In Vitro.–Enhanced Production of Podophyllotoxin in Phytohormonal-Induced and Regenerated Roots of Podophyllum hexandrum.

ABSTRACT

Sixty-day-old in vitro–induced roots and roots regenerated from totipotent calli of Podophyllum hexandrum Royle produced an enhanced quantity of podophyllotoxin. Induced root cultures originated from aseptically germinated seedlings, and regenerated roots were established on both Gamborg's B5 and Murashige and Skoog media. The podophyllotoxin content was compared with normal plant root/rhizomes by HPLC. The highest values, both in growth/proliferation and in podophyllotoxin production, were obtained using Gamborg's B5 medium.

Keywords:

• Berberidaceae
• lignan
• podophyllotoxin
• Podophyllum hexandrum. Royle
• root culture

Introduction

Recent advances in the chemistry and bioassay of plant products have led to the identification of many cytotoxic compounds that could be used in the future chemotherapy of cancer. Among them are a number of lignans isolated from genus Podophyllum..

Podophyllum hexandrum. Royle (Berberidaceae) growing in the inner ranges of Himalayas (from Kashmir to Sikkim at an altitude of 2700–4200 m), is a threatened species that produces abundant quantities of lignans in the root/rhizomes (Chatterji, 1952; Wealth of India, 1969; Jain et al., 1980). Lignans display a variety of biological activities, of which the anticancer activity is currently of greatest interest (Hartwell et al., 1958). The phenylpropanoid derived lignan podophyllotoxin, occurring in Podophyllum. species, is used as a starting material/lead compound for the semisynthesis of the more water-soluble antitumor agents like etoposide (VP-16-213) and teniposide (VM-26), which have FDA approval in the United States. These are important commercial products in the United States, Europe, and Japan (Emmenger et al., 1961; Juslen et al., 1971). Podophyllotoxin itself is particularly used against certain viral diseases and skin cancers (Kadkade, 1982). Total synthesis of etoposide and teniposide is unlikely to be commercially achievable in the foreseeable future. A continued supply of podophyllotoxin from natural sources must be assured (Sharma et al., 1988), because podophyllotoxin is biosynthesized at very low quantities in the intact plant, it becomes increasingly rare (Kadkade, 1981). Indian Podophyllum. (Podophyllum hexandrum.) contains three-times more resin and podophyllotoxin than the American species, Podophyllum peltatum., which additionally contains α.- and β.-peltatins. The peltatins do not contribute to the anticancer properties of the plant. In order to find an alternative source, suspension cultures of Podophyllum hexandrum. have been established (Woerdenbag, 1990; Van Uden et al., 19891990). Herein we report the in vitro. production of podophyllotoxin in induced roots and regenerated root cultures.

Materials and Methods

Plant material

P. hexandrum. seeds were procured from plants growing in Pahelgam (Jammu and Kashmir), India. The Department of Botany, Hamdard University, New Delhi, India, identified the plant material and voucher specimens (P'cog. and Phyto./FOP/HB Sp. No. 131 and 132) have been deposited in the herbarium of the Pharmacognosy and Phytochemistry Department, Faculty of Pharmacy, Jamia Hamdard (Hamdard University).

Determination of moisture content of different seed samples

Moisture content of seeds was determined by drying seeds to a constant weight in a hot-air oven below 60°C for 4 h.

Seed viability and vigor studies

Methods used to assess viability and vigor

Triphenyltetrazolium chloride staining method (TTC or tetrazolium salt) (Dixon et al., 1994)

Seeds were soaked in distilled water for 1 week and were split longitudinally into equal halves with the help of a scalpel. One half of each seed was kept in 1% aqueous solution of 2,3,5-triphenyltetrazolium chloride for 5–6 h. Then the seeds were washed with distilled water, and number of half-seeds stained red (viable) was recorded for calculation of percentage viability.

Germination without surface sterilization

The seeds were washed properly under running tap water, dipped into sulfuric acid (5%, 10%, 25%, 50%, and 100%, successively) for 2 min each and further washed with double-distilled water. The washed seeds were transferred into Petri dishes containing absorbent cotton covered by a filter paper. Aqueous gibberellic acid solution (1% or 2%) was poured over the paper, and the dish was covered with aluminum foil and kept in seed germinator (Hicon Instruments, Grover Enterprises, New Delhi, India) under dark at 25±2°C. The percentage seed viability was calculated. Seed vigor was determined: where a., b., c., and d. represent the number of seeds which germinated after 1, 2, 3, and 4 days, respectively, and S. represents the total number of seeds to be germinated.

Aseptic germination of seeds

Seeds were washed with tap water and subjected to concentrated sulfuric acid treatment as above followed by washing with double-distilled water. The seeds were surface sterilized by dipping into ethanol (70%) for 1 min followed by immersion for 15 min in silver nitrate (0.5%) solution and transferred into Petri dishes containing absorbent cotton pads covered with a filter paper. Gibberellic acid solution (1%) was poured onto the filter paper and the dish covered with aluminum foil. Germination was carried out as above.

Induction of rooting

Five-day-old excised roots from the germinated seedlings were cultured on Gamborg's B5 (Gamborg et al., 1968) or Murashige & Skoog (1962) (US) media supplemented with various combinations and concentrations of plant growth regulators as given in Table 1. The cultures were kept under controlled conditions of day/night regime (16 h/8 h; 1600 lux) and temperature (25±2°C).

Table 1.. Root induction in Gamborg's B5 or Murashige and Skoog's (MS) media supplemented with various plant growth regulators.

Sample no.	Hormonal combinations in Gamborg's or MS media	Observations (B5 medium)	Observation (MS medium)
1	NAA (2 mg/l)	+	+
2	IAA (2 mg/l)	+ +	+
3	IBA (2 mg/l)	+	+
4	IAA (2 mg/l) + NAA (2 mg/l)	+ + +	+ +
5	IAA (2 mg/l) + NAA (5 mg/l)	+ +	+ +
6	IAA (0.003 mg/l) + NAA (0.003 mg/l)	+ +	+
7	IAA (1 mg/l) + NAA (2 mg/l)	+	+
8	IBA (2 mg/l) + NAA (2 mg/l)	+ +	+ +
9	NAA (2 mg/l) + 2,4-D (5 mg/l)	+	−
10	IAA (2 mg/l) + KIN (2 mg/l)	+	−
11	2,4-D (5 mg/l) + KIN (2 mg/l)	−	−
12	IAA (1 mg/l) + NAA (1 mg/l) + 2,4-D (2 mg/l)	+	−
13	IAA (1 mg/l) + NAA (1 mg/l) + 2,4-D (5 mg/l)	−	−
14	IAA (1 mg/l) + NAA (1 mg/l) + 2,4-D (3 mg/l)	+	−
15	IBA (2 mg/l) + NAA (2 mg/l) + IAA (2 mg/l)	+	+

+++, excellent root induction; ++, comparatively good root induction; +, normal root induction; −, no root induction.

Regeneration of roots from developed totipotent calli

The callus initiated from roots of aseptically germinated seedlings grown in Gamborg's B5 medium supplemented with 2,4-D (2 mg/l) + KIN (0.5 mg/l). Callus was developed in Gamborg's B5 + NAA (2 mg/l) + KIN (1 mg/l) + 2,4-D (1 mg/l). For regeneration studies, the calli of the forth passage (approximately 3 months old) were aseptically cultured on MS and Gamborg's B5 media at pH (5.8 and 5.5, respectively) as given in Table 2.

Table 2.. Effects of plant growth regulators on root regeneration.

Sample no.	Hormonal combinations in Gamborg's and Murashige and Skoog's (MS) media	Observations (B5 medium)
1	Gamborg's B5 + IAA (2 mg/l)	+
2	Gamborg's B5 + IBA (2 mg/l) + IAA (1 mg/l)	+ +
3	Gamborg's B5 + IAA (2 mg/l) + NAA (2 mg/l)	+ + +
4	Gamborg's B5 + IBA (2 mg/l) + NAA (2 mg/l)	+ +
5	Gamborg's B5 + IAA (3 mg/l) + NAA (2 mg/l)	+ +
6	Gamborg's B5 + 6-BA (1 mg/l) + IAA (1 mg/l)	−
7	MS + IAA(2 mg/l) + NAA (2 mg/l)	+ +
8	MS + BA (2 mg/l) + NAA (2 mg/l)	+
9	MS + IAA (3 mg/l) + NAA (2 mg/l)	+
10	MS + 6-BA (1 mg/l) + NAA (1 mg/l)	−

+++, excellent root induction; ++, comparatively good root induction; +, normal root induction; −, no root induction.

Extraction of resin

A weighed quantity (10 g) of 5-month-old freeze-dried cultures and natural plant root/rhizome were powdered and extracted with ethanol (70% v/v) in a Soxhlet apparatus for 6 h. The ethanol extracts were filtered and concentrated on a water bath to syrupy liquid. The concentrated extracts were poured into ice-cold acidified water (1 ml concentrated hydrochloric acid in 50 ml of water) and continuously shaken for complete separation of resin from culture extracts. The resin obtained was filtered through Whatman filter paper and further washed with ice-cooled acidified water. The percentage of resin was calculated for different samples.

Isolation of podophyllotoxin by preparative thin-layer chromatography

The samples of different extracts and authentic podophyllotoxin were applied to TLC plates (wet thickness: 250 µm) and eluted with CHCl₃:MeOH (9:1). Reference podophyllotoxin on the TLC plate was sprayed with sulfuric acid:MeOH (1:1) solution, followed by heating at 110°C for 10 min, whereas samples of plant material were not sprayed. The podophyllotoxin produced dark blue-blackish blue spots. The spots of different test samples having the same R_f value (i.e., 0.55) were scraped off and the silica gel shaken vigorously with 10 ml of MeOH and filtered through Whatman filter paper. The filtrate was kept in a Petri dish overnight. Podophyllotoxin, in amorphous form, appeared after 24 h.

Characterization of podophyllotoxin

The podophyllotoxin obtained was white in color and characterized further by melting point (m.p.), solubility, UV, IR, ¹H NMR, ¹³C NMR, and mass spectral analysis.

Quantitative estimation of podophyllotoxin by HPLC (Cairnes et al., 1981)

Dilutions (20 µl) of podophyllotoxin (supplied by Indo-World Trading Corporation, New Delhi, India) in acetone were injected into a Lichrosorb RP-18 column (25 × 4.6 cm id) protected by a C₁₈-corasil precolumn. Development with acetonitrile:water (40:60 v/v) at 1 ml/min gave a scale of peak areas when monitored at 254 nm and standard curve was drawn.

Results and Discussion

Moisture content of the seeds was found to be 2.4%. The TTC assay and germination studies showed that all the seed halves were stained red and germinated within 24 h (Fig. 1). Both seed viability and vigor were found to be 100% from data recorded in TTC assay and germination studies. The concentrated sulfuric acid and gibberellic acid (1%) treatments helped in breaking the hard seed coat and seed dormancy, respectively. Further, it was also found that gibberellic acid treatment supported the callus initiation and decreased the callus initiation time to 2 weeks. Without gibberellic acid, callus initiation time was 3–4 weeks. In root induction studies, after 45 days, excised mother root explants after swelling showed vigorous proliferation of lateral roots. The induction of rooting was observed in excised root explants cultured on Gamborg's B5 medium containing auxins and cytokinins. Gamborg's B5 medium supplemented with IAA (2 mg/l) + NAA (2 mg/l) was found to be the most suitable combination for proliferation of roots and formation of lateral roots (up to 3–4 inches long) (Figs. 2 and 3). Other suitable combinations were:

• Gamborg's B5 medium + IAA (2 mg/l) + NAA (5 mg/l) (Fig. 4)

• Gamborg's B5 medium + IBA (2 mg/l) + NAA (2 mg/l) (Fig. 5)

• Gamborg's B5 medium + IAA (2 mg/l) (Fig. 6)

Figure 1 Four-day-old aseptically germinated seedling of Podophyllum hexandrum. Royle.

Figure 2 Induction of rooting after 30 days in Gamborg's B5 medium supplemented with IAA (2 mg/l) + NAA (2 mg/l).

Figure 3 Induction of rooting after 30 days in Gamborg's B5 medium supplemented with IAA (2 mg/l) + NAA (2 mg/l).

Figure 4 Induction of rooting after 30 days in Gamborg's B5 medium supplemented with IAA (2 mg/l) + NAA (5 mg/l).

Figure 5 Induction of rooting after 30 days in Gamborg's B5 medium supplemented with IBA (2 mg/l) + NAA (2 mg/l).

Figure 6 Induction of rooting after 30 days in Gamborg's B5 medium supplemented with IBA (2 mg/l) only.

The effects of different plant growth regulators on callus initiation were observed after 4 weeks of incubation. Optimal initiation of callus was obtained on roots in Gamborg's B5 as well as in Murashige and Skoog's medium supplemented with 2,4-D (2 mg/l) + KIN (0.5 mg/l) (Fig. 7).

Figure 7 Root explant showing initiation of callus after 15 days in Gamborg's B5 medium supplemented with 2,4-D (2 mg/l) + KIN (0.5 mg/l).

The Gamborg's B5 medium was found to be more suitable than MS medium for regeneration of roots from the totipotent calli. Media combinations found suitable for induction and proliferation of roots also induced the same effect in totipotent calli. The most favorable media for regeneration of roots (up to 6–10 cm in length) is the same as that for root proliferation listed above (Fig. 8).

Figure 8 Regeneration of roots from developed calli after 45 days in Gamborg's B5 medium supplemented with IAA (2 mg/l) + NAA (2 mg/l).

The resin content (percentage of dry weight) in induced roots and regenerated root cultures were found to be 18.73% and 16.68% while it was 17.54% in intact plants. The crystals of podophyllotoxin obtained were white with m.p. 183–184°C. It was found easily soluble in hot EtOH, Et₂O, CHCl₃, HAc, Me₂CO, and hot C₆H₆; sparingly soluble in cold EtOH, cold C₆H₆, and CCl₄; and almost insoluble in H₂O and light petrol. The λ_max of the sample (Hitachi UV-Vis spectrophotometer, Japan) was found to be 252 nm (abs EtOH) (Hartwell & Schrecker, 1953).

IR ν_max (KBr): 3450 (OH), 2950, 2850 (CH₃, CH₂), 1750 (C=O), 1600 (C=C), 1480 (CH₂, CH₃), 1240 (C–O, phenolic), 1050 (C–O, alcoholic), 938, 890 (C=C, aromatic), 750 cm⁻¹.

H¹ NMR (CDCl₃, 400 MHz): δ 2.81–2.77 (2H, d, J. = 14 Hz, H-6a), 3.80–3.74 (9H, bs, 3 × OMe), 4.06–4.01 (2H, t, J. = 8Hz, H-3a), 4.57–4.54 (1H, m, H-3), 4.74–4.72 (iH, bs, OH) 6.37–6.32 (1H, d, J. = 20Hz, H-1), 6.49–6.45 (1H, d, J. = 16Hz, H-4), 7.11 (2H, s, H-5, H-8), 7.18 (1H, d, J. = 8Hz, H-2′), 7.25–7.27 (1H, d, J. = 8Hz, H-6′).

C¹³ NMR (H₂O-D₂O, 100 MHz): δ 44.13 (C-3a), 45.34 (C-6a), 56.30 (C-3), 60.79 (C-2), 71.45 (C-1), 72.68 (C-4), 76.76, 77.08, 77.40 (3 × OMe), 101.45 (H-2′), 106.38 (C-6′), 109.7 (C-3′), 129.06 (C-5′), 130.53 (C-4′), 131.12 (C-8), 133.32 (C-5), 135.57 (C-6), 137.22 (C-7), 146.48 (C-9), 152.59 (C-10), 174.65 (C=O).

MS (70 ev): m./z. 414 (M, C₂₂H₂₂O₈), 397 (M-H₂O), 398 (M-16), 351 (M-2 × OMe), 400 (M-28), 247 (M-C₉H₁₁O₃), 229 (247-OH), 202 (229-C O), 149 (M-C₁₄H₅O₂, base peak), 135, 120, 111, 97, 83, 71, 57.

HPLC analysis of different culture extracts of Podophyllum hexandrum. showed the presence of podophyllotoxin. The podophyllotoxin content of induced roots and regenerated root cultures were found to be 11.625% and 10.5% of dry weight of the cultures while in plant drug it was 9.3% of the dry weight of plant root/rhizome.

Acknowledgments

Thanks are due to Dr. Bahar Ahmad and Mr. Qaiser Naeem Khan for their assistance in interpretation of spectral data and reprographical work, respectively. We are grateful to the Department of Biotechnology, Ministry of Science and Technology, for financial support. We feel deeply obliged and indebted to Mr. S.K. Katyal, Managing Director of Indo-World Trading Corporation, for providing a pure sample of podophyllotoxin.