Production of lignans in callus culture of Podophyllum

Purpose: Podophyllum hexandrum Royle, a source of highly valued podophyllotoxin has been subjected to heavy collection from the wild. The ever-increasing demand of podophyllum is mainly due to two semi synthetic derivatives of podophyllotoxin that is etoposide and teniposide, which are used in the treatment of various types of cancer. The anti cancer lignan derivative podophyllotoxin in Podophyllum hexandrum is biosynthesized at very low quantities in intact plant, so the biotechnological production of podophyllotoxin has been considered essential. Method: The aseptically germinated embryos of Podophyllum hexandrum were developed on solid nutrient agar slab. For the growth of callus culture, Murashigae and Skoog media (MS media)) with various concentrations of BAP, NAA and GA3 adjusted to pH 5.8 was used. Podophyllotoxin content in the alcoholic extract of calli and plant root was analysed by HPLC and HPTLC and was also compared with cultivated Podophllum hexandrum root extracts. Result: A fully defined MS medium supplemented with Naphthalene acetic acid and 6benzylaminopurine (BAP) were effective for both initiation and sustained growth of callus tissue. The relative proportion of callus was markedly influenced by presence of plant growth regulators. The amount of Podophyllotoxin obtained from callus was 0.78 and 0.79 percent as characterized by HPLC and HPTLC respectively. Conclusion: The study revealed that callus culture may be a fruitful tool for the production of Podophyllotoxin resin, an anticancer entity.


INTRODUCTION
Podophyllum hexandrum Royle (Berberidaceae) also known as the Indian podophyllum is a perennial herb, growing on the lower slopes of the Himalayas in scrub and forest from Afghanistan eastwards to central China 1,2,3,4 .The rhizomes of Podophyllum hexandrum are known to contain several lignans which are dimerisation product of phenylpropanoid pathway intermediates linked by central carbons of their side chain 5,6,7 .The lignans occurring in Podophyllum posses anti-tumor properties.Podophyllum being the most active cytotoxic herb contains 4.3% Podophyllotoxin on a dry weight basis.Its insecticidal and phytotoxic activities are also reported 7 .However these lignans are also toxic for the treatment of neoplastic disease in humans.Nevertheless, Podophyllotoxin is used as starting compound for the chemical synthesis of etoposide and teniposide; both being applied successfully as antitumor agents 8,9 .Their cytotoxic action is based on inhibition of topoisomerase II, while Podophyllotoxin acts as an inhibitor of the microtubule assembly.These semi synthetic analogues are indicated for small lung cell cancer, testicular cancer, neuroblastoma, hepatoma and other tumor diseases 10 .The limited availability of Podophyllum hexandrum plant due to its long juvenile phase and poor fruit setting ability as well as the time consuming collection of the plants results in shortage of Podophyllum resin.Moreover, because of the non-optimal yield after extraction, Podophyllotoxin is an expensive starting compound for the chemical synthesis of its derivatives.Therefore, the biotechnological production of Podophyllotoxin using plant cell culture derived from Podophyllum hexandrum may be an attractive alternative.

Seeds and Seedlings
Seeds were separated from pulp, washed under running tap water for 20 min dried under shade and stored at 4˚C until used.Seeds were cut with a scalpel blade, removing a section of the seed coat with two incisions around the seed hilum region, and then maintained in dark, sterile conditions on moist filter paper at 27˚C until emergence of the radical.Germinated seeds were placed on solid nutrient agar slabs (full strength MS medium, 0.8% agar, pH 5.8) in sterile culture tubes and transferred to growth room conditions, with diffuse light day/night regime (16/8 hr) as well as in the dark.

Callus initiation and growth
Aseptically germinated seed embryos were washed with double distilled sterile water and their surfaces were disinfected with aqueous solution of sodium hypochlorite for 8 min followed by four washings in sterile double distilled water under aseptic conditions so as to remove traces of sodium hypochlorite.The explants (3-4 mm) were carefully excised and transferred into MS media with various concentrations of BAP (0.5-2.5µM), NAA (0.5-5µM) and GA 3 (0.5µM-1µM).The medium was adjusted to pH 5.8.The cultures were maintained at 25±2°C in 16 hr.light and 8 hr.dark cycle and were transferred to fresh MS medium after a period of four weeks.

Lignan extraction
Obtained calli and plant root were extracted with ethanol.The lignan extract was redissolved in

HPLC Analysis
HPLC was carried out using a Column C-18 Bonda pack (250 X 4.6 mm) and solvent system such as Acetonitrile: Water: Methanol: n-  Heptane (30:40: 25: 5) was used with a flow rate of 1.2ml/min with UV detection at 280 nm.Peak areas were assessed by integrator.Retention time for authentic podophyllum lignans were as follows: standard Podophyllotoxin (6.810), root extract (6.890), and for callus extract (6.800).

HPTLC Analysis
Quantitative and qualitative analyses were carried out using the HPTLC method.Callus extracts, and plant extracts were identified using the TLC method.The sample extract was compared with standard Podophyllotoxin.HPTLC was carried out using a silica gel GF254 Precoated plate of 0.2mm thickness as a stationary phase.A Mobile Phase of Acetonitrile: Water (4: 6) was used and UV detection at 210 nm.Peak areas were assessed by integrator.
The extract made from the roots of Podophyllum hexandrum contained a mixture of potent pharmacologically active compounds.The major component was the resin, podophyllotoxin.The extract was standardized on the basis of being podophyllotoxin.

RESULTS
In the present study, experiments were carried out on the tissue culture of Podophyllum hexandrum (Berberidaceae).Tissue culture study was started from aseptic seed germination, standardization of media, callus initiation and growth study followed by extraction, and estimation of Podophyllotoxin content in the callus cultures and from cultivated roots of the plant.

Seed and Seedlings
For aseptic germination, the seeds were treated with 70% ethanol for 15 seconds followed by 3% sodium hypochlorite for 5 minutes.They were then washed several times with sterile double distilled water to remove excess of sterilants.The result showed that seed germination was within 6 days using the Petri dish method.The sprouted seedling in the immature stage showed better proliferation and growth in MS basal medium with 100 µM solution of gibberrellic acid whereas it remained stagnated if allowed to stay in the cotton bed method for a long time.

HPLC Analysis
The Qualitative tests for the identification of podophyllotoxin and the methods for their quantitative estimation were carried out by using HPLC.Root parts and callus were extracted from Podophyllum hexandrum under different conditions and these extracts were analyzed using 1 mg ml -1 sample concentration and compared with that of the standard using same concentration (100µM).Callus under different conditions contained podophyllotoxin.The HPLC data are presented in Table 2.

HPTLC Analysis
The qualitative tests for the identification of podophyllotoxin and the methods for their quantitative estimation were carried out using HPTLC.Different parts were extracted from Podophyllum hexandrum under different conditions and these extracts were analyzed using 5 mg ml -1 sample concentration and compared with that of the standard (98%) using 1mg ml -1 concentration (10µl).Callus under different conditions contained podophyllotoxin.The HPLC data are shown in Table 3.The chromatograms obtained by HPLC and HPTLC are shown in Fig. 1.

DISCUSSION
Establishment of a routine protocol for the tissue culture of Podophyllum hexandrum proved to be difficult due to erratic seed germination, problems with the sterilization of explants and the poor response of most plants to the culture regimes tested.Sterilization of explants, especially root material from soil-grown plants was unsuccessful 11,12 and germination of seeds under aseptic conditions proved the most satisfactory approach 13 .Germination was dependent on storage conditions and appropriate pre-germination treatment.A successful procedure was developed involving aseptic seed germination, dark condition at 27°C.For callus initiation, full strength MS (Murashige and Skoog) showed better results 14,15 and was therefore adopted for further studies.Root explants from seedlings derived from a single plant were tested on hormone grids.No individual auxin or cytokinin initiated callus formation at the concentrations tested, with the exception of NAA where some response was observed at 0.5 -5µM after four weeks.High concentrations of BAP (2.5µM) in the presence of NAA enhanced callus initiation.The best response, however, was observed with the addition of GA 3, which reduced callus initiation time to two weeks.Development of a friable callus was particularly associated with BAP and GA 3 and also with NAA in different concentrations.
Optimum growth response was achieved with combined GA 3 and NAA treatment, which appeared independent of GA 3 concentration in the range tested ( 0.5 -1.0µM ), but dependent on the NAA concentration ( 0.5-1.0µM).A similar high response was observed in the presence of BAP concentration (0.5-1.0µM).Higher concentrations of BAP (up to 2.5µM) were found to facilitate growth when NAA levels were low, but retarded growth when NAA levels were high.Callus growth was more effectively supported on a MS medium with full strength, rather than at half strength MS medium 16 .The effect of different growth regulators on callus culture has been summarized in Table 1.Quantitative estimation of podophyllotoxin was carried out using HPLC and qualitative estimation of podophyllotoxin was carried out using HPTLC which revealed the presence of presence of podophyllotoxin in callus culture.

CONCLUSION
Podophyllum hexandrum has been considered a rare and threatened species, large scale removal of its underground parts still continues at rates well over natural regeneration.Therefore special attention needs to be given for its propagation and conservation.The use of seeds/plant materials from different population would help to ensure the high Podophyllotoxin content by tissue culture techniques.The combined and sustained effort with seed germination, in vitro culture and estimation is required to reduce the pressure on natural population.This study was successful in standardizing the media for tissue culture, aseptic germination, callus initiation, extraction and estimation of podophyllotoxin from Callus and cultivated Podophyllum hexandrum extract.

Table 2 :
HPLC data of podophyllotoxin extracted from root and callus of Podophyllum hexandrum analytical grade methanol prior to analysis and analysis of the calli and plant root was carried out to identify and compare the Podophyllotoxin and Resin content in the callus with cultivated Podophyllum hexandrum root extracts.

Table 3 :
HPTLC data of podophyllotoxin extracted from root and callus of Podophyllum hexandrum