Chemical Fingerprinting and Hierarchical Clustering Analysis of Centella asiatica from Different Locations in China

Centella asiatica (L.) Urban is a widely distributed herbaceous plant with great medicinal value that has been extensively used in traditional Chinese medicine. For effective quality control of C. asiatica, a feasible approach and control system is necessary. In this paper, a chemical fingerprint method (column liquid chromatography) was developed for investigating and demonstrating the variance of chemical components among different populations of C. asiatica, from 14 locations in China. The LC data showed considerable variation of chemical constituents among C. asiatica populations. Four chemotypes were visually developed from the chromatographic profiles. The hierarchical clustering analysis further suggested that the samples were divided into four major groups. The grouping of 14 C. asiatica populations in hierarchical clustering analysis was in good agreement with the visual comparison of their chromatograms, as demonstrated by chemotypes. In addition, the bioactive compound asiaticoside in this herb was quantitatively determined by a validated reversed-phase liquid chromatography analysis. Chemotype I was identified as asiaticoside-rich chemotype and exhibited significantly higher amounts of asiaticoside. These findings and results provide a solid basis to establish good agriculture practice and select geo-authentic crude drug for C. asiatica. The established method was considered to be suitable for fingerprint analysis for the quality control of C. asiatica.     

Metabolomic Profiling and Antioxidant Activities of Breonadia salicina Using 1H-NMR and UPLC-QTOF-MS Analysis

Breonadia salicina (Vahl) Hepper and J.R.I. Wood is widely used in South Africa and some other African countries for treatment of various infectious diseases such as diarrhea, fevers, cancer, diabetes and malaria. However, little is known about the active constituents associated with the biological activities. This study is aimed at exploring the metabolomics profile and antioxidant constituents of B. salicina. The chemical profiles of the leaf, stem bark and root of B. salicina were comprehensively characterized using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The antioxidant activities of the crude extracts, fractions and pure compounds were determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and reducing power assays. A total of 25 compounds were tentatively identified using the UPLC-QTOF-MS. Furthermore, the ¹H-NMR fingerprint revealed that the different parts of plant had differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the root and dichloromethane leaf revealed the presence of hexadecane. Column chromatography and preparative thin-layer chromatography were used to isolate kaempferol 3-O-(2″-O-galloyl)-glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid. The crude methanol stem bark showed the highest antioxidant activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity with an IC₅₀ value of 41.7263 ± 7.6401 μg/mL, whereas the root crude extract had the highest reducing power activity with an IC_0.5 value of 0.1481 ± 0.1441 μg/mL. Furthermore, the ¹H-NMR and UPLC-QTOF-MS profiles showed the presence of hydroxycinnamic acids, polyphenols and flavonoids. According to a literature survey, these phytochemicals have been reported to display antioxidant activities. Therefore, the identified hydroxycinnamic acid (caffeic acid), polyphenol (ellagic acid) and flavonoids (catechin and (epi) gallocatechin) significantly contribute to the antioxidant activity of the different parts of plant of B. salicina. The results obtained in this study provides information about the phytochemistry and phytochemical compositions of Breonadia salicina, confirming that the species is promising in obtaining constituents with medicinal potential primarily antioxidant potential.     

Cinnamomum verum J. Presl Bark Contains High Contents of Nicotinamide Mononucleotide

The global population is aging, and intervention strategies for anti-aging and the prevention of aging-related diseases have become a topic actively explored today. Nicotinamide adenine dinucleotide (NAD⁺) is an important molecule in the metabolic process, and its content in tissues and cells decreases with age. The supplementation of nicotinamide mononucleotide (NMN), an important intermediate and precursor of NAD⁺, has increased NAD⁺ levels, and its safety has been demonstrated in rodents and human studies. However, the high content of NMN in natural plants has not been fully explored as herbal medicines for drug development. Here, we identified that the leaf of Cinnamomum verum J. Presl (C. verum) was the highest NMN content among the Plant Extract Library (PEL) with food experience, using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). To validate this result, the extraction and quantitative analysis of bark, leaf, root, and stem of fresh C. verum was conducted. The results revealed that the bark had the highest NMN content in C. verum (0.471 mg/100 g). Our study shed light on the prospects of developing natural plants in the context of NMN as drugs for anti-aging and prevention of aging-related diseases. The future should focus on the development and application of C. verum pharmaceutical formulations.     

Chemical Profile of Lipophilic Fractions of Different Parts of Zizyphus lotus L. by GC-MS and Evaluation of Their Antiproliferative and Antibacterial Activities

Zizyphus lotus L. is a perennial shrub particularly used in Algerian folk medicine, but little is known concerning the lipophilic compounds in the most frequently used parts, namely, root bark, pulp, leaves and seeds, which are associated with health benefits. In this vein, the lipophilic fractions of these morphological parts of Z. lotus from Morocco were studied by gas chromatography–mass spectrometry (GC–MS), and their antiproliferative and antimicrobial activities were evaluated. GC–MS analysis allowed the identification and quantification of 99 lipophilic compounds, including fatty acids, long-chain aliphatic alcohols, pentacyclic triterpenic compounds, sterols, monoglycerides, aromatic compounds and other minor components. Lipophilic extracts of pulp, leaves and seeds were revealed to be mainly composed of fatty acids, representing 54.3–88.6% of the total compounds detected. The leaves and seeds were particularly rich in unsaturated fatty acids, namely, (9Z,12Z)-octadeca-9,12-dienoic acid (2431 mg kg⁻¹ of dry weight) and (9Z)-octadec-9-enoic acid (6255 mg kg⁻¹ of dry weight). In contrast, root bark contained a high content of pentacyclic triterpenic compounds, particularly betulinic acid, accounting for 9838 mg kg⁻¹ of dry weight. Root bark extract showed promising antiproliferative activity against a triple-negative breast cancer cell line, MDA-MB-231, with a half-maximal inhibitory concentration (IC₅₀) = 4.23 ± 0.18 µg mL⁻¹ of extract. Leaf extract displayed interesting antimicrobial activity against Escherichia coli, methicillin-sensitive Staphylococcus aureus and Staphylococcus epidermis, presenting minimum inhibitory concentration (MIC) values from 1024 to 2048 µg mL⁻¹ of extract. Our results demonstrate that Zizyphus lotus L. is a source of promising bioactive components, which can be exploited as natural ingredients in pharmaceutical formulations.     

Extraction and TLC Desitometric Determination of Triterpenoid Acids (Arjungenin, Arjunolic Acid) from Terminalia arjuna Stem Bark Without Interference of Tannins

The stem bark of Terminalia arjuna Linn. (fam: Combretaceae), commonly known as Arjuna in Indian systems of medicine, is a reputed drug used for various cardiac disorders. T. arjuna stem bark is reported to contain different groups of chemical constituents including phenolics, tannins, saponins and triterpenoid acids. From our earlier experience with tannin containing herbal drugs, we are aware that tannins interfere in the extraction of certain compounds and hence in their quantification. In the present experiment, we report a sample preparation method to overcome the interference of the tannins by adsorbing them with carboxy methyl cellulose, which facilitates the efficient extraction of the triterpenoid acids. Further we established TLC densitometric methods for the quantification of two of the triterpenoid acids of T. arjuna stem bark viz., arjungenin and arjunolic acid using HPTLC. The methods were validated in terms of accuracy, precision and repeatability. The calibration curve showed linearity in the range of 160–480 ng spot^?1 and 160–560 ng spot^?1 for arjungenin and arjunolic acid respectively. The percentage of arjungenin and arjunolic acid were found to be 0.324% w/w and 0.524% w/w in the stem bark by this modified method of extraction, which was many times higher than when compared to that using the extraction method without CMC (0.018% and 0.049% respectively). The study reiterates the importance of sample preparation in the quantification of non polar phytochemicals from herbal raw materials, such that the compounds of interest are extracted efficiently, overcoming the interference of other compounds like tannins in the matrix of plant material.     

LC Fingerprint and Hierarchical Cluster Analysis of Crocus sativus L. from Different Locations in China

Using simple, sensitive and specific column liquid chromatography, a chemical fingerprinting method was developed for investigating and demonstrating the variance of chemical components among different populations of Crocus sativus L., from 15 locations in China. The LC assay was performed on a reversed-phase C₁₈ column with linear gradient elution using methanol and 1% aqueous acetic acid. The LC data showed considerable variation of chemical constituents among C. sativus populations. Four chemotypes were visually developed from the chromatographic profiles. The grouping of 15 C. sativus populations in hierarchical clustering analysis was in good agreement with the visual comparison of their chromatograms, as demonstrated by chemotypes. In addition, the bioactive compound crocin 1 in this herb was quantitatively determined by a validated reversed-phase LC analysis. Relative peak areas of characteristic compounds were also calculated for quantitative expression of the LC fingerprints. These results showed that the established method for fingerprinting analysis was considered to be suitable for the quality control of C. sativus.     

Accumulation Pattern of Flavonoids during Fruit Development of Lonicera maackii Determined by Metabolomics

Lonicera maackii (Caprifoliaceae) is a large, upright shrub with fruits that contain many bioactive compounds. Flavonoids are common active substances in L. maackii. However, there is a dearth of information about the accumulation of these flavonoids and their possible medicinal value. We used targeted metabolomics analysis based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze five developmental stages of L. maackii fruit. A total of 438 metabolites were identified in the five developmental stages, including 81 flavonoids and derivatives. The 81 flavonoids included 25 flavones and derivatives, 35 flavonols and derivatives, two isoflavones, three cyanidins and derivatives, eight procyanidins, and eight flavanones. In addition, we outlined the putative flavonoid biosynthesis pathway and screened their upstream metabolites. More importantly, we analyzed the accumulation patterns of several typical flavones and flavonols. The results reported here improved our understanding of the dynamic changes in flavonoids during fruit development and contributed to making full use of the medicinal value of L. maackii fruit.     

Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C₁₈ column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L⁻¹ ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.     

Establishment of a UPLC-PDA/ESI-Q-TOF/MS-Based Approach for the Simultaneous Analysis of Multiple Phenolic Compounds in Amaranth (A. cruentus and A. tricolor)

We used ultraperformance liquid chromatography coupled with a photodiode-array detector and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-PDA/ESI-Q-TOF/MS) to rapidly and accurately quantify 17 phenolic compounds. Then, we applied this method to the seed and leaf extracts of two Amaranthus species to identify and quantify phenolic compounds other than the 17 compounds mentioned above. Compounds were eluted within 30 min on a C18 column using a mobile phase (water and acetonitrile) containing 0.1% formic acid, and the specific wavelength and ion information of the compounds obtained by PDA and ESI-Q-TOF/MS were confirmed. The proposed method showed good linearity (r² > 0.990). Limits of detection and quantification were less than 0.1 and 0.1 μg/mL, respectively. Intra- and interday precision were less than 2.4% and 1.8%, respectively. Analysis of amaranth seed and leaf extracts using the established method showed that the seeds contained high amounts of 2,4-dihydroxybenzoic acid and kaempferol, and leaves contained diverse phenolic compounds. In addition, six tentatively new phenolic compounds were identified. Moreover, seeds potentially contained 2,3-dihydroxybenzaldehyde, a beneficial bioactive compound. Thus, our method was an efficient approach for the qualitative and quantitative analysis of phenolic compounds, and could be used to investigate phenolic compounds in plants.     

Effect of Holoptelea integrifolia (Roxb.) Planch. n-Hexane Extract and Its Bioactive Compounds on Wound Healing and Anti-Inflammatory Activity

The stem bark of Holoptelea integrifolia (Roxb.) Planch. has been applied for the treatment of human cutaneous diseases as well as canine demodicosis in several countries. However, no detailed mechanistic studies have been reported to support their use. In this study, thin-layer chromatography and gas chromatography were used to screen phytochemicals from the fresh stem bark extract of H. integrifolia. We found the two major bioactive compounds, friedelin and lupeol, and their activity on wound healing was further investigated in keratinocytes. Both bioactive compounds significantly reduced wound area and increased keratinocyte migration by increasing matrix metalloproteinases-9 production. Subsequently, we found that the mRNA gene expressions of cadherin 1 and desmoglobin 1 significantly decreased, whereas the gene expression involved in keratinocyte proliferation and homeostasis (keratin-17) increased in compound-treated human immortalized keratinocytes cells. The expression of inflammatory genes (cyclooxygenase-2 and inducible nitric oxide synthase) and pro-inflammatory cytokine genes (tumor necrosis factor-alpha and interleukin-6) was reduced by treatment with n-hexane extract of H. integrifolia and its bioactive compounds. Our results revealed that H. integrifolia extract and its bioactive compounds, friedelin and lupeol, exhibit wound-healing activity with anti-inflammatory properties, mediated by regulating the gene expression involved in skin re-epithelialization.     

Validation of a confirmatory method for the determination of melamine in egg by gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry

A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC-MS/MS, which was completed in 5 min for each injection. For the GC-MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC-MS and UPLC-MS/MS was 10 and 5 μg kg⁻¹, respectively. This validated method was successfully applied to the determination of melamine in real samples from market.     

Evaluation of traditional Chinese herbal medicine: Chaihu (Bupleuri Radix) by both high-performance liquid chromatographic and high-performance thin-layer chromatographic fingerprint and chemometric analysis

Chaihu (Bupleuri Radix), roots of Bupleurum chinense and B. scorzonerifolium, is an authentic Chinese Materia Medica in the Chinese Pharmacopoeia. Some other species such as the roots of B. falcatum, B.bicaule and B. marginatum var. stenophyllum similar to Chaihu can also be occasionally found in local raw herb markets. The quality of 33 lots of authenticated Chaihu samples vs. 31 lots of commercial samples was evaluated by both high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and high-performance thin-layer chromatography (HPTLC) analyses of its principal bioactive components (saikosaponins). The pre-treated data acquired from both HPLC fingerprints and HPTLC fluorescent images were processed by chemometrics for similarity and pattern recognition, including Artificial Neural Networks (ANNs), k-nearest neighbor (k-NN) and an expert’s panel. It was apparent that k-NN classifier exhibited good performance with sufficient flexibility for processing HPTLC fingerprint images which were otherwise not easily dealt with by other algorithms due to the shift of R_f values and varying hue/saturation of the band colours between different TLC plates. These two chromatographic fingerprint methods can be considered complementary measure of quality control. The roots of Chaihu from different species of the genus Bupleurum could readily be distinguished from each other so that commercial samples can easily be classified. Chaihu collected from several major herbal distribution centers was found to belong to B. chinense with great variation in the content of its major saikosaponins.     

Rapid Screening of Lipase Inhibitors from Ophiopogonis Radix Using High-Performance Thin Layer Chromatography by Two Step Gradient Elution Combined with Bioautographic Method

In this study, a high-performance thin layer chromatography (HPTLC) method by two step gradient elution with two mobile phases was developed for the simultaneous analysis of seven constituents in Ophiopogonis Radix. The chromatography was performed on silica gel 60 F₂₅₄ plate with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) and dichloromethane-methanol (300:1, v/v) as the mobile phase for two step gradient elution. Then, the HPTLC profiles were observed after derivatization with 10% sulfuric acid in ethanol solution. The obtained HPTLC images were further analyzed by chemometric approaches and the samples could be clustered based on regions and/or growth years, which were two important factors affecting the constituents in Ophiopogonis Radix. Furthermore, five compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D’, ophiopogonin C’ and methylophiopogonanone B were screened as potential lipase inhibitors from Ophiopogonis Radix by the HPTLC-bioautographic method. The binding modes and interactions between the five compounds and lipase were further explored by molecular docking analysis. The developed HPTLC method could be used for quality control of Ophiopogonis Radix and screening of the potential lipase inhibitors.     

Screening and Analysis of the Potential Bioactive Components in Rabbit Plasma after Oral Administration of Hot-Water Extracts from Leaves of <em>B</em><em>ambusa </em><em>textilis </em>McClure

Bambusa textilis McClure is a traditional Chinese medicinal plant belonging to the Bambusoideae subfamily and used to treat chronic fever and infectious diseases. To investigate the bioactive compounds absorbed in the rabbit blood after oral administration of hot-water extracts from the leaves of B. textilis McClure, a validated chromatographic fingerprint method was established using LC-Q-TOF-MS. Twenty compounds in bamboo leaves and three potential bioactive compounds in rabbit plasma were detected. Of the twenty detected compounds in vitro, fifteen of which were tentatively identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by reviewing the literature. Three potential bioactive compounds, including (E)-p-coumaric acid, (Z)-p-coumaric acid, and apigenin-8-C-β-D-(2"-O-α-L-rhamnosyl)-gluco-pyranoside, were detected in both the leaves of B. textilis McClure and rabbit plasma. Of the three compounds, apigenin-8-C-β-D-(2"-O-α-L-rhamnosyl)glucopyranoside was identified based on its UV, MS, and NMR spectra. This study provides helpful chemical information for further pharmacology and active mechanism research on B. textilis McClure.     

Isolation,Structure Elucidation and Antimicrobial Evaluation of Natural Pentacyclic Triterpenoids and Phytochemical Investigation of Different Fractions of Ziziphus spina-christi (L.) Stem Bark Using LCHRMS Analysis

Ziziphus spina-christi L. (ZSC-L) is a tree with thorny branches, belongs to the family Rhamnaceae and grows in the sub-tropics. The purpose of this research is to isolate and partially purify bioactive components from the crude ethanol extract of the stem bark of ZSC-L. Besides, bioassay-guided fractionation of ZSC-L stem bark was conducted using different solvents. The solvents were reutilized to minimize the production cost and environmental harm. In addition, the antimicrobial activities of the fractions were analyzed, followed by metabolic profiling using LC-HRMS. The n-butanol fraction showed the highest antimicrobial efficacy, so it was subjected to further purification. For the first time, two major compounds were isolated from the stem bark of ZSC-L and identified as lupane-type pentacyclic triterpenoids betulinic acid and betulin. Both compounds were used as antibacterial and anticancer agents and considered as a green product as the extraction procedure reduced the use of hazardous chemicals. Metabolic characterization of ZSC-L and its bioactive fractions were performed using LC-HR-ESI-MS and the results revealed the dereplication of 36 compounds belonging to different chemical classes. Flavonoids and triterpenes were the most prominent metabolite classes in the different fractions. The molecular docking results were obtained by studying the interaction of betulin and betulinic acid with antimicrobial receptors (4UYM, 1IYL, 1AJ2, 6J7L, 1AD4, 2VEG) to support the in vitro results. Our study highlights that Ziziphus spina-christi and its phytoconstituents, especially triterpenoids, act as a promising antimicrobial candidate in pharmaceutical and clinical applications.     

Simultaneous quantification of five marker compounds of Betula utilis stem bark using a validated high-performance thin-layer chromatography method

A sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for five marker compounds, namely betulin, lupeol, oleanolic acid, 3-acetyloleanolic acid and β-sitosterol, known for their various therapeutic activities. The marker compounds have been isolated from the stem bark of Betula utilis, well characterized by the spectral analysis, and their simultaneous quantitative determination carried out by high-performance thin-layer chromatography (HPTLC) method. The resolution of marker compounds was carried out on silica-gel 60 plates, using n-hexane:ethyl acetate (8:2 v/v) as the mobile phase. The HPTLC densitometry was performed at 500-nm wavelength after the post chromatographic derivatization with ceric ammonium sulfate reagent. The optimized method provided good linear relation (r>0.9960) for all the investigated analytes. The method is simple, and reproducible, which may be applied for quantitative analysis of the above-mentioned marker compounds.     

An Expeditious Method Combining Multi-Components Determination and Fingerprinting Based on Ultra Performance Liquid Chromatography-Photo Diode Array-Tandem Mass Spectrometry and Chemometrics: Application for Authentication and Quality Evaluation of Cornus Officinalis Sieb. et Zucc

Selective and efficient analytical methods for authentication and quality evaluation of herbal medicines are significant and necessary. An expeditious method combining multi-components determination and fingerprinting based on ultra-performance liquid chromatography-photo diode array-tandem mass spectrometry (UPLC-PDA -MS/MS) and chemometrics for authentication and quality evaluation of Cornus Officinalis Sieb. et Zucc was developed. Tandem mass spectrometer operating in multiple reaction monitoring (MRM) was used for determination of three characteristic constituents (morroniside, sweroside, and loganin) in C. Officinalis. Meanwhile, UPLC fingerprint of C. Officinalis was established and the data set was submitted for classification to a suite of chemometrics method. Combing main biologically active components content level and chemometrics analysis, the effects of cultivation area, harvesting, and storage time on the quality of C. Officinalis were investigated. The study reveals that multi-components determination coupled with fingerprinting could be applied for authentication and quality evaluation of C. Officinalis which is accurate, efficient, and reliable.     

Within-Plant Variation in Rosmarinus officinalis L. Terpenes and Phenols and Their Antimicrobial Activity against the Rosemary Phytopathogens Alternaria alternata and Pseudomonas viridiflava

This study investigated within-plant variability of the main bioactive compounds in rosemary (Rosmarinus officinalis L.). Volatile terpenes, including the enantiomeric distribution of monoterpenes, and phenols were analyzed in young and mature foliar, cortical and xylem tissues. In addition, antimicrobial activity of rosmarinic acid and selected terpenes was evaluated against two rosemary pathogens, Alternaria alternata and Pseudomonas viridiflava. Data showed that total concentration and relative contents of terpenes changed in relation to tissue source and age. Their highest total concentration was observed in the young leaves, followed by mature leaves, cortical and xylem tissues. Rosmarinic acid and carnosic acid contents did not show significant differences between leaf tissues of different ages, while young and mature samples showed variations in the content of four flavonoids. These results are useful for a more targeted harvesting of rosemary plants, in order to produce high-quality essential oils and phenolic extracts. Microbial tests showed that several terpenes and rosmarinic acid significantly inhibited the growth of typical rosemary pathogens. Overall, results on antimicrobial activity suggest the potential application of these natural compounds as biochemical markers in breeding programs aimed to select new chemotypes less susceptible to pathogen attacks, and as eco-friendly chemical alternatives to synthetic pesticides.     

Quantification of Valerenic Acid in Valeriana jatamansi and Valeriana officinalis by HPTLC

A simple, rapid, cost-effective and accurate high performance thin layer chromatographic method has been developed for quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis which is one of the stable compounds of Valeriana officinalis and designated as a key marker compound. Valerenic acid makes substantial contribution to the sedative and spasmolytic activity of the essential oil and extract of Valeriana officinalis. Separation and quantification was achieved by HPTLC using ternary mobile phase of hexane: ethyl acetate: acetic acid (80:20:0.5 v/v) on precoated silica gel 60F₂₅₄ aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent at 700 nm, in absorption-reflectance mode. The calibration curves were linear in the range of (500 ng–2.5 μg). This is the first HPTLC report for the identification and quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis.     

Dual-bioactivity-based liquid chromatography-coupled quadrupole time-of-flight mass spectrometry for NF-κB inhibitors and β2AR agonists identification in Chinese Medicinal Preparation Qingfei Xiaoyan Wan

Traditional Chinese medicine (TCM) preparations have been used as an effective multitarget strategy for the treatment of complex diseases; however, their bioactive constituents are undefined and difficult to identify. In this study, a simple and dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and β₂-adrenergic receptor) luciferase reporter assay systems was developed for the rapid determination of various bioactive compounds of TCM preparations. Qingfei Xiaoyan Wan, a TCM preparation used for the clinical therapy of asthma, was analyzed with this method. Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and β₂-adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time-of-flight mass spectrometry data. One β₂-adrenergic receptor agonist (ephedrine) and four structural types of NF-κB inhibitors (arctigenin derivatives, cholic acid derivatives, chlorogenic acid, and sinapic acid) were characterized. Tracheloside was considered a new NF-κB inhibitor. Further cytokine and chemokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. The integration of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and dual-bioactive human cell functional evaluation systems proved to be a simple and effective strategy for the rapid screening of various bioactive compounds in TCM preparations used to treat complex diseases.