Sep 15, 2010 - podophyllotoxin and related aryltetrahedronnapthalene lignans with multifaceted pharmacological applications. Etoposide and teniposide, the ...
Current Research Journal of Biological Sciences 2(5): 345-351, 2010 ISSN: 2041-0778 © M axwell Scientific Organization, 2010 Submitted Date: March 10, 2010 Accepted Date: April 16, 2010
Published Date: September 15, 2010
Isolation, Characterization and Comparative Study on Podophyllotoxin and Related Glycosides of Podophyllum heaxandrum 1
Phalisteen Sultan, 1 A.S. Shawl, 2 A.A. Abdellah and 3 P.W. Ramteke 1 Indian Institute of Integrative Medicine, Formerly, R RL -(CS IR), Sanatnagar, Srinagar, 190 005 , Jammu and Kashm ir 2 Department of Biochemistry, Faculty of Medicine, AlBeida, 919- Libya 3 Departm ent of Biological Sciences, Allahab ad A gricultural Institute, Deemed U niversity, A llahab ad, 2110 07, U ttar Pradesh , India Abstract: HPLC, column and thin layer chromatography guided studies led to the isolation of seven different compounds in methanolic extracts of Podophyllum hexandrum. The isolated compounds were analyzed using L C-MS and High Performance Liquid Chromatography (HPLC ) studies interfaced to mass spectroscopy. Isolated compounds were used successfully as chemical markers for the comparison of the twelve different accessions of Podophyllum. We hav e also shown that the variation of chemical composition in P. hexandrum agree well with their botanical phylogeny as revealed by genetic phylogeny. HPLC analysis also revealed developm ent of valuab le chemoty pes containing hig her co ncen tration of isolated mark er com pounds. Key w ords: Chemical, markers, podophyllotoxin, Podophyllum, spectroscopy INTRODUCTION Podophyllotoxin is a naturally occurring lignan, which is extracted from the rhizomes o f Podophyllum peltatum and P. hexandrum (Berberidaceae) and serves as a starting compound for the prepara tion of the sem isynthetic cytostatics etoposide (VP-16-213) and teniposide (VM-26) (Clark and Slevin, 1987; Holthuis, 1988; Stähe lin and Von W artburg , 1989 ). Pod ophyllum hexandrum, a moisture and shade lov ing erect, glabrous, succulent herb thriving from K ashm ir to Sikkim in Himalayas at altitudes ranging from 2500-4000m (Fig. 1). P. hexandrum has been extensively explo ited in Ayurvedic system of medicine for treatm ent of ailments like constipation, cold, biliary fever, septic wound s, inflammation, burning sensation, mental disorder, genital warts, mon ocytoid leukemia, Hodgkin’s and non Hod gkin’s lymphoma (Singh and Shah, 1994). Extensive chemical investigation of Podophyllum species revealed presence of a number of compounds like podophyllin, podophyllotoxin, querc etin, 4-demethylpodophyllotoxin, podophyllotoxin gluco side, 4-dimethyl podophyllotoxin glucoside, kaemp ferol, picro pod oph ylotox in, deoxypod oph yllotox in, p icro p o d o p hy lotoxin , isopicropodophyllone, 4-demethyldeoxypodophyllotoxin, "-peltatin and $- peltatin (S ingh a nd Shah, 1994 ). Natural products have long been an important source of treatments for cancer. Of the natural compounds with
anticancer properties, podoph yllotoxin occu pies a very important position (Imbert, 1998). Medicinal use of Podophyllum hexandrum Royle (Himalayan Mayapple) syn. P. emodi Wall (family, Berberidaceae) a high altitude plant species native to alpine and sub-alpine regions of Himalyas dates b ack to ancient times (Singh and Shah, 1994). The plant has been de scribed as A indri-a divine drug in the Indian traditional system of medicine, the ayurveda and has also been used in traditional Chinese system of medicine (Wong et al., 2000) for the treatment of number of ailments. In the modern allopathic system of medicine, the plant has been successfully used for treatment of various disorders, monocytoid leukemia, hodgkins lympho ma, bacterial and viral infections (Gowdey and C arpen ter, 1995; Co bb, 1990), veneral warts (Beutner and Krog, 199 0), rheu matoid arthralgia associated with n umb ness of the limbs and pyogenic infection of skin tissues, AIDS assoc iated K aposis sarcoma and different cancers of brain, lung and bladder (Blasko and Cordell, 1998). Recently P. hexandrum extracts have been found to offer radioprotection by modulating free radical flux involving the role of lignans presents (Chawla et al., 2006 ). The roots and rhizomes of P. hexandrum are known to synthesize a plethora of secondary metabolites besides podophyllotoxin and related aryltetrahedronnapthalene lignans with multifaceted pharmacological applications. Etoposide and teniposide, the two semisynthetic glycoside
Corresponding Author: Phalisteen Sultan, Indian Institute of Integrative Medicine, Formerly, RRL-(CSIR), Sanatnagar, Srinagar, 190005, Jammu and Kashmir 345
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010
Fig. 1: Medicinal herb Podophyllum hexandrum in IIIM gene bank derivatives form an integral part of the therapeu tic regimen used for che motherapy and have triggered further studies in the design and the synthesis of other potent anticancer com pounds (C anel et al., 2000; Iseel, 1982; Van et al., 1988). An analytical method for estimation and characterization of the chemical constituents of this high value medicinal plant is mandatory. Methods for identification of aryltetrahydronapthalene and related chemical marker lignans from P. hexandrum have rarely been reported. Difficulty in obtaining the reference standards is probably major reason for identification of minor constituents. It is noted that aryltetrahydronapthalene lignans occurring in nature are all built around a common basic skelton and may show under appropriate condition fragmentation pathway ame nable to straightforward structural interpretation. It is thus worth exploring the possibility of identifying these constituents using their mass spec trome tric data with the aim of registering a chem otaxo nom ic profile, w hich could be diagn ostic value to herbs. The prese nt work reports the use of LC-MS and HPL C for the identification of important lignans from P. hexandrum.
deionized by a Milli-Q purification system with a 0.2 m fiber filter (B arnstead, C A, U SA ). Collection and identification of plan t ma terial: Podophyllum plants growing in their natural habitat of Himalayan Mountains were collected and transplanted under partia l shade a t thre e diffe re nt gene banks of IIIM, Srinagar at Bonera, Yarikha and Srinagar. The plant material was identified by Department of Plant Taxonomy, Kashmir University, Srinagar, India. The voucher specimens of all the collected sam ples were deposited at the H erbariu m of IIIM , Srinag ar, India. Preparation of herbal extracts: The rhizomes samples were taken from all the accessions grown at three different location s viz. ge ne ba nk, IIIM -Srinagar, field Station Bonera (Pulwama) and Yarikha (Gulmarg)- J and K, India for preparation of phytoextacts. The dried plant material was grounded to a fine powder and stored at 4ºC. A known quantity of grounded sample was weighed and subjected to sequential hot extraction using 100% methanol. Contents were squeezed through muslin cloth, and the filtrate from aqueous extract was filtered using W hatman No.1 filter paper. The extraction process was repeated three times (4 to 6 h). Organic solven ts were clarified by centrifuga tion and then conc entrated to dryness under reduced pressure. The known residue of the extract was dissolved in HPLC prepa ratory tubes w ith methanol (Dw ivedi et al., 1997).
MATERIALS AND METHODS Ch em icals and reagents used: The major marker compounds as described above were isolated in the Natural Product Chem istry section of IIIM, Srinagar in the year 2004 by rou tine chromatography techniques. Identity and purity was confirmed by chromatographic (TLC, HPLC) and spectral (IR, 1D- and 2D-NMR) methods (Bastos et al., 1995). Solvents (water and methanol,) were of HPLC grade and purchased from Ranbaxy Fine Chemicals Limited (Okhla, New Delhi, India). The structures were confirmed by their UV, MS, 1 H NMR and 1 3 C NM R data compared with the authe ntic data from literature. Acetonitrile of HPLC grade (Aldrich, USA) and M illex syringe filter unit were purchased from Reagent, New D elhi, India. Water for preparation of samples and HPLC A D analysis was
Extraction and isolation of compoun ds: The dried and pulverized roots of Podophyllum hexandrum (120 g) were extracted with MeOH (300 mL) in a soxhlet over water bath for 6 h. The extract was filtered and solvent was removed in Rotavapor at 50ºC. The concentrated extract was redissolved in HPLC grade methanol and volume was adjusted to 1ml each. Injection volume 5uL and column temperature 30ºC, flow rate 1m l/ minu te and wavelen gth was set at 283 nm . The extracted portions were combined and con centrated by evaporation under reduced pressure
346
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010
Fig. 2: HPLC chromatograms showing peaks of major marker compounds to give a crude extract (18.5 g), which w as dissolved in MeOH (100 m l).
Analytical HPLC cond itions: The chem ical analysis was done on ThermoF innigan HPLC machine equipp ed w ith auto sampler, column apartment and U V detector. Acquiring and analysis of data was controlled by Shemstation software (Agilent Tech, USA). A RP-18 column (0.3x150 mm) from E. merck was employed at 30ºC column temperature. Separation was done in the isocratic mode using methanol and water (60:40) at a flow rate of 0.8 ml/min with injection volume of 5 :L, UV detection was set at 290nm. Prior to use, solvents w ere filtered through a 0 .22 mm diameter m embrane filters. Equal volum e of the standard solution was mixed and injected in the HPLC system in volumes of 2, 4, 6, 8 and 10 :L for plotting calibration curves. Solutions w ere injected in triplicate and the calibration curve s were constructed by plotting value for concentration of each analyte. Satisfactory separation was obtained as shown in the chrom atograms (Fig. 2).
HPLC analysis: Podophyllotoxin and its glycosides were identified by HPLC based on the comparison of retention time and UV spectrum with the reference compound. HPLC analysis was performed on a ThermoFinnigan HPLC mac hine w ith pum p syste m eq uippe d with a 966-photodiode-array detector, with the detection wavelen gth set at 283 nm. Satisfactory separation was obtained with reverse phase column utilizing a E. Merck RP-18 column (250×4 mm, 5 :m) with a diode array detector (SPDM-10 A VP/RF-10 AXL fluorescent detector) and auto-injector STL-10 AD VP. Elution was done with the mo bile phase (M eoH , H 2 O; 60:40) for 30 min. at a flow rate of 0.8 ml/min. A standardized mixture of two marker compounds with known concentration of podophyllotoxin and podophyllotoxin $-D glycoside were used to create calibration curves (percentage area with respect to the quantity of the pure compounds). Both the marker com pounds exhibited eno ugh differences in th eir retention times, which made their quantification easier. LC-UV (DAD) chromatogram of the samples showed the presence of two m arkers has been observed. The data was statistically analysed for significant results.
RESULTS AND DISCUSSION The plant material was extracted with solvent system methanol. Colum followed by thin layer chromatography lead to the isolation of seven marker compounds. The isolated compounds were labelled as pH-1, pH-2, pH-3,
347
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010
Podophyllotoxin 1-O--$ -D-glycoside
Quercetin 3-O-$ -D-glycoside
PPT, 1-O--$ -D-glycoside
Fig. 3: Various compounds isolated from phytoextracts of Podophyllum hexandum
Fig. 4: Fragmentation pathway of podophyllotoxin 1-0-$ -D glycoside pH-4, pH-5, pH -6 and pH-7. After LC -MS analysis, the identified compounds were found to be podophyllotoxin, 4 Demethyl podo- phyllotoxin, podophyllotoxin 1-0-"-D glycoside, podophyllotoxin 1-0-$-D glycoside and quercetin 3-0-$-D glycoside (Fig. 3) In the LC separation
it was found that gradient of methanol and water was the optimal mobile phase. Different marker compounds were identified by the HPLC analysis in all the collected accessions. Considerable variation in the chemical composition was
348
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010 Table 1: H PLC analysis of different sam ples of Podophyllum hexandrum Accession code R1 R2 R3 R4 PH 18-S 2.17 2.37 2.18 1.98 PH 18-S 1.91 2.09 2.06 1.95 PH 18-S 1.36 2.25 3.15 2.15 PH 18-S 2.35 4.00 2.96 3.44 PH 18-S 1.76 2.59 2.61 2.33 PH 18-S 1.66 2.96 2.03 3.41 PG r-S 2.06 1.99 1.89 2.23 PG r-S 2.06 2.11 2.17 2.03 PG r-S 2.21 1.19 2.72 1.76 PG r-S 2.89 2.21 2.32 2.97 PG r-S 2.40 2.78 2.39 1.99 PG r-S 2.61 2.47 2.29 2.00 PS-S 3.96 4.83 3.86 4.21 PS-S 4.79 5.96 5.02 4.02 PS-S 3.03 4.91 3.99 4.38 PS-S 5.31 3.42 4.23 5.65 PS-S 3.00 4.84 4.36 3.76 PS-S 4.29 4.33 3.98 4.49 Pp-B 2.20 2.68 2.93 2.88 Pp-B 3.19 4.11 3.17 3.51 Pp-B 2.52 3.72 2.55 3.47 Pp-B 3.09 3.24 4.11 2.45 Pp-B 2.79 3.09 3.11 4.21 Pp-B 3.12 4.09 3.78 2.68 PG -S 4.98 5.23 3.91 5.36 PG -S 4.12 5.13 4.76 5.78 PG -S 6.91 6.87 7.59 7.32 PG -S 5.21 4.91 6.12 5.03 PG -S 5.12 6.09 4.24 3.91 PG -S 5.75 6.29 6.33 4.92 PS-B 3.20 3.06 1.93 3.24 PS-B 4.98 2.07 3.84 2.95 PS-B 3.00 2.41 3.92 4.91 PS-B 3.55 5.92 3.28 5.84 PS-B 3.40 3.93 4.11 3.32 PS-B 3.55 3.41 3.92 3.65 PG -B 3.01 2.95 3.12 3.31 PG -B 2.33 3.93 3.01 3.97 PG -B 2.90 3.01 3.46 3.23 PG -B 3.06 2.23 4.22 3.07 PG -B 2.37 2.81 3.41 3.18 PG -B 3.53 4.02 1.26 4.51 PW -S 1.91 1.71 2.58 2.82 PW -S 2.21 1.47 1.63 2.06 PW -S 2.56 2.18 1.87 2.34 PW -S 1.94 3.09 2.41 1.88 PW -S 2.11 2.09 2.91 2.78 PW -S 3.32 2.19 4.49 2.98 PV -S 1.81 1.92 2.09 2.07 PV -S 1.49 1.85 2.18 2.11 PV -S 2.02 2.81 3.09 3.17 PV -S 1.91 2.53 2.71 3.02 PV -S 2.41 2.81 3.12 2.01 PV -S 2.02 1.82 1.94 3.29 PY -S 1.57 1.84 2.09 2.26 PY -S 3.11 2.19 1.77 3.94 PY -S 1.12 0.98 2.13 2.53 PY -S 2.80 1.19 1.98 3.30 PY -S 2.41 3.03 2.71 1.71 PY -S 3.09 3.09 3.31 2.69 PY -B 1.91 2.38 4.11 3.68 PY -B 2.08 1.83 2.08 1.93 PY -B 1.55 2.60 2.59 1.93 PY -B 2.48 2.92 2.44 2.57 PY -B 2.64 2.79 2.37 2.24 PY -B 2.77 2.62 1.94 2.42 PSH -B 6.11 5.91 5.12 5.77 PSH -B 5.12 3.68 4.41 4.09 PSH -B 6.24 5.92 6.48 6.56 PSH -B 6.31 5.81 6.12 6.25 PSH -B 6.19 6.41 7.09 6.52 PSH -B 7.03 7.28 7.41 7.12
R5 2.20 1.89 3.58 1.95 2.26 2.35 1.68 1.98 2.07 2.06 2.19 1.98 2.78 5.16 3.04 3.19 5.84 4.36 3.21 3.32 2.59 3.31 3.85 4.30 4.07 4.06 6.93 5.18 5.31 5.11 2.37 3.41 3.21 4.41 4.19 3.22 2.16 3.01 4.04 2.22 2.98 3.33 1.13 2.38 1.30 2.28 2.36 2.62 2.01 2.12 2.66 1.28 2.90 1.85 2.14 3.24 1.99 2.68 2.39 2.42 2.83 1.58 1.58 1.84 3.21 1.85 5.09 3.25 6.29 5.86 5.94 7.81
R1 4.19 7.49 6.12 5.21 5.91 5.72 4.50 2.09 2.64 2.87 2.81 4.25 2.25 2.06 3.18 1.91 3.37 1.87 6.68 5.91 2.53 4.61 5.49 4.92 7.09 5.41 7.11 6.49 4.12 4.92 1.15 2.00 5.89 2.08 4.22 6.3 3.24 2.54 1.36 0.79 4.21 3.91 0.69 2.79 1.10 1.99 2.80 1.29 0.86 0.69 2.41 2.44 2.40 3.12 3.29 2.40 1.08 0.96 2.11 3.43 2.95 1.96 2.92 1.17 0.94 1.68 2.10 1.82 3.03 1.58 2.61 0.81
R2 5.33 6.33 7.21 5.51 7.12 4.68 3.10 3.02 2.19 2.47 4.70 4.61 2.41 1.95 2.12 3.18 1.94 2.61 5.36 3.33 5.44 4.84 5.25 5.33 6.83 6.03 4.91 5.12 5.33 5.31 3.90 2.51 5.09 3.70 5.21 3.50 3.61 2.21 2.41 3.47 2.94 2.68 1.47 0.92 2.07 1.22 1.98 1.02 2.65 2.68 1.34 3.32 2.18 2.09 2.18 3.10 2.33 2.96 0.99 3.28 3.13 1.59 1.41 1.24 0.41 1.84 1.19 1.94 2.46 0.91 2.12 0.94
R3 5.56 5.34 6.03 4.98 5.24 5.82 3.02 2.51 3.10 3.25 1.99 5.03 1.87 2.07 1.91 1.86 2.09 0.78 4.12 5.09 5.45 5.41 4.48 5.22 5.47 5.68 4.71 5.41 6.37 4.97 3.21 2.41 3.23 4.28 3.06 5.60 2.94 2.81 2.91 2.68 2.90 1.36 1.90 0.91 1.85 0.95 2.11 1.68 2.68 2.33 1.01 1.22 2.03 1.39 1.89 2.58 1.85 1.29 4.02 2.12 2.37 0.21 0.92 1.87 3.21 1.25 1.47 2.37 2.81 1.03 1.98 1.61
R4 6.30 6.53 5.26 6.11 5.79 3.99 4.10 1.84 1.86 3.51 3.92 2.98 2.03 2.35 2.95 2.74 2.56 1.31 3.18 4.41 4.99 5.23 4.60 4.27 4.99 5.91 5.16 6.19 5.98 5.71 2.78 2.91 5.41 5.52 2.09 4.20 1.91 2.78 2.36 4.09 3.68 4.91 2.93 -1.66 4.01 3.39 1.16 2.41 1.42 0.92 0.50 1.86 1.61 2.11 1.43 2.71 2.53 3.68 1.81 3.74 1.22 3.46 1.11 2.91 0.92 0.91 2.94 3.47 2.59 1.88 1.23
R5 6.23 5.21 5.29 5.59 6.14 5.09 3.78 2.44 2.70 5.05 2.28 3.23 1.99 1.47 2.74 2.36 1.74 0.83 6.13 3.86 4.84 4.91 6.23 5.21 6.33 4.92 6.11 7.39 5.50 5.81 1.96 1.68 4.88 4.22 2.97 3.86 3.52 3.01 3.31 4.22 1.28 3.49 2.61 2.93 2.43 3.23 4.22 -2.00 2.23 0.90 1.12 0.92 2.04 3.68 2.09 0.93 1.86 2.45 1.86 1.91 0.92 0.84 2.16 2.68 1.46 2.78 2.03 1.68 2.49 1.86 0.96
Podophyllum hexandrum samples obtained w ere a na lyze d. It w as found that the patte rns of their LC-MS
recorded in the analysed accessions as shown in Table 1. Using the experimental conditions reported above the 349
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010 Table 2: L ignans identified using LC-M S profiling of Podophyllum hexandrum S a mp le na m e Retention time Percentage area of (major peaks) major peak Stand ard P odo phyllo toxin 39.848 90.99 Standard (4 Demethyl podophyllotoxin) 21.623 100 .0 PH -1 4 - D eme thyl po dop hylloto xin 21.634 80.75 PH-2 podophyllotoxin $-D-glycoside 25.882 100.00 PH-3 podophyllotoxin " - D-glycoside 32.380 86.00
Mass (m/z) of major peaks 423 .1,43 7,39 7.1,3 13.1 423 ,383 .1, 28 8.9,1 85.1 423 ,383 .2, 24 6.5 599 .2,39 7.1,3 13.1 599.2,397.1,313
PH-4 Quercetin 3-0-$-D-glycoside PH-5 un identified
34.128 32.803
94.14 58.00
PH-6 un identified
26.23
PH-7 podophyllotoxin "-D-glycoside
39.82
R e te nt io n ti me (minor peak) 30.495
Mass (m/z) of minor peaks 313.1,246
15.14
585 .2 413 .0 383 .0 217 .2
504 .2,32 5.1,2 99.1 599 .2,39 7.1,2 29.0
39.756
71.11
599 .2,39 7.1,3 13.0
32.67
599.2, 437, 397 .4 247 .0 599 .2 397.1, 365 .1, 21 7.0
98.00
437,397.2,313.0,247 184 .9
reconstructed chromatograms were more or less similar except for som e variation in the relative intensity of peaks. By studying, the fragmentation pattern as revealed in corresponding LC-MS spectras, a number of podophyllotoxin and related lignan marker compounds were identified in various extracts (Fig. 4). The major fragment ions observed in the mass spectra are summarised in Table 2. The compound identification was possible on the basis of the different fragmentation pathways (Issel et al., 1998; Rahman et al., 1995 ; Clark and Slevin, 1987). To ensure that observed (M+Na) ion fragm ents of glycosylated lignans was indeed the same as observed in previous spectras. Due to the high sensitivity of instrument, this method was particularly advantageous for the sam ples of limited quantity. Usin g this methodology, detailed structural information was obtained for lignans, lignan glycosides and other second ary metabolites. HPLC analysis prov ides a re producible retention time using stand ardised conditions for development of lignan database. UV spectra collected on line provided evidence for general classification and sub structures of each comp ound.
previous reports for isolation and spectral identification of iridoids from the studied species. Due to diversity of species and h abitats, the results may be much more complex. Therefore, it seems that more attention should be paid to the scrutiny of various toxic ingredients rather than the determination of a few kinds of compounds or only one with high sensitivity. This conclusion is also supported by the following data from the herbal preparations. The HPLC technique appeared particularly useful for screening the UV absorbing glycosides in P. heaxandrum lead to the detection of these compounds. Detailed metabolite p rofiles for selected com pounds in these P. heaxandrum samples demo nstrated that the chemical compositions of these accessions was infact quantitatively different because these plants were not grown under identical conditions at the same time or in the sam e location. ACKNOWLEDGMENT Autho rs are highly thankful to Dire ctor, IIIM, Srinagar for financial support of the project. Autho rs are highly thankful to Miss Safeera of GLC section for computer help in drawing chemical structures. Also like to thank to the contribution of Maxwell Scientific Organization for providing extensive su pport financially to publish it online.
DISCUSSION The prime objective of the stud y was to inv estigate the chem otaxo nom ic studies of different P. heaxandrum extracts. W ith this distribution p attern, popdophyllotoxin and its glycosides are potential taxonomic markers although they apparently. Pod ophyllum has been exten sively investigated due to its use in Ayurvedic and Chinese medicine. Roots of P. heaxandrum have given a number of glycosides. Plant samples from different regions were analyzed and the occurrence of the glyco sidic compounds was confirmed by a combined set of criteria including retention time, mass spectra of both positive and negative mode. All major peaks that could be recognized as glycosides w ere taken into consideration. The results were summarized and compared with the
REFERENCES Bastos, J.K., C.L. Burandt, N.P. Nanayakkara, L. Bryant and J.D. M cChesn ey, 19 95. Q uantitation of aryltetralin lignans in plan t parts and among different populations of Podophyllum peltatum by reverse phase high performance liquid chromatography. J. Nat. Prod., 59: 406-408. Beutner, K.R . and V . Krog, 1990. Current status of podophyllotoxin for the treatment of warts. Semin. Dermatol., 9: 148.
350
Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010 Blasko, G. and G.A. Cordell, 1998. Recent Develop men ts in Chemistry of Plant Derived Anticancer Agents. In: W agner, H., H. Hiroshi and N.R. Fransw orth (Eds.), Economics a nd M edicinal Plant Research. Academics Press, London. Canel, C., R.M. Moraes, F. Dayan and D. Ferreria, 2000. Podophyllotoxin, Phytochemistry, 54: 115-120. Chawla, R., R. Arora, S. Singh, A.S. Shawl, P. Sultan, T. Krishan and G.N. Q azi, 2006. Podophyllum hexandrum offers radioprotection by modulating free radical flux: role of Aryl-Tetralin lignans. eCAM, 3(4): 50 3-511. Clark, P.J. and M.L. Slevin, 1987. The clinical pharmac ology of etoposide and teniposide. Clin. Pharmacokinet., 12: 223-252. Cobb, M.W ., 1990. Human Pap iloma Virus infection. J. Am. Acad. Dermatol., 22: 547. Dw ivedi, A.K., D. Kulkarni and S. Singh, 1997. Sensitive high-performance liquid chromatographic assay method for the determination of picroside I in plasma. J. Chromatogr. B, 698: 317-320. Gowdey, G., R.K. Lee and W.M. Carpenter, 1995. Traetise of HIV - related hairy Leuoplak ia with Podophyllum resin 25% solution. Oral. Pathol. Oral Radiol. Endocrinol., 79: 64.
Holthuis, J.M., 1988. Etoposide and teniposide. B i o a n a l y s i s , m e t a b o l is m a n d c l i n i c a l pharmacokinetics Pharm. Weekbl. Sci. Ed., 10: 101-116. Imbert, F., 1998. Discovery of podophyllotoxins. Biochimie., 80: 207-222. Iseel, B.F., 1982 . Podophyllotoxin derivatives VP16-213 and VM 26. Cancer Chemother. Pharmacol., 7: 72. Issel, B.F., F.H. Muggia and S.K. C arter, 1998. Etoposide (VP-16), Current Status and New Developm ents, Academic Press, Orland. Rahman, M., M.I. Ashraf, H. Choudhary and M.H. Kazmi, 1995. Antifunga l aryltetralin lignans from le a ves of Podophyllum hexandrum . Phytochemistry, 40: 427- 431. Singh, J. and N.C . Shah , 1994 . Podophyllum: A review. Curr. Res. Med. Arom. Plants, 16: 53-83. Stähelin, H.F. and A. Von Wartburg, 1989. From podophyllotoxin gluco side to etoposide. Progr. Drug Res., 33: 169-266. Van Mannen, J., J. Retel, J.D. Vries and H.M. Pinedo, 1988. Mechanism of action of antitumor drug etoposise: A review. J. Natrl. Cancer. Inst., 80: 1526. W ong, S.K., S.K. Tsui, S.Y. Kwan, X.L. Su and R.C. Lin, 2000. Identification and characterization of P. emodi by API-L C/MS/M S. J. Mass Spectrum, 35: 1246-1251.
351
