Extraction Of Podophyllotoxin Research Articles

Development of hydroxypropyl cellulose and graphene oxide modified molecularly imprinted polymers for separation and enrichment of podophyllotoxin

A novel hydroxylpropyl cellulose (HPC) modified graphene oxide (GO)-based molecularly imprinted polymers (HPC-GO-MIP) have been developed as a solid phase extraction (SPE) material for the selective separation and extraction of podophyllotoxin. In this strategy, the cellulose with rich hydroxyl groups was introduced to form bi-functional monomers with methacrylic acid to provide more recognition sites for the improving of extraction efficiency, then GO was added as a two-dimensional substrate for MIP to improve the material morphology and surface area. The extraction performances of obtained HPC-GO-MIP material were tested, and the results prove its high efficiency and selectivity for podophyllotoxin extraction. The saturated adsorption capacity reached 23.1 μg/mg, and high enrichment efiiciency of 463.8 folds was realized under the premise of ensuring the recovery rate. The selective imprinting factor was much higher than those of kaempferol and quercetin, which were the main compounds in podophyllum fruit. Under the optimized SPE conditions, the HPC-GO-MIP based SPE-HPLC method showed the detection limit of 14.2 ng/mL for podophyllotoxin assay. When applied to podophyllum fruit samples, the material showed excellent ability of selective separation and enrichment of podophyllotoxin, and the relative standard deviations (RSD) of intra and inter batches were less than 8.1 % and 5.7 % in real samples detection. The HPC-GO-MIP SPE method broadened the application for high multiple extraction in trace analyte samples and provided a valuable solution to improve the selective separation and detection.

Green and efficient extraction of podophyllotoxin from Sinopodophyllum hexandrum by optimized subcritical water extraction combined with macroporous resin enrichment

Sinopodophyllum hexandrum is an important traditional Tibetan medicinal plant with great development potential in pharmaceutical industry because of the presence of major bioactive lignans such as podophyllotoxin (PT) that are widely used in combination therapy of cancer. In this study, a green and efficient method for the extraction of PT from S. hexandrum was established by optimized subcritical water extraction (SWE) combined with macroporous resin enrichment. Response surface methodology was introduced for SWE optimization to obtain the maximum yield of PT. The maximum extraction efficiency was achieved under the following conditions: extraction pressure 4 MPa, extraction solvent to feed ratio 18 mL/g, extraction temperature 180 °C, and SW flow rate 2.5 mL/min. Subsequently, an efficient macroporous resin enrichment method was developed to further purify PT in the crude extract obtained from SWE. D101 resin was selected as a proper adsorbent owing to its adsorption and desorption properties, whereas its adsorption process was found to be consistent with a pseudo-second-order model. The optimized loading amount was 3 BV of extract solution, and the desorption conditions were as follows: First, the product was eluted with deionized water until no sugar was detected in the effluent, followed by 6 BV of 20% aqueous ethanol elution and then by 15 BV of 60% aqueous ethanol elution. After the treatment with D101 resin, the concentration of PT increased from 8.3% in the crude extract to 61.5% in the final product. Moreover, the recovery yield of PT extracted with SW combined with D101 resin enrichment was 74.6%. The results show that SWE combined with macroporous resin enrichment is a green, effective, and low-cost technique for the extraction of PT from S. hexandrum.

The application of SVR model in the improvement of QbD: a case study of the extraction of podophyllotoxin

In order to make a further optimization of process design via increasing the stability of design space, we brought in the model of Support Vector Regression (SVR). In this work, the extraction of podophyllotoxin was researched as a case study based on Quality by Design (QbD). We compared the fitting effect of SVR and the most used quadratic polynomial model (QPM) in QbD, and an analysis was made between the two design spaces obtained by SVR and QPM. As a result, the SVR stayed ahead of QPM in prediction accuracy, the stability of model and the generalization ability. The introduction of SVR into QbD made the extraction process of podophyllotoxin well designed and easier to control. The better fitting effect of SVR improved the application effect of QbD and the universal applicability of SVR, especially for non-linear, complicated and weak-regularity problems, widened the application field of QbD.

Vacuum-powered bubble-assisted solvent extraction followed by macroporous resin enrichment for isolation of podophyllotoxin from Sinopodophyllum emodi.

A vacuum-powered bubble-assisted solvent extraction (VBE) technique was used to extract podophyllotoxin from the root of Sinopodophyllum emodi. We optimized the VBE procedure and showed it had the highest efficiency of extraction compared to other conventional extraction techniques. Based upon the results of single-factor experiments, a three-factor, three-level experiment design was developed by application of a Box-Behnken design. The method was validated by stability, repeatability and recovery experiments. The optimal conditions were: solvent, 60% (v/v) ethanol; particle size of the sample, 60-80 mesh; soak time, 2h; liquid/solid ratio, 21L/kg; air flow, 32mL/min; vacuum-powered bubble extraction time, 65min. The VBE method we developed achieved efficient extraction of podophyllotoxin from S. emodi. The podophyllotoxin extracted can be enriched and separated by an HPD300 macroporous resin adsorption and desorption process. The results indicated that VBE is a convenient, rapid and efficient sample preparation technique.

Extraction of podophyllotoxin from Podophyllum peltatum using pulsed electric field treatment

Pulsed electric field technology (PEF) was applied to Podophyllum peltatum as a novel approach to enhance the extraction of podophyllotoxin. Podophyllotoxin is a valuable for the treatment of cancer and venereal warts. However, the demand for podophyllotoxin exceeds the supply and conventional extraction methods are inefficient. A low energy pulse generator was used to perform PEF treatment with pulse frequency of 840Hz and electric field intensity of 17.7 and 19.4kV/cm for 5, 6, and 8min of apparent treatment time. PEF treatment significantly changed the color and apparent viscosity of the samples. PEF treatment with 840Hz rectangular pulses of 19.4kV/cm for 6min was successful in significantly raising the concentration of podophyllotoxin by up to 47%. Although the exact mechanism of electroplasmolysis of plant cells is unknown, the synergistic effect between temperature and PEF treatment enhanced the extraction of podophyllotoxin from P. peltatum.

An efficient regeneration system via direct and indirect organogenesis for the medicinal plant Dysosma versipellis (Hance) M. Cheng and its potential as a podophyllotoxin source

Dysosma versipellis (Hance) M. Cheng is an endangered plant due to overharvesting for the extraction of podophyllotoxin. Thus, the in vitro technique is valuable for the propagation of this species. When the explants of rhizome buds were cultured on Murashige and Skoog’s (MS) medium with 6-benzyladenine (BA) (1.0 mg l−1), gibberellic acid (GA3) (0.5 mg l−1) and zeatin (Zea) (0.5 mg l−1), multiple buds were regenerated directly on the explants without callusing within 6 weeks. Callus was induced from the leaf segment cultures on MS basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg l−1) and BA (0.2 mg l−1) within 4 weeks. The adventitious buds were differentiated when the calli were subcultured on MS medium supplemented with BA (1.0 mg l−1) and thidiazuron (TDZ) (0.2 mg l−1) within 6 weeks. The adventitious buds obtained from callus and the rhizome-buds rooted with a frequency of 100% on half strength MS medium fortified with indole-3-butyric acid (IBA) 0.5 mg l−1 and activated charcoal (AC) 0.5 g l−1 for 4 weeks. The rooted shoots were successfully transplanted from a mixture of vermiculite:soil (1:1 v/v) to the field with a survival rate of 85%. Podophyllotoxin production in calli, cultured rhizomes, rhizomes of transplanting plants from the garden and rhizomes in the wild field was confirmed by high-performance liquid chromatography (HPLC) analysis. Our results suggest that calli, cultured rhizomes and rhizomes of transplanting plants would be the potential sources of podophyllotoxin.

Podophyllotoxin and Deoxypodophyllotoxin in Juniperus bermudiana and 12 Other Juniperus Species: Optimization of Extraction, Method Validation, and Quantification

The lignans podophyllotoxin and deoxypodophyllotoxin are secondary metabolites with potent pharmaceutical applications in cancer therapy. However, the supply of podophyllotoxin from its current natural source, Podophyllum hexandrum, is becoming increasingly problematic, and alternative sources are therefore urgently needed. So far, podophyllotoxin and deoxypodophyllotoxin have been found in some Juniperus species, although at low levels in most cases. Moreover, extraction protocols deserve optimization. This study aimed at developing and validating an efficient extraction protocol of podophyllotoxin and deoxypodophyllotoxin from Juniperus species and applying it to 13 Juniperus species, among which some had never been previously analyzed. Juniperus bermudiana was used for the development and validation of an extraction protocol for podophyllotoxin and deoxypodophyllotoxin allowing extraction yields of up to 22.6 mg/g DW of podophyllotoxin and 4.4 mg/g DW deoxypodophyllotoxin, the highest values found in leaf extract of Juniperus. The optimized extraction protocol and HPLC separation from DAD or MS detections were established and validated to investigate podophyllotoxin and deoxypodophyllotoxin contents in aerial parts of 12 other Juniperus species. This allowed either higher yields to be obtained in some species reported to contain these two compounds or the occurrence of these compounds in some other species to be reported for the first time. This efficient protocol allows effective extraction of podophyllotoxin and deoxypodophyllotoxin from aerial parts of Juniperus species, which could therefore constitute interesting alternative sources of these valuable metabolites.

Application of ultrasound as pretreatment for extraction of podophyllotoxin from rhizomes of Podophyllum peltatum

The effect of high-power ultrasound pretreatment on the extraction of podophyllotoxin from Podophyllum peltatum was investigated. Direct sonication by an ultrasound probe horn was applied at 24 kHz and a number of factors were investigated: particle size (0.18–0.6 mm), type of solvent (0–100% aqueous ethanol), ultrasonic treatment time (2–40 min), and power of ultrasound (0–100% power intensity, maximum power: 78 W). The optimal condition of ultrasound was achieved with 0.425–0.6 mm particle size, 10 min sonication time, 35 W ultrasound power, and water as the medium. There was no obvious degradation of podophyllotoxin with ultrasound under the applied conditions, and an improvement in extractability was observed. The SEM microscopic structure change of treated samples disclosed the effect of ultrasound on the tissue cells. The increased pore volume and surface area after ultrasonic treatment also confirmed the positive effect of ultrasound pretreatment on the extraction yield of podophyllotoxin from the plant cells.

Resources of survey in rare and endangered medicinal plant Sinopodophyllum emodi

The resource of Sinopodophyllum emodi in Tibet, Qinghai, Gansu, Sichuan and Yunnan province were surveyed by the ways of documents, interview, quadrat and market investigation. The results indicated that S. emodi were mainly distributed in central part from Taibai Shan of Shanxi province along the middle and high mountain on either side of Hengduan mountain which from north to south and in Tibetan plateau of China, the suitable eco-environment of S. emodi was dominated by valleys, wet forests and low bush-woods vegetation which comparatively dry on plateau with the altitude focused on 1 5004 500 meters, and the distribution density of simple alpine meadow was relatively lower. Illumination, water, soil, temperature and altitude had significant influences on the growth, distribution and reserves of S. emodi from different angles. In recent years, there was sharp increase of market requirement in Xiaoyelian (fruit) which were used in Tibetan medicine and the underground part which was used for the extraction of podophyllotoxin, excess collection of the underground part was the primary cause of the rapid decreasing resource of S. emodi. It is suggested that the management of rational collection should be strengthen and the development of culturing and production should be meanwhile accelerated.

Review on biosynthesis of podophyllotoxin

Podophyllotoxin (PTOX) is an extremely important plant-derived natural product, of which derivatives, like etoposide and teniposide, have been widely applied in therapies for cancers and venereal wart. A durable, intense plant extraction of podophyllotoxin posed a severe pressure on wild resources; researchers consequently sought to explore new sources, like cultivation, plant cell or organ culture, and chemical synthesis. Understanding biosynthesis of PTOX is one of the basic necessary steps for standard cultivation of medicinal plants and metabolite engineering. An important progress has been made in this field during the last two decades, particularly in the last ten years. Although a number of reviews concerning the related topic have existed, we specifically deal with biosynthesis of podophyllotoxin with an emphasis on the literatures of the past decade, highlighting characterization of genes encoding synthetic enzymes and down-stream metabolism of PTOX. The present review focuses on several key biosynthesis processes, important metabolites, function of related enzymes, and characterization of cDNA encoding the enzymes. Finally, the author proposed a hypothetical biosynthetic scheme of podophyllotoxin and perspectives.

Application of ionic liquids in the microwave-assisted extraction of podophyllotoxin from Chinese herbal medicine

A micelle-mediated extraction technique, i.e. ionic liquid-based microwave-assisted extraction (ILs-MAE) technique has been developed for the effective extraction of podophyllotoxin from three Chinese medicinal plants. Several operating parameters were successively optimized by single-factor and L(9) (3(4)) orthogonal array experiments. 1-Butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]), 1-decyl-3-methylimidazolium tetrafluoroborate ([demim][BF(4)]) and 1-allyl-3-methylimidazolium tetrafluoroborate ([amim][BF(4)]) were selected as the optimal surfactants for Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis, respectively. Compared with other extraction techniques, such as ionic liquids-based maceration extraction (ILs-ME), heat extraction (ILs-HE) and ultrasound-assisted extraction (ILs-UAE), the ILs-MAE technique not only took a shorter time but also afforded a higher extraction rate of podophyllotoxin from the herbs. Reversed phase high performance liquid chromatography was employed for the analysis of podophyllotoxin. The results showed that the linearity for analyzing podophyllotoxin in all three herbs was in the concentration range of 0.005-0.4 mg mL(-1) with the correlation coefficient between 0.9993 and 0.9996. LODs were 2.05-2.58 μg mL(-1) and RSDs of inter-day stability were less than 5.8%. Repeatability and intermediate precision were separately lower than 3.3% and 6.3%. The recoveries for podophyllotoxin extracted with the ILs-MAE technique were in the range of 97.1-102% and all RSDs were lower than 3.0%. Furthermore, the mechanism of ILs-MAE was preliminarily studied by means of kinetic mechanism, surface structures and chemical compositions of samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures and high solubility of ILs, the ILs-MAE technique eventually got the maximum yield value.

Heat transfer modeling of radio frequency assisted packed bed extraction of an anticancer agent (podophyllotoxin)

Podophyllotoxin is considered as an outstanding anti-tumor agent against lung cancer which is commercially extracted by the percolation of warm ethanol through packed beds of dried rhizomes of Podophyllum emodi or Podophyllum peltatum. Radio frequency (RF) heating can be used for uniform heating of the packed bed during the extraction. Based on local volume averaging (LVA) and local thermal equilibrium (LTE), a mathematical model with variable properties was developed for transient heat transfer during RF-assisted packed bed extraction of podophyllotoxin. The validity of LVA and LTE was assessed by evaluating the length scale, time scale, and convective constraint. A control function was included in the model to take into account the effect of a temperature controller functioning during the actual RF-assisted packed bed extraction. For a vertical packed bed reactor subjected to RF heating, two governing equations associated with the packed bed and the solvent phase at the top of the bed were numerically solved using implicit-finite difference and Gauss–Seidel methods. The simulation results had a good agreement with the experimental data. Heat transfer simulation was performed for different flow rates of ethanol/water solution with different dielectric properties during the RF-assisted packed bed extraction process.

Radio frequency-assisted extraction of podophyllotoxin: Prototyping of packed bed extraction reactors and experimental observations

Radio frequency (RF) heating was used for uniform and noncontact heating of a packed bed of rhizome of Podophyllum peltatum while an ethanol/water solution was continuously flowing through the bed during solvent extraction of podophyllotoxin. RF-assisted packed bed extraction was carried out at 27.12 MHz for five solvent flow rates and three levels of dielectric loss factor of ethanol/water solution provided by three fractions of sodium chloride in the solvent. The performances of RF heating on horizontally and vertically oriented packed bed reactor were assessed. RF heating provided a very small temperature gradient across the height of the vertically oriented packed bed with a thickness of 10 cm at the stationary state of the bed; however, the temperature gradient across the horizontally oriented packed bed was significant. A sodium chloride concentration of 2.5 g/L in the solvent at a flow rate of 160 ml/min provided a relatively uniform temperature of 50 °C across the height of the bed during the extraction process. The constant rate and falling rate period of extraction took place within 2 and 8 min, respectively.

Bioprospection of Eastern red cedar from nine physiographic regions in Mississippi

Juniperus virginiana L. (Family Cupressaceae), commonly called Eastern red cedar, is a widely distributed species in the United States and parts of Canada. It produces two important chemical products, the anticancer compound podophyllotoxin and essential oil. The objective of this study was to evaluate variations in podophyllotoxin concentration in Eastern red cedar, across nine physiographic regions in Mississippi: Tishomingo Hills, Bluff Region, Blackland Prairie, Jackson Prairie, Delta, Pontotoc Ridge, Flatwoods, North Central Plateau, and Piney Woods. The concentration of podophyllotoxin in red cedar needles across different physiographic regions varied from 0.11% to 0.36%. This variation was not statistically significant in 2007 nor in 2008, suggesting that soil nutrient concentrations and topography within Mississippi may not affect the synthesis and the accumulation of podophyllotoxin in Eastern red cedar. Podophyllotoxin accumulation seems to be a stable trait. The results demonstrated that J. viriginiana could be used as a consistent source of podophyllotoxin and that the collection of plant material for commercial extraction of podophyllotoxin can be done independent of the site of collection within the state.

Dielectric properties of a packed bed of the rhizome of P. Peltatum with an ethanol/water solution for radio frequency-assisted extraction of podophyllotoxin

The dielectric properties of a packed bed of the rhizome particles of Podophyllum peltatum and an ethanol/water solution were measured from 10 to 30 MHz using a precision LCR meter and a liquid test fixture. The effects of temperature, particle moisture content, volumetric concentration of ethanol and bed porosity on the dielectric constant, dielectric loss factor and power penetration depth were investigated. The dielectric constant of ethanol decreased with temperature but the effect of frequency was not significant. The dielectric constant of packed beds with 100% and 70% ethanol increased with the moisture content. For a packed bed with 30% ethanol, the dielectric constant was not significantly affected by particle moisture content. Frequency and temperature had no significant effect on the dielectric constant of the packed bed. The dielectric loss factor significantly increased with the particle moisture content for the beds with 100% and 70% ethanol but not with 30% ethanol. The dielectric loss factor was proportional to temperature directly and to frequency inversely. With 30% ethanol (and therefore 70% water) the dielectric loss factor of the bed dramatically increased compared to 70% and 100% ethanol. Porosity had a significant effect on the dielectric constant but not on the dielectric loss factor. The power penetration depth of a packed bed with 100% ethanol was significantly larger than that of the packed bed with 30% and 70% ethanol. Empirical regression equations were developed for simulation and design of an RF-assisted packed bed extraction of podophyllotoxin.

An optimum ethanol–water solvent system for extraction of podophyllotoxin: Experimental study, diffusivity determination and modeling

Solvent extraction of podophyllotoxin was carried out with different ethanol–water solvent systems at various extraction conditions. Maximum extraction rate and yield were obtained with a solvent system of 30 ethanol/70 water (volumetric ratio) and 53 °C for all particle moisture contents. When temperature increased from 53 to 80 °C, the extraction yield of podophyllotoxin significantly decreased due to podophyllotoxin degradation. Particle moisture content had significant effect on yield only at 100% ethanol. Also radio frequency heating had no significant effect on yield and extraction rate within 30–55 °C. A diffusional mathematical model was developed and used to determine the effective diffusivity and partition coefficient of podophyllotoxin for 14 extraction conditions. A very good agreement between predictions and observations led to reliable estimations of the effective diffusivity and partition coefficient of podophyllotoxin.

Identification of High Podophyllotoxin Producing Biotypes of Podophyllum hexandrum Royle from North-Western Himalaya

Fifty plants of Podophyllum hexandrum were collected from the interior ranges of western Himalaya of India from 8000 to 10000 ft above mean sea level for the analysis of podophyllotoxin content using HPLC. Two methods were used for the extraction of podophyllotoxin from powdered roots to standardize an extraction procedure for getting high yield of podophyllotoxin content. Extraction of podophyllotoxin was done efficiently in hot ethanol with continuous stirring. Highest podophyllotoxin content (7.84% on dry root wt basis) was obtained from P. hexandrum plants collected from Jalori Pass. In general, 24 per cent of the plant population from Jalori Pass contained higher amount of podophyllotoxin (4.0–7.84%). There was considerable reduction in the podophyllotoxin content in the roots of plants, planted at Palampur (4000 ft amsl) for one year. Embryogenic callus cultures obtained from root explants of P. hexandrum yielded 0.393% podophyllotoxin on dry wt basis.