Optimization of indole acetic acid produced by plant growth promoting fungus, aided by response surface methodology.

Indole acetic acid (IAA) is one of the prime communicator playing a chief role in the interaction between host plant and endophytes. IAA produced by the endophytes primarily contributes to plant growth and development. Here, we optimized IAA production by an endophytic fungus Diaporthe terebinthifolli GG3F6 isolated from the asymptomatic rhizome of Glycyrrhiza glabra employing response surface methodology (RSM) and exploring its effect on the host plant biology. The methodology revealed 1.1 fold increases in IAA accumulation. The maximum IAA (121.20 µg/mL) was achieved using tryptophan substrate (1 mg/mL) in Potato dextrose broth (48 g/L) adjusted to pH 12 and incubated at 35 °C for 7 days. The significantly low p-value (p < 0.0001) of the experiment propounded that the model best fits the experimental data, and the independent variables have considerable effects on the production of IAA. Morphologically, the in-vitro grown G. glabra plants showed enhanced root and shoot growth when co-cultivated with the isolated endophytic fungal strain (GG3F6) relative to the control plants. Also, the enhanced accumulation of total phenolic (10.7 %) and flavonoid (10.2 %) in the endophyte treated plants was observed. The optimization of IAA production by an endophytic fungus using (RSM) has not been reported so far. Interestingly, 2.1 fold increase in glycyrrhizin content was recorded in GG3F6 treated in-vitro host plants as compared to the control plants. This suggested a potential use of D. terebinthifolli as a biostimulator for plant and enhanced accumulation of glycyrrhizin. The study highlights the dynamic host-endophyte interaction for exploitation in agricultural and pharmaceutical applications.

In-vitro antioxidant and anti-inflammatory potential along with p.o. pharmacokinetic profile of key bioactive phytocompounds of Snow Mountain Garlic: a comparative analysis vis-à-vis normal garlic.

Snow mountain garlic (SMG) is a trans-Himalayan medicinal plant used in the traditional medicine system for several ailments, including inflammatory arthritis. Research studies are insufficient to validate its folk medicinal applications. In the present study, the comparative abundance of its key bioactive phytocompounds, viz., S-allyl-L-cysteine (SAC), alliin, and S-methyl-L-cysteine (SMC) against normal garlic were assessed using the LC-MS/MS-MRM method. In addition, the study also explored the antioxidant and anti-inflammatory potency of crude extract of SMG and purified signature phytocompounds (i.e., SMC, SAC, and alliin) in comparison with normal garlic and dexamethasone in LPS-stimulated RAW264.7 macrophage cells. The LC-MS/MS-MRM study revealed significant differences among SMG and normal garlic, viz., alliin 22.8-fold higher in SMG, and SMC could be detected only in SMG. In the bioassays, SMG extract and purified signature phytocompounds significantly downregulated oxidative damage in activated macrophages, boosting endogenous antioxidants' activity. SMG extract-treated macrophages significantly suppressed NF-κB expression and related inflammatory indicators such as cytokines, COX-2, iNOS, and NO. Notably, the observed anti-inflammatory and antioxidant bioactivities of SMG extract were comparable to signature phytocompounds and dexamethasone. In addition, SAC being uniformly found in SMG and normal garlic, its comparative pharmacokinetics was studied to validate the pharmacodynamic superiority of SMG over normal garlic. Significantly higher plasma concentrations (Cmax), half-life (t1/2), and area under curve (AUC) of SAC following SMG extract administration than normal garlic validated the proposed hypothesis. Thus, the abundance of bioactive phytocompounds and their better pharmacokinetics in SMG extract might be underlying its medicinal merits over normal garlic.

Costunosides A-C: cytotoxic sesquiterpene lactones from the rhizomes of Aucklandia costus Falc.

Three new eudesmane type rare sesquiterpene lactone galactosides, costunosides A-C (1-3) were isolated from the rhizomes of Aucklandia costus along with ten known compounds (4-13). Costunosides A-C (1-3) are the first example of naturally eudesmane glycosides containing a ß-galactopyranoside moiety. The structure and relative configurations of these compounds were established by comprehensive analysis of MS and, in particular 1D/2D NMR spectroscopic data. The isolated compounds were tested against a panel of human cancer cell lines, where compounds 3, 6 and 7 have shown promising cytotoxic activity against PC-3, HCT-116 and A549 cell lines with IC50 values in the range of 3.4 µM to 9.3 µM, respectively. Costunosides A-C (1-3) were also screened for inhibition assay of acetyl-cholinesterase (AChE), and butyrylcholinesterase (BChE) and found inactive at a concentration of 10 µM.

Endophytic fungal community of Rosa damascena Mill. as a promising source of indigenous biostimulants: Elucidating its spatial distribution, chemical diversity, and ecological functions.

The role of endophytes in maintaining healthy plant ecosystems and holding promise for agriculture and food security is deeply appreciated. In the current study, we determine the community structure, spatial distribution, chemical diversity, and ecological functions of fungal endophytes of Rosa damascena growing in the North-Western Himalayas. Culture-dependent methods revealed that R. damascena supported a rich endophyte diversity comprising 32 genera and 68 OTUs. The diversity was governed by climate, altitude, and tissue type. Species of Aspergillus, Cladosporium, Penicillium, and Diaporthe were the core endophytes of the host plant consisting of 48.8% of the endophytes collectively. The predominant pathogen of the host was Alternaria spp., especially A. alternata. GC-MS analyses affirmed the production of diverse arrays of volatile organic compounds (VOC) by individual endophytes. Among the primary rose oil components, Diaporthe melonis RDE257, and Periconia verrucosa RDE85 produced phenyl ethyl alcohol (PEA) and benzyl alcohol (BA). The endophytes displayed varied levels of plant growth-promoting, colonization, and anti-pathogenic traits. Between the selected endophytes, P. verrucosa and D. melonis significantly potentiated plant growth and the flavonoids and chlorophyll content in the host. The potential of these two endophytes and their metabolites PEA and BA was confirmed on Nicotiana tabacum. The treatments of the metabolites and individual endophytes enhanced the growth parameters in the model plant significantly. The results imply that P. verrucosa and D. melonis are potential plant growth enhancers and their activity may be partially due to the production of PEA and BA. Thus, R. damascena harbors diverse endophytes with potential applications in disease suppression and host growth promotion. Further investigations at the molecular level are warranted to develop green endophytic agents for sustainable cultivation of R. damascena and biocontrol of leaf spot disease.

HPLC-PDA Method for Quantification of Bioactive Compounds in Crude Extract and Fractions of Aucklandia costus Falc. and Cytotoxicity Studies against Cancer Cells.

Aucklandia costus Falc. (Synonym: Saussurea costus (Falc.) Lipsch.) is a perennial herb of the family Asteraceae. The dried rhizome is an essential herb in the traditional systems of medicine in India, China and Tibet. The important pharmacological activities reported for Aucklandia costus are anticancer, hepatoprotective, antiulcer, antimicrobial, antiparasitic, antioxidant, anti-inflammatory and anti-fatigue activities. The objective of this study was the isolation and quantification of four marker compounds in the crude extract and different fractions of A. costus and the evaluation of the anticancer activity of the crude extract and its different fractions. The four marker compounds isolated from A. costus include dehydrocostus lactone, costunolide, syringin and 5-hydroxymethyl-2-furaldehyde. These four compounds were used as standard compounds for quantification. The chromatographic data showed good resolution and excellent linearity (r2 ˃ 0.993). The validation parameters, such as inter- and intraday precision (RSD < 1.96%) and analyte recovery (97.52-110.20%; RSD < 2.00%),revealed the high sensitivity and reliability of the developed HPLC method. The compounds dehydrocostus lactone and costunolide were concentrated in the hexane fraction (222.08 and 65.07 µg/mg, respectively) and chloroform fraction (99.02 and 30.21 µg/mg, respectively), while the n-butanol fraction is a rich source of syringin (37.91 µg/mg) and 5-hydroxymethyl-2-furaldehyde (7.94 µg/mg). Further, the SRB assay was performed for the evaluation of anticancer activity using lung, colon, breast and prostate cancer cell lines. The hexane and chloroform fractions show excellent IC50 values of 3.37 ± 0.14 and 7.527 ± 0.18 µg/mL, respectively, against the prostate cancer cell line (PC-3).

Redox-Responsive Hyaluronic Acid-Tacrolimus Conjugate: Synthesis, Characterization, and In Vitro Immunosuppressive Activity.

A redox-responsive macromolecular prodrug of tacrolimus, HA-ss-Tac, was constructed by conjugation of tacrolimus (TAC, FK506) through its succinate ester to cystamine-modified hyaluronic acid (HA-Cys), and its physicochemical properties and immunosuppressive activity were studied. The synthesized HA-ss-TAC was determined to contain 8% of chemically loaded TAC with significantly enhanced water solubility. The release study showed a sustained release of drug through slow degradation of linker-drug bonds. In vitro inhibition of proliferation of T- and B-lymphocytes was almost comparable to that of TAC, implying that the biologically active compound could be released from the conjugate. The polymeric prodrug lacks obvious cytotoxicity on Raw 264.7 macrophages and significantly suppressed the production of inflammatory cytokines IL-2 and IL-1ß by LPS-activated cells. Additionally, the cellular uptake study of the FITC-labeled conjugate confirmed the HA receptor-mediated internalization of the conjugate into targeted cells, thus avoiding systemic side effects. Taken together, the HA-ss-TAC prodrug could be an optimal prodrug for intravenous administration based on this preliminary data and can be expected to have improved therapeutic efficacy.

Correction: Chrysomycins A-C, antileukemic naphthocoumarins from Streptomyces sporoverrucosus.

[This corrects the article DOI: 10.1039/C3RA42884B.].

Molecular dissection of genes and promoters involved in glycyrrhizin biosynthesis revealed phytohormone induced modulation in Glycyrrhiza glabra L.

The study reports cloning and characterization of complete biosynthetic gene cluster committed to glycyrrhizin biosynthesis along with their corresponding promoter regions from Glycyrrhiza glabra. The identified genes namely, ß-amyrin synthase, ß-amyrin-11-oxidase, 11-oxo-beta-amyrin 30-oxidase and UDP-dependent glucosyltransferase, were hetrologously expressed in Nicotiana benthamiana for functional validation. The phyto-hormone, naphthalene acetic acid was shown to prompt maximum up regulation (1.3-14.0 folds) of all the genes, followed by gibberellic acid (0.001-10.0 folds) and abscisic acid (0.2-7.7 folds) treatments. The promoter-GUS fusion constructs infiltrated leaves of the identified genes exhibited enhanced promoter activity of ß-amyrin synthase (3.9 & 3.0 folds) and 11-oxo-beta-amyrin 30-oxidase (3.6 & 3.2 folds) under the GA3 and NAA treatments, respectively as compared to their respective untreated controls. The transcriptional control of the three phytohormones studied could be correlated to the cis-responsive elements present in the upstream regions of the individual genes. The study provided an insight into the intricate interaction between hormone-responsive motifs with the corresponding co-expression of the glycyrrhizin biosynthetic pathway genes. The study will help in understanding the phytohormones-mediated regulation of glycyrrhizin biosynthesis and its modulation in the plant.

Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.

Preclinical development of gastro-protective botanical candidate from Woodfordia fruticosa (Linn.) Kurz: Chemical standardization, efficacy, pharmacokinetics and safety pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Woodfordia fruticosa is traditionally used in the Ayurvedic system of medicine for the treatment of diarrhoea, poisoning, menstrual disorders, ulcers and fertility. In the present study, we report a standardized extract preparation through modern scientific approach for anti-ulcer activity. MATERIALS AND METHODS: The hydro-alcoholic extract of flowers of W. fruticosa was standardized using four chemical markers. The standardized extract was coded as ICB014. HPLC method was developed for identification and quantification of Gallic Acid, Oenothein-C, Quercetin and Kaempferol. Based on the prior published H+, K+-ATPase activity and Anti-bacterial activity against Helicobacter pylori of ICB014, was evaluated for its in-vivo efficacy in gastric ulcers models in rats followed by regulatory safety studies. RESULTS: The extract demonstrated efficacy at 31.25-62.5 mg/kg in gastric ulcer models. The extract was safe by oral route up to 2000 mg/kg in a single dose and NOAEL of 800 mg/kg in 28 days repeat study. Bioequivalent capsule formulation was prepared. CONCLUSIONS: The extract showed anti-ulcer potential and is ready for clinical evaluation.

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L.

Chalcone synthase constitutes a functionally diverse gene family producing wide range of flavonoids by catalyzing the initial step of the phenylpropanoid pathway. There is a pivotal role of flavonoids in pollen function as they are imperative for pollen maturation and pollen tube growth during sexual reproduction in flowering plants. Here we focused on medicinally important fruit-bearing shrub Grewia asiatica. It is a rich repository of flavonoids. The fruits are highly acclaimed for various putative health benefits. Despite its importance, full commercial exploitation is hampered due to two drawbacks which include short shelf life of its fruits and larger seed volume. To circumvent these constraints, seed abortion is one of the viable options. Molecular interventions tested in a number of economic crops have been to impair male reproductive function by disrupting the chalcone synthase (CHS) gene activity. Against this backdrop the aim of the present study included cloning and characterization of two full-length cDNA clones of GaCHS isoforms from the CHS multigene family. These included GaCHS1 (NCBI acc. KX129910) and GaCHS2 (NCBI acc. KX129911) with an ORF of 1176 and 1170 bp, respectively. GaCHSs were heterologously expressed and purified in E. coli to validate their functionality. Functionality of CHS isoforms was also characterized via enzyme kinetic studies using five different substrates. We observed differential substrate specificities in terms of their Km and Vmax values. Accumulation of flavonoid constituents naringenin and quercetin were also quantified and their relative concentrations corroborated well with the expression levels of GaCHSs. Further, our results demonstrate that GaCHS isoforms show differential expression patterns at different reproductive phenological stages. Transcript levels of GaCHS2 were more than its isoform GaCHS1 at the anthesis stage of flower development pointing towards its probable role in male reproductive maturity.

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: bioactivity-guided isolation and LCMS-based quantification.

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.

Capsaicin production by Alternaria alternata, an endophytic fungus from Capsicum annum; LC-ESI-MS/MS analysis.

Alternaria alternata, an endophytic fungus capable of producing capsaicin (1) was isolated from Capsicum annum. The endophyte was found to produce capsaicin upto three generations. Upscaling of the fermentation broth led to the isolation of one known and one compound characterized as 2,4-di-tert-butyl phenol (2) and alternariol-10-methyl ether (3) respectively. Compound 1 and 3 were identified and quantified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system through multiple reaction monitoring (MRM). Furthermore, compound 3 displayed a range of cytotoxicity against a panel of human cancer cell lines and was found to induce apoptosis evidenced by Hoechst staining and loss of mitochondrial-membrane potential in HL-60 cells.

Induction of apoptosis in human pancreatic MiaPaCa-2 cells through the loss of mitochondrial membrane potential (ΔΨm) by Gentiana kurroo root extract and LC-ESI-MS analysis of its principal constituents.

The objective of the current study was to evaluate the methanolic root extract of Gentiana kurroo for antioxidant and antiproliferative activities as well as to study the effect of the extract on the induction of apoptosis in human pancreatic cancer cell line (MiaPaCa-2). The extract exerted significant antioxidant activity as verified by DPPH, hydroxyl radical, lipid peroxidation and protective oxidative DNA damage assays. The results were comparable to standard antioxidants like α-tocopherol, catechin and BHT used in such experiments. Antioxidant potential of G. kurroo may be attributed to the presence of high phenolic and flavonoid content (73±1.02 and 46±2.05 mg/g extract respectively). The anti-proliferative property of Gentiana kurroo root extract was determined by sulphorhodamine B (SRB) assay against Human colon cancer cell line (HCT-116), Lung carcinoma cell line (A-549), Pancreatic cancer cell line (MiaPaCa-2), Lung cancer cell line (HOP-62) and acute monocytic leukaemia cell line (THP-1). G. kurroo root extract inhibited cancer cell growth depending upon the cell line used and in a dose dependent manner. The extract induced potent apoptotic effects in MiaPaCa-2 cells. The population of apoptotic cells increased from 11.4% in case of control to 49.6% at 100 µg/ml of G. kurroo root extract. The extract also induced a remarkable decrease in mitochondrial membrane potential (ΔΨm) leading to apoptosis of cancer cells used. The main chemical constituents identified by the liquid chromatography-tandem mass spectrometry (LC-ESI-MSMS) were found to be iridoid glucosides (iridoids and secoiridoids), xanthones and flavonoids. Iridoid glucosides are the bitter principles of Gentiana species. Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4″-O-ß-D-glucosyl-6'-O-(4-O-ß-D-glucosylcaffeoyl)-linearoside and Swertisin were the principal compounds present in the methanol root extract of G. kurroo.

Direct rhizogenesis, in vitro stolon proliferation and high-throughput regeneration of plantlets in Glycyrrhiza glabra.

Direct rhizogenesis from leaf explants and establishment of an in vitro stolon culture system and subsequent plant regeneration for Glycyrrhiza glabra have been described. MS liquid medium supplemented with 0.01 mg l-1 of NAA was most effective for stolon proliferation. Extensive proliferation of stolon and shoot regeneration was achieved on medium containing 3 % sucrose with 0.01 mg l-1 NAA. Stolons with nodes showing growth was transferred under light for plantlet regeneration in the same medium. This paper is the first report in G. glabra describing a complete regeneration procedure via in vitro stolon proliferation along with quantitative data for glycyrrhizin and genetic fidelity of plant regenerated in vitro there from. In vitro stolon proliferation described here would be an efficient way for regeneration of plants for functional genomics studies and better understanding of glycyrrhizin (GA) metabolism.

ortho-Amidoalkylation of phenols via tandem one-pot approach involving oxazine intermediate.

A new and efficient method for ortho-amidoalkylation of phenols via Mannich-type condensation with formaldehyde and lactams using recyclable solid acid catalyst is described. This is the first report for ortho-amidoalkylation of phenols by lactams via Mannich-type condensation. LC-ESI-MS/MS based mechanistic study revealed that reaction proceeds through o-quinone methide (o-QM) and an oxazine intermediate via tandem Knoevenagel condensation, formal [4 + 2]-Diels-Alder cycloaddition and acid catalyzed oxazine ring-opening.

Ultrafast UPLC-ESI-MS and HPLC with monolithic column for determination of principal flavor compounds in vanilla pods.

In this study, two novel chromatographic methods based on monolithic column high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were developed for the ultrafast determination of principal flavor compounds namely vanillin, vanillic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde in ethanolic extracts of Vanilla planifolia pods. Good separation was achieved within 2.5 min using Chromolith RP18e column (100 mm x 4.6 mm) for HPLC and Acquity BEH C-18 (100 mm x 2.1 mm, 1.7 microm) column for UPLC. Both methods were compared in terms of total analysis time, mobile phase consumption, sensitivity, and validation parameters like precision, accuracy, LOD, and LOQ. Further, system suitability test data including resolution, capacity factor, theoretical plates, and tailing factor was determined for both the methods by ten replicate injections. Monolithic column based HPLC gave better results for most of the selected parameters while UPLC was found to be more eco-friendly with low mobile phase consumption and better sensitivity. Both methods may be used conveniently for the high throughput analysis of large number of samples in comparison to traditional particulate column.

Simultaneous densitometric determination of shikonin, acetylshikonin, and beta-acetoxyisovaleryl-shikonin in ultrasonic-assisted extracts of four Arnebia species using reversed-phase thin layer chromatography.

A simple, precise, and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin (1) together with its derivatives acetylshikonin (2), and beta-acetoxyisovalerylshikonin (3) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP-18 F(254S )TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100-600 ng for shikonin and acetylshikonin, and 100-1800 ng for beta-acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1-3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.

Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species.

A rapid, simple, sensitive, robust, and improved HPLC method was developed and validated for determination of 10 polyphenols, namely gallic acid, catechin, epicatechin, rutin, m-coumaric acid, quercitrin, myricetin, quercetin, apigenin, and kaempferol in fresh flowers of Rosa bourboniana and R. brunonii and in both fresh flowers and marc (left after industrial distillation of rose oil) of R. damascena. Six polyphenols, gallic acid, rutin, quercitrin, myricetin, quercetin, and kaempferol, were detected and quantified in all extracts. The chromatographic separation of 10 polyphenols was achieved in less than 16 min by RP-HPLC (Phenomenex, Luna C18 (2) column, 5 microm, 250 mm x 4.6 mm) using linear gradient elution of water and acetonitrile (0.02% trifluroacetic acid) with a flow rate of 1 mL/min at lambda 280 nm. Standard calibration curves were linear in the range of 0.39-500 microg/mL. Good results were achieved with respect to repeatability (RSD <3%) and recovery (98.6-100.8%). The method was validated for linearity, accuracy, repeatability, LOD, and LOQ.