267
J. Crop Sci. Biotech. 2009 (December) 12 (4) : 267 ~ 274 DOI No. 10.1007/s12892-009-0066-z RESEARCH ARTICLE
Germplasm Conservation and Quantitative Estimation of Podophyllotoxin and Related Glycosides of Podophyllum hexandrum Phalisteen Sultan1*, AS Shawl1, Arif Jan1, Hamid B2, Irshad H1 1 2
Division of Biotechnology, Indian Institute of Integrative Medicine,(CSIR) - Srinagar - 190005, J&K, India Jawaharlal Nehru Cancer Hospital and Research Center, Bhopal, India - 462001
Received: May 5, 2009 / Revised: December 5, 2009 / Accepted: December 15, 2009 Ⓒ Korean Society of Crop Science and Springer 2009
Abstract We have done comparative morphological and chemotaxonomic studies to investigate the phylogenetic relationship of different accessions within the Podophyllum species. HPLC profiles revealed that all Podophyllum hexandrum accessions collected from different geographical locations are chemically highly diverse. Also, we have observed that chemotaxonomic studies clearly demonstrated that chemical characters of the Podophyllum hexandrum are suitable to generate essentially the same relationship as revealed by RAPD analysis. Key words: accessions, quantitative, glycoside, morphological
Introduction Medicinal use of Podophyllum hexandrum Royle syn. P. emodi Wall (Himalayan Mayapple; family, Berberidaceae), a high altitude plant species is native to alpine and sub-alpine regions of Himalyas. Podophyllum hexandrum Royle grows wild in the interior Himalayan ranges of India. Rhizomes contain antitumor lignans such as podophyllotoxin and podophyllotoxin β-D glycoside (Tyler et al.1988). Among the lignans, podophyllotoxin is most important for its use in the semisynthesis of anticancer drugs etoposide and teniposide. Recently P. hexandrum extracts have been found to offer radioprotection by modulating free radical flux involving the role of lignans present. As podophyllotoxin and its glycoside are highly toxic compounds, the use of Podophyllum hexandrum as a herbal medicine is potentially hazardous and needs to be carefully controlled. An analytical method for estimation and characterization of the chemical constituents of this high value medicinal plant is mandatory. A number of plant products have been evaluated for Dr. Phalisteen Sultan ( ) Email:
[email protected] Tel: +91- 99-0652-4590
The Korean Society of Crop Science
protection against a lethal dose of radiation including Podophyllum hexandrum (Goel et al.1998). Pre-radiation administration of the extracts of Podophyllum hexandrum mitigated radiation induced postnatal and physiological alterations (Goel et al. 2002). Traditionally, dried rhizomes of the plant are mixed with liquid and taken as a laxative or to get rid of intestinal worms as a powerful purgative. Podophyllotoxin is the major lignan present in the resin and is a dimerized product of the intermediates of the phenylpropanoid pathway (Panda et al.1989). The Indian Podophyllum hexandrum is superior to its American counterpart, Podophyllum peltatum, in terms of its higher podophyllotoxin content (5%) in dried roots in comparison to only 0.25% of Podophyllum peltatum (Panda et al. 1992). The podophyllotoxin derivatives etoposide, etopophos (etoposide phosphate), and teniposide are successfully utilized in the treatment of a variety of malignant conditions. Also, it is used as a precursor for the chemical synthesis of anticancer drug etoposide, teniposide and etopophos (Farkya et al. 2004). These compounds have been used for the treatment of lung and testicular cancers as well as certain leukemias (Imbert 1998 ; Stahelin and
268
Podophyllotoxin and Related Glycosides of Podophyllum hexandrum
Wartburg 1991). The patent for the use of etoposide in such therapies expired in 1995 and since then, etoposide has been tested in 167 clinical trials for the use as new investigative cancer treatments or as positive control (Ajani et al. 1999; Ekstrom et al. 1998; Holm et al. 1998). In addition, podophyllotoxin is also the precursor to a new derivative CPH 82 that is being tested for rheumatoid arthritis in Europe and other derivatives for the treatment of psoriasis and malaria (Leander and Rosen 1988; Lerndal and Svensson 2000). Podophyllotoxin preparations are also in the market for dermatological use to treat genital warts (Beutner 1996) and recently, immune-stimulatory activities of podophyllotoxin have been reported (Pugh et al. 2001). Total synthesis of podophyllotoxin is an expensive process and availability of the compound from natural resources is an important issue for pharmaceutical companies that manufacture these drugs (Canel et al. 2000). Pre-irradiation administration of the extract of Podophyllum hexandrum -mitigated radiation induced postnatal physiological alterations. Bioactive molecules of mayapple (Podophyllum spp.) are of interest to the pharmaceutical industry as cheapest source of podophyllotoxin. Radiation protection is provided by extracts of Podophyllum hexandrum through expression of various cytoprotective proteins that have been evidenced by immunoblotting (Raj et al. 2005). Although radiotherapy is an effective treatment for management of diseases, radiation morbidity does develop in certain patients with normal radiosensitivity (Nair et al. 2004). The traditional medicinal uses of Podophyllum hexandrum are for the treatment of colds, constipation, septic, wounds, burning sensation, erysipelas, mental disorders, rheumatism, plague, allergic and inflammatory conditions of the skin, cancer of the brain, bladder and lung, venereal waste, Hodgkin's disease and non-Hodgkins lymphoma (Beutner and Von 1990; Blasko and Codeu 1998; Singh and Shah 1994). This study was conducted with the main objective to determine quantitative evaluation of the major marker compounds like podophyllotoxin and its glycosides and to evaluate phytochemical diversity in Podophyllum hexandrum populations collected from highly diverse geographical zones. Since the germplasm of this prioritized medicinal plant has been exploited from natural habitat, a conservation protocol using micropropagation studies is highly useful.
Materials and Methods Chemicals and reagents used All the standard compounds used in the present investigation were isolated in the Natural Product Chemistry section by routine 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, acetonitrile, and methanol) were of HPLC grade and purchased from Ranbaxy Fine Chemicals Limited (Okhla, New Delhi). Ultra-pure distilled water was used. All organic solvents used in the study were of analytical grade.
JCSB 2009 (December) 12 (4) : 267 ~ 274
Isolation of podophyllotoxin and its related glycosides Dried plant material (1 kg) was extracted with 100% methanol in a soxhlet for 6 h at room temperature. The extract was stripped of methanol and the aqueous left behind was successively extracted with chloroform and benzene-methanol. The extract was crystallized with benzene-methanol to give a mixture consisting of podophyylotoxin and podophyllotoxin β-D glycoside. The light brown-colored residue obtained after solvent evaporation was dissolved in methanol (400 ml) and filtered. The filtrate refluxed repeatedly with neutral alumina (1050 g). The methanol extract on removal of solvent gives a solid which is re-crystallized by ethyl acetate to give a white amorphous solid (9.5 g). The solid was subjected to column chromatography using silica gel and the fraction that got eluted with hexane: EtOA (2:8) gave podophyllotoxin β-D- glycoside. These compounds were then characterized on the basis of their 1HNMR, 13C-NMR, 13C- DEPT (45 and 135o) and MS spectral data. All compounds were identified by 1H-, and 13C-NMR and spectroscopic data (Bergeron et al. 2000).
diode array detector (SPD M-10 A VP/RF-10 AXL fluorescent detector) and auto-injector STL-10 AD VP. Elution was done with the mobile phase [MeOH & H2O; 60: 40] for 30 min. at a flow rate of 0.8 ml/min. A standardized mixture of two marker compounds with known concentrations podophyllotoxin and podophyllotoxin β-D glycoside was used to create calibration curves (percentage area with respect to the quantity of the pure compounds. Using the above conditions, good separations were achieved. Both the marker compounds exhibited enough differences in their retention times, which made their quantification easier. Fig.1 shows the LC-UV (DAD) chromatogram of the sample where the presence of both the two markers has been observed.
Tissue culture studies through embryogenesis
Preparation of phytoextracts The rhizomes and leaf samples of Podophyllum hexandrum were collected from all the accessions cultivated at three different locations viz. gene bank, RRL-Srinagar, field Station Bonera (Pulwama) and Yarikha (Gulmarg) J&K (Table 1).The rhizome samples were thoroughly washed under running tape water to free the plant material of soil, dust, and extrinsic contamination. The samples after proper washing were air dried in a hot oven at 30 °C overnight. The dried samples were powdered and subjected to methanolic extraction in a soxhlet by hot extraction (two washes for 3 h each). The solvent was removed and concentrated under vacuum using rotavapor at 50 °C. Removal of the solvent from the extract under reduced pressure gave a semi-solid gum which was resuspended in HPLC grade methanol for analysis. The lignans were separated and quantified by HPLC (Canel et al. 2001). Statistical analysis of data was carried out using SAS, 1999 software. Table 1. Location, altitude, accession code for various collected populations of Podophyllum hexandrum. Accession Accession Location Altitude (ft) Location Altitude (ft) code code Gurez Veerinag-15 Gulmarg Phalagam W. Khrew Haftnar
PGr-A PV15-A PG-A PP-A PW-A PHh-A
12,000 9,000 13,000 10,500 6,000 9,500
Keller Khilanmarg Veerinag-16 Yousmarg Sonamarg Himachal
PKs-A PK-A PV16-A PY-A PS-A PH-A
10,500 11,500 7,500 9,500 10,000 12,000
High-performance liquid chromatography system HPLC analysis was performed on a Thermofinnignan HPLC machine with pump system equipped with a 966 photodiode array detector with the detection wavelength set at 240 nm. Satisfactory separation was obtained with reverse phase column utilizing E. Merck RP-18 column (250×4 mm, 5 µm) with a
taneously under similar HPLC conditions for better results. The marker compounds were identified by their respective retention time. Samples were analyzed and processed for determining quantitative evaluation of marker compounds with respect to their retention time. The percentage of marker compounds in all the samples was determined. This analysis was done twice in a year. The data was statistically analyzed for significant results. We multiplied peak area by the appropriate response factor to give ㎍/ml extract then divided by the original concentration of extracted dried root/ml sample and multiplied by 106 to reach g/gm dry wt. of the sample and determined the percentage. The concentration of these marker compounds in the extract solution was determined exactly. The mass spectra of all peaks were examined and compared with literature data or reference compounds mentioned in the section.
Fig. 1. HPLC separation of lignans extracted from P. hexandrum (A) Chromatograms of standard marker compounds showing two major peaks (B) Sample extract of Podophyllum hexandrum.
Preparation of recovery studies The follow-up extractions and HPLC analysis were accomplished in the same manner as detailed above. The recovery was determined as follows. Recovery (%) = (A-B)/C×100. where, A is the amount of detections above, B is the amount of sample without added standards, C is the added amount of the standards. To evaluate the accuracy of the method the recovery test was used. Equal amounts of standard solutions were added to P. hexandrum samples and extracted and analyzed.
Quantitative estimation of podophyllotoxin and its glycosides The standard solutions were run with sample extract preparations filtered through fine mesh. All the samples were run simul-
In vitro propagated plantlets of Podophyllum hexandrum were produced from excised zygotic embryos of surface disinfected seeds with a solution of mercuric chloride (0.1% w/v for 3 min.) followed by 5 min. washing in sterile distilled water under aseptic conditions to remove any traces of mercuric chloride. The seeds were imbibed for 72 h in double distilled water prior to the inoculation of zygotic embryos. The zygotic embryos were carefully excised with a sharp aseptic needle and excised embryos were then transferred to a tissue culture medium composed of Murashige and Skoog (Murashige and Skoog, 1962) containing 3% sucrose and activated charcoal (0.5-1%), 0.8% agar and various combinations of growth regulators (cytokinin: BA, 0.5-4.0 mg/1), auxins; IAA, 1.0-4.0 mg/1, NAA, 1.0-3.0 mg/1. The pH of the medium was adjusted to 5.8. The medium was dispensed in 100 ml glass vessels which were steam sterilized at 121˚C for 20 min. The experiment was repeated more than once and 10 flasks were used for each treatment. The culture vessels were incubated 25 ± 2 ˚C in 16 h light with cool fluorescent tubes (Philips TI 40/54). The cultures were transferred to fresh medium after a five-seven week interval.
Hardening of rooted plants The rooted plants of Podophyllum hexandrum were washed in running tap water and finally transferred to jiffy pots containing sand, soil and vermiculite in 1:1:1 ratio. Before transferring to soil, plants were exposed to higher humidity and gradually reduced i.e from 90-65% during first 20 days. The potted plants were finally taken to grow under ex vitro conditions.
Results and Discussion The mean levels of major marker compounds in representative samples from 12 different Podophyllum populations presented show significant quantitative variation (ANOVA, p< 0.05) between mean levels of all the major compounds across all the 12 populations. In order to investigate potential variation in concentration of major marker compounds in Podophyllum
269
268
Podophyllotoxin and Related Glycosides of Podophyllum hexandrum
Wartburg 1991). The patent for the use of etoposide in such therapies expired in 1995 and since then, etoposide has been tested in 167 clinical trials for the use as new investigative cancer treatments or as positive control (Ajani et al. 1999; Ekstrom et al. 1998; Holm et al. 1998). In addition, podophyllotoxin is also the precursor to a new derivative CPH 82 that is being tested for rheumatoid arthritis in Europe and other derivatives for the treatment of psoriasis and malaria (Leander and Rosen 1988; Lerndal and Svensson 2000). Podophyllotoxin preparations are also in the market for dermatological use to treat genital warts (Beutner 1996) and recently, immune-stimulatory activities of podophyllotoxin have been reported (Pugh et al. 2001). Total synthesis of podophyllotoxin is an expensive process and availability of the compound from natural resources is an important issue for pharmaceutical companies that manufacture these drugs (Canel et al. 2000). Pre-irradiation administration of the extract of Podophyllum hexandrum -mitigated radiation induced postnatal physiological alterations. Bioactive molecules of mayapple (Podophyllum spp.) are of interest to the pharmaceutical industry as cheapest source of podophyllotoxin. Radiation protection is provided by extracts of Podophyllum hexandrum through expression of various cytoprotective proteins that have been evidenced by immunoblotting (Raj et al. 2005). Although radiotherapy is an effective treatment for management of diseases, radiation morbidity does develop in certain patients with normal radiosensitivity (Nair et al. 2004). The traditional medicinal uses of Podophyllum hexandrum are for the treatment of colds, constipation, septic, wounds, burning sensation, erysipelas, mental disorders, rheumatism, plague, allergic and inflammatory conditions of the skin, cancer of the brain, bladder and lung, venereal waste, Hodgkin's disease and non-Hodgkins lymphoma (Beutner and Von 1990; Blasko and Codeu 1998; Singh and Shah 1994). This study was conducted with the main objective to determine quantitative evaluation of the major marker compounds like podophyllotoxin and its glycosides and to evaluate phytochemical diversity in Podophyllum hexandrum populations collected from highly diverse geographical zones. Since the germplasm of this prioritized medicinal plant has been exploited from natural habitat, a conservation protocol using micropropagation studies is highly useful.
Materials and Methods Chemicals and reagents used All the standard compounds used in the present investigation were isolated in the Natural Product Chemistry section by routine 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, acetonitrile, and methanol) were of HPLC grade and purchased from Ranbaxy Fine Chemicals Limited (Okhla, New Delhi). Ultra-pure distilled water was used. All organic solvents used in the study were of analytical grade.
JCSB 2009 (December) 12 (4) : 267 ~ 274
Isolation of podophyllotoxin and its related glycosides Dried plant material (1 kg) was extracted with 100% methanol in a soxhlet for 6 h at room temperature. The extract was stripped of methanol and the aqueous left behind was successively extracted with chloroform and benzene-methanol. The extract was crystallized with benzene-methanol to give a mixture consisting of podophyylotoxin and podophyllotoxin β-D glycoside. The light brown-colored residue obtained after solvent evaporation was dissolved in methanol (400 ml) and filtered. The filtrate refluxed repeatedly with neutral alumina (1050 g). The methanol extract on removal of solvent gives a solid which is re-crystallized by ethyl acetate to give a white amorphous solid (9.5 g). The solid was subjected to column chromatography using silica gel and the fraction that got eluted with hexane: EtOA (2:8) gave podophyllotoxin β-D- glycoside. These compounds were then characterized on the basis of their 1HNMR, 13C-NMR, 13C- DEPT (45 and 135o) and MS spectral data. All compounds were identified by 1H-, and 13C-NMR and spectroscopic data (Bergeron et al. 2000).
diode array detector (SPD M-10 A VP/RF-10 AXL fluorescent detector) and auto-injector STL-10 AD VP. Elution was done with the mobile phase [MeOH & H2O; 60: 40] for 30 min. at a flow rate of 0.8 ml/min. A standardized mixture of two marker compounds with known concentrations podophyllotoxin and podophyllotoxin β-D glycoside was used to create calibration curves (percentage area with respect to the quantity of the pure compounds. Using the above conditions, good separations were achieved. Both the marker compounds exhibited enough differences in their retention times, which made their quantification easier. Fig.1 shows the LC-UV (DAD) chromatogram of the sample where the presence of both the two markers has been observed.
Tissue culture studies through embryogenesis
Preparation of phytoextracts The rhizomes and leaf samples of Podophyllum hexandrum were collected from all the accessions cultivated at three different locations viz. gene bank, RRL-Srinagar, field Station Bonera (Pulwama) and Yarikha (Gulmarg) J&K (Table 1).The rhizome samples were thoroughly washed under running tape water to free the plant material of soil, dust, and extrinsic contamination. The samples after proper washing were air dried in a hot oven at 30 °C overnight. The dried samples were powdered and subjected to methanolic extraction in a soxhlet by hot extraction (two washes for 3 h each). The solvent was removed and concentrated under vacuum using rotavapor at 50 °C. Removal of the solvent from the extract under reduced pressure gave a semi-solid gum which was resuspended in HPLC grade methanol for analysis. The lignans were separated and quantified by HPLC (Canel et al. 2001). Statistical analysis of data was carried out using SAS, 1999 software. Table 1. Location, altitude, accession code for various collected populations of Podophyllum hexandrum. Accession Accession Location Altitude (ft) Location Altitude (ft) code code Gurez Veerinag-15 Gulmarg Phalagam W. Khrew Haftnar
PGr-A PV15-A PG-A PP-A PW-A PHh-A
12,000 9,000 13,000 10,500 6,000 9,500
Keller Khilanmarg Veerinag-16 Yousmarg Sonamarg Himachal
PKs-A PK-A PV16-A PY-A PS-A PH-A
10,500 11,500 7,500 9,500 10,000 12,000
High-performance liquid chromatography system HPLC analysis was performed on a Thermofinnignan HPLC machine with pump system equipped with a 966 photodiode array detector with the detection wavelength set at 240 nm. Satisfactory separation was obtained with reverse phase column utilizing E. Merck RP-18 column (250×4 mm, 5 µm) with a
taneously under similar HPLC conditions for better results. The marker compounds were identified by their respective retention time. Samples were analyzed and processed for determining quantitative evaluation of marker compounds with respect to their retention time. The percentage of marker compounds in all the samples was determined. This analysis was done twice in a year. The data was statistically analyzed for significant results. We multiplied peak area by the appropriate response factor to give ㎍/ml extract then divided by the original concentration of extracted dried root/ml sample and multiplied by 106 to reach g/gm dry wt. of the sample and determined the percentage. The concentration of these marker compounds in the extract solution was determined exactly. The mass spectra of all peaks were examined and compared with literature data or reference compounds mentioned in the section.
Fig. 1. HPLC separation of lignans extracted from P. hexandrum (A) Chromatograms of standard marker compounds showing two major peaks (B) Sample extract of Podophyllum hexandrum.
Preparation of recovery studies The follow-up extractions and HPLC analysis were accomplished in the same manner as detailed above. The recovery was determined as follows. Recovery (%) = (A-B)/C×100. where, A is the amount of detections above, B is the amount of sample without added standards, C is the added amount of the standards. To evaluate the accuracy of the method the recovery test was used. Equal amounts of standard solutions were added to P. hexandrum samples and extracted and analyzed.
Quantitative estimation of podophyllotoxin and its glycosides The standard solutions were run with sample extract preparations filtered through fine mesh. All the samples were run simul-
In vitro propagated plantlets of Podophyllum hexandrum were produced from excised zygotic embryos of surface disinfected seeds with a solution of mercuric chloride (0.1% w/v for 3 min.) followed by 5 min. washing in sterile distilled water under aseptic conditions to remove any traces of mercuric chloride. The seeds were imbibed for 72 h in double distilled water prior to the inoculation of zygotic embryos. The zygotic embryos were carefully excised with a sharp aseptic needle and excised embryos were then transferred to a tissue culture medium composed of Murashige and Skoog (Murashige and Skoog, 1962) containing 3% sucrose and activated charcoal (0.5-1%), 0.8% agar and various combinations of growth regulators (cytokinin: BA, 0.5-4.0 mg/1), auxins; IAA, 1.0-4.0 mg/1, NAA, 1.0-3.0 mg/1. The pH of the medium was adjusted to 5.8. The medium was dispensed in 100 ml glass vessels which were steam sterilized at 121˚C for 20 min. The experiment was repeated more than once and 10 flasks were used for each treatment. The culture vessels were incubated 25 ± 2 ˚C in 16 h light with cool fluorescent tubes (Philips TI 40/54). The cultures were transferred to fresh medium after a five-seven week interval.
Hardening of rooted plants The rooted plants of Podophyllum hexandrum were washed in running tap water and finally transferred to jiffy pots containing sand, soil and vermiculite in 1:1:1 ratio. Before transferring to soil, plants were exposed to higher humidity and gradually reduced i.e from 90-65% during first 20 days. The potted plants were finally taken to grow under ex vitro conditions.
Results and Discussion The mean levels of major marker compounds in representative samples from 12 different Podophyllum populations presented show significant quantitative variation (ANOVA, p< 0.05) between mean levels of all the major compounds across all the 12 populations. In order to investigate potential variation in concentration of major marker compounds in Podophyllum
269
270
Podophyllotoxin and Related Glycosides of Podophyllum hexandrum
hexandrum, a detailed chemoprofiling was done based on marker compounds. The highest content of podophyllotoxin was found in the accession PKs-A, location, Keller, Shopian, while the highest content of podophyllotoxin β-D-glycoside was found in accession PK-A, Location, Khilanmarg, Baramulla. There were significant differences in the active content of samples collected from different ecozones (Table 2), which suggests that environmental and genetic factors may play role in determining the active compound contents. In chemical analysis of Podophyllum hexandrum, calibration curves were derived from five independent injections of two major marker compounds i.e PPT and PPT-β-D-glycoside in the concentration of 2, 4, 6, 8 and 10 ㎕. The content of lignans, in Podophyllum hexandrum samples, Table 2. Detailed HPLC analysis data of different Podophyllum populations collected from diverse geographical locations. Year of Mean Podophyllotoxin Mean Location Podophyllotoxin ±SD -β-D-gylcoside ±SD Analysis Phalgam Verinag-15 Khrew Khilanmarg Yousmarg Sonamarg Gurez Himachal Haftnar Gulmarg Veeronag-16 Keller
2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006
2.56 2.29 2.74 2.51 2.25 1.81 4.29 4.62 3.98 5.52 4.53 6.19 2.94 3.34 3.42 3.24 3.55 4.01 2.95 3.10 3.33 2.01 2.35 2.61 2.60 2.20 2.09 2.09 2.00 3.10 1.97 2.20 2.67 4.60 5.98 7.33
2.53±0.18 2.19±0.28 4.29±0.26 5.41±0.68 3.23±0.20 3.60±0.31 3.12±0.15 2.32±0.24 2.29±0.21 2.39±0.49 2.28±0.29 5.97±1.11
5.74 6.18 5.20 3.70 2.79 3.10 2.14 2.37 1.96 6.15 5.68 5.34 5.09 4.68 4.96 2.33 3.93 4.69 2.47 3.04 3.27 1.92 1.78 2.03 2.12 1.30 2.05 2.63 1.78 2.50 2.82 1.18 1.43 1.69 2.96 7.33
2.53±0.40 3.19±0.37 2.15±0.16 5.72±0.33 4.91±0.17 3.65±0.98 2.92±0.33
JCSB 2009 (December) 12 (4) : 267 ~ 274
metabolite compositions could be attributed to environmental factors. Also, it appears that genetic variation must be responsible for differential accumulation of these major bioactive principles. Variation in expression or activity of genes/enzymes involved in the production of these bioactive molecules can explain these differences. Further more, the present investigation supports the contention that Podophyllum from different sources is infact likely to possess different properties in potential bioactivity and health benefits. Identification of genes, involved in regulating the production of these compounds is a major goal of future research in this area. To assess the precision of the method, we injected standard solution of PPT, PPT-β-D-glycoside on the same day of analysis. The precision as well as reproducibility of the method was satisfactory. The results of the recoveries of PPT, PPT-β-D-glycoside ranged from 97.6 to 102.3%. The relative standard deviations (R.S.D) of recoveries of these two constituents ranged between 1.35 and 2.64% (Table 3). The peaks of the sample solution were identified by comparison of the retention time with their corresponding authentic samples. Regarding the extraction efficiency, three times work up was sufficient since it allowed an over 95% extraction of these two marker compounds. The content of the two marker constituents in rhizomes and leaves of Podophyllum hexandrum growing in different locations was also analyzed demonstrating that all of these phytochemicals in the target plant are interestingly dependent of the locality. Detailed morphological studies revealed the presence of variTable 3. Recovery of two major marker compounds, podophyllotoxin and pod. β-D glycoside. Standard Spiked (mg) Found (mg) Recovery % Recovery % (R.S.D) podophyllotoxin
1.91±0.10 1.82±0.37
podophyllotoxin β-D-glycoside
0.0038 0.0046 0.1771 0.0680 0.807 0.0462
0.0080 0.0086 0.3944 0.1333 0.1027 0.1423
105.2 102.9 98.8 98.2 96.2 99.5
102.3 (2.64)
97.96 (1.35)
Table 4. Morphological characteristics of Podophyllum hexandrum variants collected from different locations. Characters Plant height Leaf size Length Across Stem size Length Diameter Girth Petiole size Length Diameter Girth Fruit size Length Diameter Girth Leaf lobe size Length Breadth Herbage/ Rhizome Herbage/hac
Keller
Pahalgam Yousmarg Sonamarg
Khrew
Himachal Verinag - 16
Haftnar
Gulmarg
Verinag-15
Gurez
Khilanmarg
34 cm
32cm
30cm
26cm
31cm
28cm
30cm
32cm
36cm
35cm
32cm
30cm
25 cm
24 cm
24.5 cm
17 cm
16cm
24cm
27cm
28cm
21cm
26cm
27cm
25cm
20 cm
21 cm
20 cm
16 cm
16cm
17cm
20cm
21cm
20cm
19cm
23cm
29cm
18cm
15.7 cm
16.7cm
10 cm
21cm
16.4 cm
18 cm
23 cm
12 cm
17 cm
18 cm
19 cm
0.5cm
0.4 cm
0.4 cm
0.4 cm
0.3 cm
0.4 cm
0.4 cm
0.5 cm
0.4 cm
0.5 cm
0.4 cm
0.5 cm
1.7cm
1.5 cm
1.6 cm
1.6 cm
1.7 cm
1.7 cm
1.6 cm
1.5 cm
1.4 cm
1.6 cm
1.5 cm
1.5 cm
10.5cm
10.5cm
13 cm
11 cm
13 cm
14 cm
10.5 cm
11.5 cm
13.5 cm
12.6 cm
11 cm
12.5 cm
0.3 cm
0.4 cm
0.4 cm
0.4 cm
0.3 cm
0.4 cm
0.4 cm
0.5 cm
0.3 cm
0.4 cm
0.4 cm
0.4 cm
1.4 cm
1.5 cm
1.6 cm
1.4 cm
1.6 cm
1.4 cm
1.5 cm
1.5 cm
1.5 cm
1.4 cm
1.5 cm
1.6 cm
6 cm
3.9 cm
0.5 cm
4 cm
4.5 cm
4.9 cm
6.1 cm
3.9 cm
4.8 cm
5.0 cm
5.4 cm
6.0 cm
3 cm
2.5 cm
2.7 cm
2.5 cm
2.4 cm
2.3 cm
2.7 cm
2.3 cm
2.4 cm
2.6 cm
2.4 cm
2.1 cm
6 cm
5.9 cm
5 cm
4.7 cm
4.5 cm
6.0 cm
5.3 cm
5.2 cm
5.1 cm
6.3 cm
4.9 cm
5.4 cm
13 cm
11 cm
9 cm
7.5 cm
11 cm
14 cm
13 cm
11 cm
9 cm
6.9 cm
7.2 cm
8.2 cm
7 cm
9 cm
8 cm
7cm
7 cm
6 cm
9 cm
10 cm
4 cm
5.5 cm
4.9 cm
1.5 cm
55g
40g
47g
30g
36.55 g
39.33 g
36.15 g
41.3 g
42.1 g
44.25 g
32.3 g
41.3 g
22g
16g
18.8 g
12g
14.6 g
15.72 g
14.46 g
16.52 g
16.84
17.7 g
12.92 g
16.52 g
6105kg
4440 kg
5217 kg
333 kg
4061 kg
4379 kg
4016 kg
4588 kg
467 kg
4588 kg
3588kg
4588 kg
2500 kg
1760 kg
2087 kg
1333 kg
1624 kg
1748 kg
1606 kg
1835 kg
1868 kg
1952 kg
1435 kg
1835 kg
Table 5. Effect of different hormonal combinations on seed germination of Podophyllum hexandrum seeds. Chemical Treatment Time taken Condition conc. Mean mg/l in germination (Days) germination%age ± SD Dark
Light Dark Light Dark Dark
GA3+BA (1.0 each) GA3+BA (2.0 each) GA3+BA (4.0 mg/l) GA3+BA (1.0 each) GA3+BA (2.0 each) GA3+BA (4.0 mg/l) Conc. Sulphuric acid Conc. Sulphuric acid GA3+BA(4.0)-unscarified GA3+BA(4.0)-scarified
50-60 35-45 20-30 60-70 60-70 25-35 25-35 25-35 65-75 40-50
20.33± 3.78 25.33± 5.86 64.66± 7.05 07.66± 1.52 10.33± 2.70 15.66± 4.00 15.33± 6.02 50.33± 2.08 11.33± 1.13 50.00± 3.57
mg/l = milligram per litre , SD = standard deviation
2.30±0.37 1.81±0.72 1.92±0.77
as summarized in Table 2, were determined by HPLC for 12 accessions that were collected from different geographical locations. The total podophyllotoxin content varied from 2.28 to 5.97% on dry wt. basis and PPT-β-D-glycoside ranging from 1.81 to 5.72%. The observed differences were significant for most of the comparisons, as determined by ANOVA analysis (p
