Raman Spectroscopy Detects Changes in Carotenoids on the Surface of Watermelon Fruits During Maturation.

A non-invasive and non-destructive technique, Raman spectroscopy, was explored to distinguish different maturity stages (20, 30, 40, and 50 days after anthesis) of watermelon (Citrullus lanatus) fruits from four cultivars: Fascination, Orange Crisp, Amarillo and Crimson Sweet. Spectral acquisition from the fruit surface was carried out at the wavelength range of 400-2,000 cm-1 using a handheld Raman spectrometer equipped with 830 nm laser excitation source. The spectra were normalized at 1,438 cm-1 which was assigned to CH2 and CH3 vibration. Detecting changes in the spectral features of carotenoids on the surface of watermelon fruits can be used as a marker to monitor the maturity of the fruit. The spectral analysis confirmed the presence of two major carotenoids, lutein and ß-carotene, and their intensity decreased upon maturity on the fruit surface. Identification of these pigments was further confirmed by resonance Raman spectra and high-performance liquid chromatography analysis. Results of partial least square discriminant analysis of pre-processed spectra have demonstrated that the method can successfully predict the maturity of watermelon samples with more than 85% accuracy. Analysis of Variance of individual Raman bands has revealed a significant difference among the stages as the level of carotenoids was declined during the ripening of the fruits. Thus, Raman spectral signatures can be used as a versatile tool for the non-invasive determination of carotenoid changes on the watermelon fruits' surface during ripening, thereby enabling effective monitoring of nutritional quality and maturity indices before harvesting the watermelon.

Comparative Profiling of Four Lignans (Phyllanthin, Hypophyllanthin, Nirtetralin, and Niranthin) in Nine Phyllanthus Species from India Using a Validated Reversed Phase HPLC-PDA Detection Method.

BACKGROUND: Phyllanthus species exhibit a wide range of in vitro and in vivo pharmacological activities; however, little is known about the compounds present in the extracts that are responsible for such actions. OBJECTIVE: Development and validation of a simple reversed phase HPLC-PDA method for profiling of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of Phyllanthus species was carried out. METHODS: Separation was achieved using an XBridge column® (150 × 4.6 mm, 5.0 µm id) in an isocratic elution mode with mobile phase comprising of a mixture of acetonitrile and water with TFA (0.05%, v/v, pH = 2.15) at ambient temperature with a flow rate of 1 mL/min. RESULTS: Phyllanthin, hypophyllanthin, nirtetralin, and niranthin were eluted at mean retention times of 10.47, 11.10, 13.67, and 14.53 min, respectively. LOD and LOQ for all four analytes were 0.75 and 3.00 µg/mL, respectively. RSDr values for intraday and interday precision for phyllanthin, hypophyllanthin, nirtetralin, and niranthin were 0.38-1.32 and 0.45-1.77%; 0.22-3.69 and 0.24-3.04%, 0.73-2.37 and 0.09-0.31%, and 1.56-2.77 and 0.12-0.68%, respectively. CONCLUSIONS: The developed and validated HPLC-PDA method was applied for identification and quantification of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of different plant parts of selected Phyllanthus species. The outcome of the present investigation could be useful for selection of best species to promote its commercial cultivation and suitable extraction solvent for preparation of lignan-enriched fractions. This HPLC-PDA method could be useful for quality control of herbal formulations containing plants from Phyllanthus species.

Development and Validation of a Reversed Phase High Performance Liquid Chromatography-Photodiode Array Detection Method for Simultaneous Identification and Quantification of Coumarin, Precocene-I, ß-Caryophyllene Oxide, α-Humulene, and ß-Caryophyllene in Ageratum Conyzoides Extracts and Essential Oils from Plants.

BACKGROUND: Ageratum conyzoides is an aromatic plant. It is considered as an invasive and cosmopolite weed, widely spread in tropical and subtropical regions. Phytochemicals such as benzopyrenes, flavonoids, and terpenoids are reported from A. conyzoides. OBJECTIVE: Development and validation of a reversed-phase HPLC-photodiode array (PDA) detection method for simultaneous identification and quantification of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene in extracts of A. conyzoides and essential oils was carried out. METHODS: Separation of analytes was achieved on a RP-18 (250 mm × 4.6 mm, 5 µm) column using a solvent system comprising of a mixture of acetonitrile and water with 0.05% trifluoroacetic acid in gradient elution mode at ambient temperature with flow rate of 1 mL/min. RESULTS: The retention time of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 4.38, 12.86, 20.10, 33.34, and 35.11 min, respectively. Limits of detection for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene were 2.5, 2.5, 2.5, 0.025, and 2.5 µg/mL, respectively. Similarly, LOQ were 10, 10, 10, 0.10, and 10 µg/mL for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß- caryophyllene, respectively. Repeatabilities (RSD, %) values for intraday and interday precision for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 0.765-2.086 and 0.886-2.128; 0.879-1.672 and 0.979-1.825; 0.696-2.418 and 0.768-2.592; 1.728-2.362 and 1.965-2.378; 1.615-2.897 and 1.658-2.906, respectively. CONCLUSIONS: The separation of five analytes was achieved within 50 min. The developed and validated HPLC-PDA method was successfully applied for identification and quantification of above five analytes in A. conyzoides extracts and essential oils. The method could be used for meeting the characterization criteria of phytoformulations.

Simultaneous Identification and Quantification of Three Xanthones and Two Polyisoprenylated Benzophenones in Eight Indian Garcinia Species Using a Validated UHPLC-PDA Method.

Background: Xanthones and polyisoprenylated benzophenones (PIBs) are two important classes of plant secondary metabolites with a wide range of bioactivities. Garcinia species synthesize numerous xanthones and PIBs. As per the literature, no data claiming simultaneous identification and quantification of three xanthones, α-mangostin, ß-mangostin, γ-mangostin, and two PIBs, xanthochymol, isoxanthochymol, were found. Methods: A validated ultra-HPLC (UHPLC)-photodiode array (PDA) method for the simultaneous identification and quantification of five compounds in different extracts of eight Indian Garcinia species was developed. The compounds were separated on a Waters ACQUITY™ UPLC H-Class column using a mobile phase consisting of solvents 0.1% formic acid in water (A) and methanol (B) in gradient elution mode. The total run time was 9 min. Results: From fruit rinds of eight Indian Garcinia species, namely Garcinia cambogia, G. cowa, G. indica, G. loniceroides, G. mangostana, G. morella, G. pedunculata, and G. xanthochymus, extracts were prepared using solvents of varying polarity. These extracts were analyzed for five biologically important compounds, namely α-mangostin, ß-mangostin, γ-mangostin, xanthochymol, and isoxanthochymol. The results revealed that there is a wide variation in concentration of these compounds in extracts of Garcinia species. Conclusions: The developed and validated UHPLC-PDA method could be used for simultaneous identification and quantification of these five compounds for bioprospection of other Garcinia species.

Ovicidal and larvicidal effects of extracts from leaves of Andrographis paniculata (Burm. f.) Wall.ex Nees against field isolates of human hookworm (Ancylostoma duodenale).

ETHNOPHARMACOLOGICAL RELEVANCE: The whole plant of Andrographis paniculata (Burm. f.) Wall.ex Nees is used traditionally in different forms by the local people of Asian countries owing to its myriad medicinal properties. Its use as an anthelmintic has been mentioned in literature but has not been well elucidated. AIM OF THE STUDY: To determine anthelmintic effects of extracts from leaves of A.paniculata against human hookworm species based on a standard assay system and to establish the effects of major active compounds responsible for the effects. MATERIALS AND METHODS: Ovicidal and larvicidal activities of extracts of leaves of A.paniculata in different solvents ethanol (Et), methanol (Met), ethyl acetate (EA) and petroleum ether (PE) was studied against field isolates of Ancylostoma duodenale collected and cultivated from hookworm infected human stool samples by egg hatch and larval motility assays. Major active compounds namely andrographolide (AP1), neoandrographolide (AP2) and andrograpanin (AP3) were estimated quantitatively in all the extracts by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Anthelmintic effects (ED50, LC50) and presence of the marker compounds in each extract was statistically analyzed by principal component analysis (PCA). Further, biological activities of pure compounds of AP1, AP2, AP3 were assessed to validate the results of the study. RESULTS: Extracts in ethanol and methanol showed highest activity in inhibition of egg hatching with lowest ED50 values (0.017 and 0.02 mg/mL respectively) while ethyl acetate extract had the highest activity against larval motility (0.001 mg/mL) followed by ethanol (0.019 mg/mL). On HPLC analysis, andrographolide content (%), the major diterpene compound, in Met and Et was 0.85 and 1.43 respectively. On PCA, andrographolide component in the extracts was associated with significant inhibitory effects both on egg hatching and larval motility. Pure compound AP1 also showed significant ovicidal and larvicidal activities at concentrations 0.125 µg/mL and 0.019 mg/mL respectively. CONCLUSION: Andrographolide is one of the main phytochemical responsible for significant ovicidal and larvicidal activity against field isolates of A.duodenale from human infections and can be developed as a potential therapeutic choice.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC-Photodiode Array Detection Method.

Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera-enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

Extraction optimization of gallic acid, (+)-catechin, procyanidin-B2, (-)-epicatechin, (-)-epigallocatechin gallate, and (-)-epicatechin gallate: their simultaneous identification and quantification in Saraca asoca.

The objective of the present investigation was to optimize extraction conditions for maximum recovery of bioactive phenolics from different parts of Saraca asoca. Extraction recovery was optimized using a mixture of methanol and water in different proportions. For identification and quantification of six analytes, a rapid reversed phase ultra-performance liquid chromatography (UPLC) photo diode array detection method was developed. UPLC separation was achieved in a gradient elution mode on a C18 column with acetonitrile and aqueous phosphoric acid (0.1%, pH = 2.5). Extraction solvent for maximum recovery of analytes varied depending on the nature of matrices. The developed UPLC method was validated in accordance with International Council for Harmonisation (ICH) guidelines. Wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase composition implied that the method could be suitable for routine analysis of all six analytes with high precision and accuracy. The uniqueness of this study is the determination of the distribution of these compounds in the various parts of S. asoca.

Comparison on extraction yield of sennoside A and sennoside B from senna (Cassia angustifolia) using conventional and non conventional extraction techniques and their quantification using a validated HPLC-PDA detection method.

Senna is an important medicinal plant and is used in many Ayurvedic formulations. Dianthraquinone glucosides are the main bioactive phytochemicals present in leaves and pods of senna. The extraction efficiency in terms of yield and composition of the extract of senna prepared using both conventional (cold percolation at room temperature and refluxing) and non conventional (ultrasound and microwave assisted solvent extraction as well as supercritical fluid extraction) techniques were compared in the present study. Also a rapid reverse phase HPLC-PDA detection method was developed and validated for the simultaneous determination of sennoside A and sennoside B in the different extracts of senna leaves. Ultrasound and microwave assisted solvent extraction techniques were more effective in terms of yield and composition of the extracts compared to cold percolation at room temperature and refluxing methods of extraction.

A validated high-performance liquid chromatography method for determination of tannin-related marker constituents gallic acid, corilagin, chebulagic acid, ellagic acid and chebulinic Acid in four Terminalia species from India.

A validated rapid HPLC-PDA method was developed for identification and quantification of five tannin-related constituents gallic acid (GA), corilagin (CL), chebulagic acid (CB), ellagic acid (EA) and chebulinic acid (CN) in the extracts prepared from the bark and fruits of four Terminalia species available in India. The separation of the five analytes was achieved on an RP-18 column (4.6 × 250 mm, 5 µm) at 25°C using a solvent mixture comprising of acetonitrile and (0.05%) trifluoroacetic acid-water in a gradient elution mode. Limit of detection was 1.0, 0.5, 1.0, 0.5 and 1.0 µg/mL for GA, CL, CB, EA and CN, respectively. Similarly, limit of quantification was 2.5, 1.0, 2.5, 1.0 and 2.5 µg/mL for GA, CL, CB, EA and CN, respectively. Good linearity (r(2) > 0.992) was observed for all the five compounds in wide concentration range. Using the developed HPLC method, the five analytes were identified and quantified in bark and fruit extracts of Terminalia chebula, Terminalia bellirica, Terminalia arjuna and Terminalia catappa. This is the first report of identification and quantification of the five tannin-related marker constituents in the bark and fruit extracts of T. chebula, T. bellirica, T. arjuna and T. catappa.

Extraction of three bioactive diterpenoids from Andrographis paniculata: effect of the extraction techniques on extract composition and quantification of three andrographolides using high-performance liquid chromatography.

Andrographis paniculata (Burm.f.) wall.ex Nees (Acanthaceae) or Kalmegh is an important medicinal plant finding uses in many Ayurvedic formulations. Diterpenoid compounds andrographolides (APs) are the main bioactive phytochemicals present in leaves and herbage of A. paniculata. The efficiency of supercritical fluid extraction (SFE) using carbon dioxide was compared with the solid-liquid extraction techniques such as solvent extraction, ultrasound-assisted solvent extraction and microwave-assisted solvent extraction with methanol, water and methanol-water as solvents. Also a rapid and validated reverse-phase high-performance liquid chromatography-diode array detection method was developed for the simultaneous determination of the three biologically active compounds, AP, neoandrographolide and andrograpanin, in the extracts of A. paniculata. Under the best SFE conditions tested for diterpenoids, which involved extraction at 60°C and 100 bar, the extractive efficiencies were 132 and 22 µg/g for AP and neoandrographolide, respectively. The modifier percentage significantly affected the extraction efficiency.

Validated HPLC method for identification and quantification of p-hydroxy benzoic acid and agnuside in Vitex negundo and Vitex trifolia.

A high performance liquid chromatography coupled with photodiode array detection method was developed for the identification and quantification of p-hydroxy benzoic acid and agnuside in the extracts of Vitex negundo and Vitex trifolia. The separation was achieved using acetonitrile and O-phosphoric acid-water (0.5%, v/v) as the mobile phase in an isocratic elution mode. Mean retention times of standard p-hydroxy benzoic acid and agnuside were 6.14 and 11.90 min respectively. The developed method was validated as per the ICH guidelines for limit of detection, limit of quantification, linearity, accuracy and precision. Good linearity (r2≥0.999) was observed for both the compounds in wide concentration range. Relative standard deviation values for intra-day and inter-day precision studies were less than 2%. The analytical recoveries of p-hydroxy benzoic acid and agnuside by the developed HPLC method were 93.07% and 106.11% respectively. Two compounds were identified and quantified in leaves and bar extracts of V. negundo and V. trifolia using the developed HPLC method.

Development and validation of a rapid high performance liquid chromatography - photodiode array detection method for estimation of a bioactive compound wedelolactone in extracts of Eclipta alba

Following optimization of extraction, separation and analytical conditions, a rapid, sensitive and simple reverse-phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed for the identification and quantification of wedelolactone in different extracts of Eclipta alba. The separation of wedelolactone was achieved on a C18 column using the solvent system consisting of a mixture of methanol: water: acetic acid (95: 5: 0.04) as a mobile phase in isocratic elution mode followed by photo diode array detection at 352 nm. The developed method was validated as per the guidelines of the International Conference on Harmonization (ICH). Calibration curve presented good linear regression (r²>0.998) within the test range and the maximum relative standard deviation (RSD, %) values for intra-day assay were found to be 0.15, 1.30 and 1.1 for low (5 µg/mL), medium (20 µg/mL) and high (80 µg/mL) concentrations of wedelolactone. For inter-day assay the maximum RSD (%) values were found to be 2.83, 1.51 and 2.06 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 2 and 5 µg/mL respectively. Analytical recovery of wedelolactone was greater than 95%. Wedelolactone in different extracts of Eclipta alba was identified and quantified using the developed HPLC method. The validated HPLC method allowed precise quantitative analysis of wedelolactone in Eclipta. alba extracts.

Desenvolveu-se método rápido, sensível e simples de Cromatografia Líquida de Alta Eficiência em fase reversa, utilizando-se arranjo de fotodiodo (HPLC-PDA), visando à separação, extração e às condições analíticas para a identificação e quantificação de wedelolactona em diferentes extratos de Eclipta alba. A separação de wedelolactona foi efetuada por meio de uma coluna C18, utilizando mistura de metanol:água:ácido acético (95:5:0.04) como fase móvel, em sistema de eluição isocrática, seguida de detecção por arranjo de fotodiodo a 352 nm. O método desenvolvido foi validado de acordo com as diretrizes da Conferência Internacional de Harmonização (ICH). As curvas de calibração apresentaram boa regressão linear (r²>0,998), dentro dos intervalos de teste, e os valores máximos de desvio padrão relativo (RSD,%) dos ensaios intra-dia foram 0,15, 1,30 e 1,1 para concentrações de wedelolactona baixa (5 µg/mL), média (20 µg/mL) e elevada (80 µg/mL) Para o ensaio inter-dia,os máximos de RSD (%) foram 2,83, 1,51 e 2,06 para as concentrações baixa, média e alta, respectivamente. O Limite de Detecção (LD) e o Limite de Quantificação (LOQ) foram de 2 e 5 µg/mL, respectivamente. A recuperação analítica de wedelolactona foi maior do que 95%. A wedelolactona em diferentes extratos de Eclipta alba foi identificada e quantificada pelo método de HPLC desenvolvido. O método de HPLC validado permitiu a análise quantitativa precisa de wedelolactona em extratos de Eclipta alba.