Chemical and enzymatic hydrolysis of anthraquinone glycosides from madder roots

For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin and the anthraquinone compositions of the suspensions were analysed by HPLC. A cheap and easy method to hydrolyse ruberythric acid in madder root to alizarin without the formation of lucidin turned out to be the stirring of dried madder roots in water at room temperature for 90 min: this gave a suspension containing pseudopurpurin, munjistin, alizarin and nordamnacanthal. Native enzymes are responsible for the hydrolysis, after which lucidin is converted to nordamnacanthal by an endogenous oxidase.     

Simultaneous determination of naphthalene and anthraquinone derivatives in Rumex nepalensis Spreng. roots by HPLC: comparison of different extraction methods and validation

Introduction – Rumex nepalensis contains mainly anthraquinone and naphthalene derivatives. Although HPLC methods have been reported for the analysis of anthraquinones, neither a phytochemical analysis of Rumex species nor the simultaneous determination of anthraquinone and naphthalene derivatives in other samples has been reported so far. Objective – To develop and validate a HPLC method for the simultaneous determination of anthraquinone and naphthalene derivatives in R. nepalensis roots. Methodology – Anthraquinones and naphthalenes were extracted from R. nepalensis roots by three methods (reflux, ultrasonication and pressurized liquid extraction) using methanol. Separation was achieved on an RP C₁₈ column with a gradient mobile phase consisting of 0.05% orthophosphoric acid in water (solvent A) and methanol (solvent B) using a UV detector (254 nm). Results – Small differences were observed in the contents of anthraquinone and naphthalene derivatives extracted by the three methods. Chrysophanol‐8‐O‐β‐D‐glucopyranoside and nepodin were detected as major constituents. The method showed a good linearity (r² > 0.9992), high precision (RSD < 5%) and a good recovery (97–105%) of the compounds. The lowest detection limit was found to be 0.97 ng and the method was found to be robust. Conclusion – Reflux and ultrasonication were found to be the best suited methods for the extraction of glycosides and aglycones, respectively. The developed and validated HPLC method is simple, precise and accurate; and can hence be recommended as the method of choice for the analysis of anthraquinones and naphthalenes in R. nepalensis and other Rumex species for both quality control as well as routine analytical purposes. Copyright © 2010 John Wiley & Sons, Ltd.     

Two validated HPLC methods for the quantification of alizarin and other anthraquinones in Rubia tinctorum cultivars

Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol-water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran-water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content.     

The mutagenic potential of madder root in dyeing processes in the textile industry

The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated.     

Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe‐emodine,emodine, rheine,chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae)

Introduction – Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the flora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of different methods of extraction. Objective – After a partial validation of an HPLC method for the simultaneous determination of five anthraquinones, aloe‐emodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three different methods of extraction. Methodology – Anthraquinones were extracted from the bark of R. alpinus using different techniques (methanol maceration, ultrasonic and supercritical CO₂ extraction). Separation and quantification of anthraquinones were accomplished using a reversed‐phase C₁₈ column with the mobile phase of H₂O–methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm. Results – All calibration curves were linear over the concentration range tested (10–200 mM) with the determination coefficients ≥0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All five anthraquinones were found in the samples tested at concentrations reported in experimental data. Conclusion – The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantification of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of response surface methodology to optimise ultrasonic-assisted extraction of four chromones in Radix Saposhnikoviae

Introduction – Radix Saposhnikoviae is one of the most famous Chinese herbal medicines with many pharmacological activities towards inflammatory symptoms and antioxidation. Chromones are considered as one of the effective components. It is important to find a reasonable method to extract the chromones in S. divaricata. Objective – To develop an ultrasonic‐assisted extraction (UAE) to extract chromones in Radix Saposhnikoviae and to optimise extraction conditions. Methodology – Four chromones (prim‐O‐glucosylcimifugin, cimifugin, 5‐O‐methylvisammioside and sec‐O‐glucosylhamaudol) were extracted by the UAE method combined with response surface methodology (RSM). Box–Behnken design (BBD) was applied to evaluate the effects of three independent variables (ethanol concentration, extraction time and extraction temperature) on the chromones yield of Radix Saposhnikoviae. Results – Correlation analysis of the mathematical‐regression model indicated that a quadratic polynomial model could be employed to optimise the extraction of chromones by UAE method. The optimal conditions to obtain the highest chromones yield of Radix Saposhnikoviae were a solvent of 75% ethanol, an extraction time of 48 min and an extraction temperature of 67°C. Conclusion – Under these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance indicated a high goodness of model fit and the success of RSM method for optimising chromones extraction in Radix Saposhnikoviae. Copyright © 2011 John Wiley & Sons, Ltd.     

Influence of the extraction mode on the yield of hyperoside, vitexin and vitexin-2'-O-rhamnoside from Crataegus monogyna Jacq. (hawthorn)

Introduction –  The extract of Crataegus monogyna shows sedative, hypotensive, vasodilator and cardio‐tonic actions. Although several papers dealing with the extraction of metabolites from Crataegus have been published, the plant productivity in terms of bioactive compounds is not easily understandable as yet. Objective –  To investigate the influence of the extraction mode on the yield of bioactive compounds from Crataegus monogyna Jacq. in order to evaluate plant productivity. Methodology –  Samples were prepared by extraction of powdered material obtained from top branches, flowers and leaves. Soxhlet extraction, maceration and ultrasound‐ and microwave‐assisted extraction at different experimental conditions were investigated for the exhaustive extraction of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside. The phytocomponents were identified and quantified by HPLC‐UV/PAD, comparing HPLC retention times and UV spectra of individual peaks with those of the standards analysed under the same conditions. Results –  An easy‐to‐use HPLC isocratic method suitable for the quantification of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside in raw plant extracts was developed. The optimised HPLC methodology was applied to evaluate different extraction procedures. The ultrasound and microwave‐assisted extraction protocols showed higher extraction efficiency than the others. In particular, the optimised microwave protocol gave rise to the highest extraction efficiency with high reproducibility. Conclusions –  A microwave protocol combined with isocratic HPLC analysis is proposed for the rapid screening of plant materials collected in different environmental conditions in order to evaluate the productivity of Crataegus monogyna Jacq. and to find out the best ecological conditions to cultivate hawthorn in Northern Italy. Copyright © 2008 John Wiley & Sons, Ltd.     

Microwave‐assisted extraction of total bioactive saponin fraction from Gymnema sylvestre with reference to gymnemagenin: a potential biomarker

Objective – To develop a fast and ecofriendly microwave assisted extraction (MAE) technique for the effective and exhaustive extraction of gymnemagenin as an indicative biomarker for the quality control of Gymnema sylvestre. Methodology – Several extraction parameters such as microwave power, extraction time, solvent composition, pre‐leaching time, loading ratio and extraction cycle were studied for the determination of the optimum extraction condition. Scanning electron micrographs were obtained to elucidate the mechanism of extraction Results – The final optimum extraction conditions as obtained from the study were: 40% microwave power, 6 min irradiation time, 85% v/v methanol as the extraction solvent, 15 min pre‐leaching time and 25 : 1 (mL/g) as the solvent‐to‐material loading ratio. The proposed extraction technique produced a maximum yield of 4.3% w/w gymnemagenin in 6 min which was 1.3, 2.5 and 1.95 times more efficient than 6 h of heat reflux, 24 h of maceration and stirring extraction, respectively. A synergistic heat and mass transfer theory was also proposed to support the extraction mechanism Conclusion – Comparison with conventional extraction methods revealed that MAE could save considerable amounts of time and energy, whilst the reduction of volume of organic solvent consumed provides an ecofriendly feature. Copyright © 2009 John Wiley & Sons, Ltd.     

Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Staining human lymphocytes and onion root cell nuclei with madder root.

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5-10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

蛇床子有效成分的RP—HPLC测定及其4种提取方法的比较研究

以蛇床子素、欧前胡素、佛手柑内酯为指标成分,建立了蛇床子有效成分分离测定的HPLC新方法,并对95%乙醇冷浸、超声、回流、索氏提取蛇床子所得的香豆素成分进行HPLC分析与比较。结果表明,HPLC新方法适合于蛇床子成分的质量控制,其中蛇床子不同提取液中各香豆素成分含量测定差异较大,且以超声提取为最佳。     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

Effect of chitosan elicitation and media components on the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture

To increase the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture, the effects of elicitation on the colorant production were investigated. Chitosan was the best biotic elicitor among nine plant derived and microbial derived polysaccharides. When elicited with 25 mg/L chitosan, the total production was increased approximately two times in a seven-day culture as compared to that in the unelicited cells. Anthraquinone production was increased in proportion to the contact period up to day 3. Maximum anthraquinone colorants were obtained with 3-day treatment of chitosan. During chitosan elicitation, the total production was increased 1.3 times in MS medium containing galactose as compared to that containing sucrose. The degree of deacetylation in chitosan and the use of growth regulator or addition of precursor did not affect the production of anthraquinone colorants. When madder cells were elicited at optimum condition, anthraquinone concentration and specific anthraquinone content increased 1.3 times (0.69 g/L) and 2.2 times (0.32 g/g DCW), respectively.     

Production and release of anthraquinone pigments by hairy roots of madder (Rubia tinctorum L.) under improved culture conditions

Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O₂ starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 mg/dm³, representing an average production rate of 0.72 mg/(dm³·d).     

Ultrasonication assistance increases the efficiency of isoflavones extraction from kudzu (Pueraria lobata Ohwi) roots waste

This study assesses the use of ultrasonication to improve the extraction process of classical solvent extraction methods for extracting isoflavones from the kudzu roots waste. The kudzu roots waste was produced after squeezing fresh kudzu roots to make juice. The effects of extraction time, extraction temperature, ultrasonic power, and ethanol concentration in ethanol/water mixtures were investigated. The extraction yield was found to increase with extraction time and temperature. The application of ultrasonication-assisted extraction (UAE) increased the extraction yield of water/ethanol mixture (20:80) at 25°C 3 fold. A maximum amount (7.28 g) of isoflavone was obtained from 100 g of dried kudzu roots waste by UAE with water/ethanol mixture (20:80) for 6 h at 80°C. Combining the use of ultrasonication with conventional vacuum evaporation method also reduced the concentration time for extracts from 45 to 24 min.     

Simultaneous analysis of three bioactive compounds in Artemisia annua hairy root cultures by reversed-phase high-performance liquid chromatography-diode array detector

Introduction – Artemisia annua is a rich source of biologically active substances such as terpenoids, coumarins and polyacetylenes. These chemicals have been reported to show beneficial pharmacological properties such as antitumor and antibacterial activities. In genetically transformed root cultures of A. annua, three bioactive metabolites, namely, ponticaepoxide (an insecticidal polyacetylene, 1 ), drimartol A (an anticancer sesquiterpene coumarin, 2 ) and (Z)‐7‐acetoxy‐methyl‐11‐methyl‐3‐methylene‐dodeca‐1,6,10‐triene (a new anticancer sesquiterpene, 3 ) were isolated and identified in our recent work. However, no quantitative analysis methods for any of them are yet available, nor for their simultaneous analysis. Objective – To develop an HPLC‐PAD method for simultaneous determination of 1 , 2 and 3 in hairy root cultures of A. annua. Methodology – HPLC operating conditions were optimised and the chromatographic separation was performed on a C₁₈ column with a gradient acetonitrile : water as mobile phase. Results – Linear relationships within the range of investigated concentrations were observed for the three metabolites with their correlation coefficients greater than 0.997. The method was validated for repeatability (RSD <3.59%) and intra‐ and inter‐day precision (RSD <3.1%) with recovery between 94.8 and 107.6% and the RSD less than 3.40%. The method was successfully applied to the time‐course of accumulation of the bioactive compounds in genetically transformed root cultures of A. annua. Conclusion – The HPLC‐PAD method developed for the simultaneous determination of three bioactive metabolites 1 , 2 and 3 was simple, reproducible and sensitive. Copyright © 2010 John Wiley & Sons, Ltd.     

Solid-phase extraction of liquiritin and glycyrrhizin from licorice using porous alkyl-pyridinium polymer sorbent

Introduction – Liquiritin and glycyrrhizin are valuable components of licorice. An effective separation and determination procedure is needed to separate the liquiritin and glycyrrhizin from the licorice extract. Methodology – A polymer‐confined, ionic liquid sorbent was developed using a process involving polymerisation and modification. The obtained porous particles were used as a sorbent in a solid‐phase extraction process to isolate liquiritin and glycyrrhizin from licorice with different washing and elution solvents. The porous alkyl‐pyridinium polymer sorbent was compared with the C₁₈ sorbent. Results – A simple and convenient method was established to the selectively separate and determinate of liquiritin and glycyrrhizin using a porous ionic liquid‐based polymer coupled with HPLC. Additionally, this study evaluated the application of this sorbent for the detection of these two compounds in commercial medicines. Conclusion – This method was a viable tool that was compatible with the existing HPLC methods and was used to separate and analyse the content of liquiritin and glycyrrhizin in licorice. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

Permeabilization of elicited suspension culture of madder (Rubia akane Nakai) cells for release of anthraquinones

To enhance the productivity of anthraquinone colorants during madder (Rubia akane Nakai) cell cultures, the effects of permeabilizing agents on the production of anthraquinone colorants were investigated. Tween 80 was the best among the permeabilizing agents tested. Addition of 1% Tween 80 increased the total and released concentrations of anthraquinones about 1.6 times (159 mg l–1) and 14 times (71 mg l–1), respectively. In addition, anthraquinone production was increased to 220 mg l–1, 2.2 times as the level of control culture by simultaneous use of 1% Tween 80, 5 mg chitosan/l and 2% (w/v) XAD-7. Also, 47% (105 mg l–1) of total anthraquinones was released to medium or adsorbed on XAD-7.