, and pharmaceutical industries. The oil-exhausted biomasses were extracted and the total polyphenol and flavonoid contents were, respectively, 19.22 ± 4.16 and 1.56 ± 0.21 mg/g for LA extract and 17.06 ± 3.31 and 1.41 ± 0.10 mg/g for LI extract. The qualitative analysis by liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS) revealed that both the extracts were rich in phenolic acids and glycosylated flavonoids. The extracts exhibited radical scavenging, chelating, reducing activities, and inhibitory capacities on acetylcholinesterase and tyrosinase. The IC50 values against acetylcholinesterase and tyrosinase were, respectively, 5.35 ± 0.47 and 5.26 ± 0.02 mg/mL for LA, and 6.67 ± 0.12 and 6.56 ± 0.16 mg/mL for LI extracts. In conclusion, the oil-exhausted biomasses demonstrated to represent important sources of bioactive compounds, suitable for several applications in the food, cosmetic, and pharmaceutical industries.

Effects of Biostimulants on the Chemical Composition of Essential Oil and Hydrosol of Lavandin (Lavandula x intermedia Emeric ex Loisel.) Cultivated in Tuscan-Emilian Apennines.

In recent years, it has been shown that biostimulants can efficiently enhance plant metabolic processes, leading to an increased production of essential oil (EO) in aromatic plants. The present study aimed to evaluate the effects of two different commercial biostimulants composed of amino acids and seaweed extract, normally used for food organic crops, on the production and composition of EO and hydrosol of Lavandula x intermedia, cultivar "Grosso". The products were applied during 2020 growing season on lavender crops in three different locations of the Northern Italian (Emilia-Romagna Region) Apennines. Plants were harvested and EOs extracted by steam distillation and analyzed by gas chromatography. Both biostimulants affected the yield of EO per plant (+11% to +49% depending on the treatment/farm combination) without significantly changing the chemical composition of EOs and hydrosols. Conversely, the composition of EOs and hydrosols are related to the location, and the main compounds of "Grosso" cultivar, limonene, 1,8-cineole, cis-ocimene, linalool, camphor, borneol, terpinen-4-ol, and linalyl acetate, show different ratios at the experimental test sites. The differences might be due to the sunlight exposure and various maintenance of the crops over the years. In conclusion, these results suggest that the employment of biostimulants on lavandin crops do not endanger the quality of the EO while increasing biomass production and promoting the sustainability of the crop.

A Novel qNMR Application for the Quantification of Vegetable Oils Used as Adulterants in Essential Oils.

Essential oils (EOs) are more and more frequently adulterated due to their wide usage and large profit, for this reason accurate and precise authentication techniques are essential. This work aims at the application of qNMR as a versatile tool for the quantification of vegetable oils potentially usable as adulterants or diluents in EOs. This approach is based on the quantification of both 1H and 13C glycerol backbone signals, which are actually present in each vegetable oil containing triglycerides. For the validation, binary mixtures of rosemary EO and corn oil (0.8-50%) were prepared. To verify the general feasibility of this technique, other different mixtures including lavender, citronella, orange and peanut, almond, sunflower, and soy seed oils were analyzed. The results showed that the efficacy of this approach does not depend on the specific combination of EO and vegetable oil, ensuring its versatility. The method was able to determine the adulterant, with a mean accuracy of 91.81 and 89.77% for calculations made on 1H and 13C spectra, respectively. The high precision and accuracy here observed, make 1H-qNMR competitive with other well-established techniques. Considering the current importance of quality control of EOs to avoid fraudulent practices, this work can be considered pioneering and promising.

Separation and non-separation methods for the analysis of cannabinoids in Cannabis sativa L.

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.

Novel Strategy for the Recognition of Adulterant Vegetable Oils in Essential Oils Commonly Used in Food Industries by Applying 13C NMR Spectroscopy.

Essential oils (EOs) are valuable products commonly employed in the food industry and intensively studied as biopreservatives for the extension of food shelf-life. Unfortunately, EOs might be counterfeit to increase industrial profits. Among the possible adulterants, vegetable oils (VOs) must be considered for their characteristics and low costs. We aimed to apply nuclear magnetic resonance (NMR) spectroscopy for the detection and identification of VOs in mixtures with EOs. This innovative strategy is based on comparing the peak area ratio matrices of characteristic VO 13C NMR fatty acid signals with those of adulterated EOs. The identification of the VOs was achieved by calculating the matrix similarity at different confidence levels. The strategy demonstrated the capacity to efficiently recognize the presence of adulteration and the type of VO adulterant in mixtures. Thus, the method was applied to 20 commercial EOs, and VOs were detected and then identified in four samples.

HR-1 H NMR spectroscopy and multivariate statistical analysis to determine the composition of herbal mixtures for infusions.

INTRODUCTION: The ever-growing diffusion and consumption of herbal teas, due to their sensory attributes and well-known health benefits exposes them to the real risk of adulteration, especially in the case of commercial mixtures already minced for infusion. Therefore, novel and suitable tools for the control of these valuable products are increasingly required. OBJECTIVES: This work provides new insights for the authenticity study of infusions. The main objective was verifying the potential of proton nuclear magnetic resonance (1 H-NMR) combined with partial least square (PLS) regression to build highly predictive models, useful for the determination of the real amounts of herbs in mixtures, by the simple analysis of the related infusion. MATERIALS AND METHODS: Peppermint, fennel, lemon balm, and passiflora were chosen to set-up an experimental plan according to a central composite design (CCD). One-dimensional nuclear Overhauser effect spectroscopy (1D-NOESY) spectra were properly pretreated and then analysed by chemometrics to extract significant information from the raw data. RESULTS: Venetian-blind cross-validation and different chemometric indicators (RMSEC, RMSECV, RMSEP, R2 CAL , R2 CV, R2 PRED ) were used to establish the best model, which include four factors explaining 88.70 and 83.77% of the total variance in X and Y, respectively. CONCLUSIONS: These promising results have laid the basis for further development of the method, to extend its applicability and make it more scalable. This tool could replace expensive separative techniques and protect the rights of consumers with particular attention to safety issues and quality assurance.

New Insights into Bioactive Compounds from the Medicinal Plant Spathodea campanulata P. Beauv. and Their Activity against Helicobacter pylori.

The medicinal plant Spathodea campanulata P. Beauv. (Bignoniaceae) has been traditionally applied for the prevention and treatment of diseases of the kidney and urinary system, the skin, the gastrointestinal tract, and inflammation in general. The present work shows for the first time how chemical components from this plant inhibit Helicobacter pylori growth by urease inhibition and modulation of virulence factors. The crude extract and the main fractions of S. campanulata bark were tested on H. pylori isolated strains and the active ones were further fractionated. Fractions and sub-fractions of the plant crude extract were characterized by ultra-high-performance liquid chromatographic tandem high resolution-mass spectrometry detection (UHPLC-HRMS). Several phenolics and triterpenoids were identified. Among the sub-fractions obtained, SB2 showed the capacity to inhibit H. pylori urease in a heterologous bacterial model. One additional sub-fraction (SE3) was able to simultaneously modulate the expression of two adhesins (HopZ and BabA) and one cytotoxin (CagA). The flavonol kaempferol was identified as the most interesting compound that deserves further investigation as a new hit for its capacity to modulate H. pylori virulence factors.

Innovative methods for the preparation of medical Cannabis oils with a high content of both cannabinoids and terpenes.

Cannabis-based medications are being increasingly used for the treatment of different clinical conditions. Among all galenic formulations, olive oil extracts from medical Cannabis are the most prescribed ones for their easy preparation and usage. A great variety of methods have been described so far for the extraction of medical Cannabis oils to reach a high yield of Δ9-tetrahydrocannabinol (Δ9-THC), but poor attention has been paid to the preservation of the terpene fraction from the plant, which may contribute to the overall bioactivity of the extracts. In this context, the present study was aimed at the chemical characterization of different medical Cannabis oils prepared by following both innovative and existing extraction protocols, with particular attention to cannabinoids and terpenes, in order to set up a suitable method to obtain an extract rich in these chemical classes. In particular, six different extraction procedures were followed, based on different techniques, of which all but one included a decarboxylation of the plant material. The profile of cannabinoids was studied in detail by means of HPLC-ESI-MS/MS, while terpenes were characterized by means both GC-MS and GC-FID techniques coupled with solid-phase microextraction operated in the head-space mode (HS-SPME). An innovative method that is based on the extraction of the oil by dynamic maceration at room temperature from plant inflorescences, which were partially decarboxylated in a closed system at a moderate temperature and partially pre-extracted with ethanol, produced similar yields of bioactive compounds as that obtained by using a microwave-assisted distillation of the essential oil from the plant material, in combination with a maceration extraction of the oil from the residue. Both these new methods provided a higher efficiency over already existing extraction procedures of medical Cannabis oils and they can be applied to obtain a product with a high therapeutic value.

Development of a new method for the analysis of cannabinoids in honey by means of high-performance liquid chromatography coupled with electrospray ionisation-tandem mass spectrometry detection.

Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male inflorescences. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its biological effects attributable to its antioxidant, neuroprotective and anti-inflammatory properties. The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources. In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products. The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance.

Use of 13C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis sativa L. (Hemp).

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of 13C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 µg/mL, which is easily achievable with concentrated extracts, without affecting the quality of 13C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety.

Identification and determination of bioactive phenylpropanoid glycosides of Aloysia polystachya (Griseb. et Moldenke) by HPLC-MS.

Aloysia polystachya (Griseb. et Moldenke) has not been deeply investigated in past years and currently data about its chemical composition are limited. Phenolic compounds characterization can be very difficult in vegetable matrices, owing to bonds to sugar moieties or conjugation, giving rise to complex structures. In this work, methanolic extracts of Aloysia polystachya leaves were analyzed by HPLC-ESI-MS, the favourite technique for the separation and quantification of their polyphenols. To assess the complete characterization and quantification of the phenylpropanoid fraction, three different MS techniques have been coupled to HPLC: ion trap mass spectrometry (Ion Trap LC/MS), quadrupole-time of flight high resolution mass spectrometry (Q-TOF HRMS) and triple-quadrupole (TQ LC/MS) for the quantification. Eleven phenylpropanoid glycosides were identified and quantified and, among them, the compounds forsythoside A, plantainoside C, purpureaside D, martynoside and its two isomers were detected for the first time to the best of our knowledge. The results presented here could be helpful to assess the quality of this plant and could further contribute to the chemotaxonomy of the genus.

Analytical methods for the study of bioactive compounds from medicinally used Echinacea species.

Echinacea purpurea (L.) Moench, Echinacea angustifolia DC. var. angustifolia and Echinacea pallida (Nutt.) Nutt. are frequently used as medicinal plants and their preparations are among the most widely used herbal medicines. The extracts from these species have shown a highly complex chemical composition, including polar compounds (caffeic acid derivatives, CADs), non-polar compounds (alkylamides and acetylenic secondary metabolites; essential oil) and high molecular weight constituents (polysaccharides and glycoproteins). All these chemical classes of compounds have demonstrated to possess interesting biological activities. In the light of all the above, this paper is focused on the analytical techniques, including sample preparation tools and chromatographic procedures, for the chemical analysis of bioactive compounds in medicinally used Echinacea species. Since sample preparation is considered to be a crucial step in the development of analytical methods for the determination of constituents present in herbal preparations, the strength and weakness of different extraction techniques are discussed. As regards the analysis of compounds present in Echinacea plant material and derivatives, the application of different techniques, mainly HPLC, HPLC-ESI-MS, HPLC-ESI-MS/MS, HPCE, HPTLC and GC, is discussed in detail. The strength, weakness and applicability of the different separation tools are stated.

Nuclear magnetic resonance and high-performance liquid chromatography techniques for the characterization of bioactive compounds from Humulus lupulus L. (hop).

Humulus lupulus L. (hop) represents one of the most cultivated crops, it being a key ingredient in the brewing process. Many health-related properties have been described for hop extracts, making this plant gain more interest in the field of pharmaceutical and nutraceutical research. Among the analytical tools available for the phytochemical characterization of plant extracts, quantitative nuclear magnetic resonance (qNMR) represents a new and powerful technique. In this ambit, the present study was aimed at the development of a new, simple, and efficient qNMR method for the metabolite fingerprinting of bioactive compounds in hop cones, taking advantage of the novel ERETIC 2 tool. To the best of our knowledge, this is the first attempt to apply this method to complex matrices of natural origin, such as hop extracts. The qNMR method set up in this study was applied to the quantification of both prenylflavonoids and bitter acids in eight hop cultivars. The performance of this analytical method was compared with that of HPLC-UV/DAD, which represents the most frequently used technique in the field of natural product analysis. The quantitative data obtained for hop samples by means of the two aforementioned techniques highlighted that the amount of bioactive compounds was slightly higher when qNMR was applied, although the order of magnitude of the values was the same. The accuracy of qNMR was comparable to that of the chromatographic method, thus proving to be a reliable tool for the analysis of these secondary metabolites in hop extracts. Graphical abstract Graphical abstract related to the extraction and analytical methods applied in this work for the analysis of bioactive compounds in Humulus lupulus L. (hop) cones.

An efficient chemical analysis of phenolic acids and flavonoids in raw propolis by microwave-assisted extraction combined with high-performance liquid chromatography using the fused-core technology.

A closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of polyphenols from raw propolis. The results obtained by means of response surface experimental design methodology showed that the best global response was reached when the extraction temperature was set at 106 °C, the solvent composition close to EtOH-H2O 80:20 (v/v), with an extraction time of 15 min. In comparison with other techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a gradient mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by DAD and MS. The method validation indicated that the correlation coefficients were >0.999; the limit of detection was in the range 0.5-0.8 µg/ml for phenolic acids and 1.2-3.0 µg/ml for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in propolis extracts to be used in apitherapy.

Chemical and functional characterization of Italian propolis obtained by different harvesting methods.

The composition and antioxidant activity of Italian poplar propolis obtained using three harvesting methods and extracted with different solvents were evaluated. Waxes, balsams, and resins contents were determined. Flavones and flavonols, flavanones and dihydroflavonols, and total phenolics were also analyzed. To characterize the phenolic composition, the presence of 15 compounds was verified through HPLC-MS/MS. The antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl radical and reducing power assays. The ability of propolis to inhibit lipid oxidation was monitored by analyzing hydroperoxide and TBARS formation in lipids incorporated into an oil-in-water (O/W) emulsion. Acetone shows the highest extraction capacity. Wedge propolis has the highest concentration of active phenolic compounds (TP = 359.1 ± 16.3 GAEs/g; TFF = 5.83 ± 0.42%; TFD = 7.34 ± 1.8%) and seems to be the most promising for obtaining high-value propolis more suitable to prepare high-quality dietary supplements (TBARS = 0.012 ± 0.009 mmol std/g; RP = 0.77 ± 0.07 TEs/g).

¹H-NMR simultaneous identification of health-relevant compounds in propolis extracts.

INTRODUCTION: Propolis is a resinous substance collected by bees from exudates of different plants that is rich in well-known health-relevant phenolic compounds such as flavonoids and phenolic acids. Propolis extracts are very complex matrices difficult to study. Different analytical methods are usable to analyse propolis extracts and to obtain chemical fingerprint but to our knowledge NMR has not previously been used for this purpose. OBJECTIVE: This study aims to demonstrate that it is possible to use ¹H-NMR for the simultaneous recognition of phenolic compounds in complex matrices, such as propolis extracts, using appropriate tools for spectra pre-treatment and analysis. METHODOLOGY: In this work 12 typical phenolic propolis compounds (apigenin, chrysin, galangin, kaempferol, quercetin, naringenin, pinocembrin, pinostrobin, caffeic acid, cinnamic acid, p-coumaric acid and ferulic acid) were considered as reference compounds and their presence in samples was verified by HPLC-MS. A simple ¹H-NMR sequence was used to obtain spectra of samples. Spectra were pre-treated by using an appropriate tool for spectra alignment and analysed by using software for the study of spectra originated from complex matrices. Sixty-five propolis samples were used to test the proposed identification procedure. RESULTS: Ten out of 12 considered compounds were identified as statistically significant in most of the samples. CONCLUSION: This work suggests that it is possible to efficiently use ¹H-NMR, coupled with appropriate spectral analytical tools, for the simultaneous detection of phenolic compounds in complex matrices.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.