Electronic Journal of Biology

Keywords

P.hexandrum seed; Dormancy mechanism; Hydroquinone; Bioactivity identification; Auxin.

Abbreviations

GC-MS - Gas chromatograph mass spectrometer; MS – Murashige and Skooge medium; IAA - Indole-3-acetic acid; GA3 - Gibberellic acid; NAA - Naphthaleneacetic acid; BAP - Benzyladenine; RL-Radical Length; SL=Seedling Length; RFW - Radical fresh weight; SFW - Seedling fresh weight; RDW - Radical dry weight; SDW - Seedling dry weight.

1. Introduction

P.hexandrum is a perennial herb,which grows in the Himalayan region and western of China [1,2]. Podophyllotoxin is a natural product mainly existed in the rhizome and roots of P.hexandrum and as well as its congeners and derivatives has pronounced biological activity mainly as anticancer,antineoplastic and anti-HIV drugs [3-5]. As the market demand of podophyllotoxin is increasing and the natural resource has been facing endangered [6,7]. In addition,the chemical synthesis of podophyllotoxin is very complicated and rather difficult [8,9]. So podophyllotoxin will ultimately depend upon the supply of raw materials. Both of cultivation and biotechnological are two mainly means for production of podophyllotoxin [10,11].

However,plants grown from rhizome cutting of P.hexandrum were estimated to take at least to produce rhizome in fair sizes and plants raised from seedling would take even longer [12]. In addition,the seed dormancy period is very long and the germination percentage is unhealthy [12,13]. In our early research the seed dormancy could be broken in 500mg/L GA3 solution 36h following cold stratification 90d with germination percentage 81.11% [13]. Although the dormancy has been broken,how to hasten the growth and development of P.hexandrum is a key to meet the demand of podophyllotoxin.

The main purpose of this study was to separate the ethyl ether extraction from seed of P.hexandrum by GC-MS in order to find the materials and methods that could overcome the difficult of slow growing and hasten the growth and development of P.hexandrum.

2. Materials and Methods

2.1 Seed collection

P.hexandrum seeds were collected from the forest (2100m) located in Huichuan,Weiyuan area,Gansu province,China,1000-seed weight 20.65g.

2.2 Extraction by ethyl ether

Seeds weighted 3.0g were grinded to powder,first the power was packed and soaked by ethyl ether at 4° C,12 h and then extracted by apparatus,Soxhlet's at 40° C,24h. At last the extraction was condensed to 3mL for preparation.

2.3 Separation of ethyl ether extraction

Instrument: Ammerican Finnigan Company Trace DSQ GC-MS. Condition of GC: 1N NOWAX quartz capillary column with 30m×0.32cm×0.25mm; column temperature 50~190° C; procedure temperature 5/min° C; carrier gas He; vaporizer temperature 280° C. Condition of MS: Ion source was EI and temperature was 200° C; ionizing voltage 70eV; electric current of collection 300μA; electric current of emission 1mA; resolution 600; mass range 10~500.

2.4 Bioactivity identification of Hydroquinone on wheat

The wheat seeds were soaked in tap water for 1h,then rinsed in 70% ethanol for 10s and five times in sterile water and subsequently immersed in 0.1% HgCl₂ for 5min. After rinsing them again in sterile water five times they were inoculated on the basic medium (MS) supplemented with 3.0% sucrose and 0.5% agar with different concentrations of Hydroquinone,IAA,NAA,BAP and GA3,the pH of the medium was adjusted to 5.8 and the temperature 25±1° C with 24 h natural light. After 4 days note and measure the wheat RL,SL,RFW,SFW,RDW and SDW. Each treatment inoculated three wheat seeds and repeats three times at the same condition. The data and results were analyzed by SPSS11.5.

2.5 Bioactivity of Hydroquinone on vitro culture of seed embryo of P.hexandrum

The seeds of P.hexandrum were firstly cleaned and soaked in tap water 6h and then rinsed in 70% ethanol for 10s and then five times in sterile water,subsequently immersed in 0.1% HgCl₂ for 20min. Then soaked in sterile 400mg/L GA3 for 36h to break the seed dormancy and make the embryo developed so as to separate them easily [13],then the seeds were rinsed as before. At last the seeds embryo were separated and inoculated on MS medium supplemented with 2.0% sucrose,0.4% agar with different growth regulators in the following concentrations[mg/L]: M1: 0.5 IAA; M2: 0.5 GA3; M3: 0.5 Hydroquinone. The pH of the mediums was adjusted to 5.8 with the temperature 22±1° C and 24 h lights (1000LX).

3. Results and Discussion

3.1 The separation and identification results of ethyl ether extraction

Nineteen compounds were separated and identified from the extraction of ethyl ether by GC-MS (Figure 1) after the treatments of extraction from P.hexandrum seed and there were seven kinds fatty acid,three kinds paraffin hydrocarbon,three kinds alcohol aldehyde,etc existed in the seed,majority of which had been proved to have inhibition bioactives to all of plants seed germination,that including (Z,Z)-9,12-Octadecadienoic acid,Hexadecanoic acid,Oleic acid and 1-Phenanthrenemethanol [14],which relative content in P.hexandrum seed reached 26.708%,17.162%,8.312% and 5.815%,respectively (Table 1),the only four of which occupied the content of 57.997%. When raising the relative content of germinating inductions such as GA3 and cold stratification that could change the relative ratio of inhibition and induction could promote the seed germination of P.hexandrum [13]. It fully demonstrated that the relative highly content of inhibition compounds of seed germination could be the primary reason of unhealthy P.hexandrum seed germination.

Figure 1: Ion proceeding of ethyl ether extract by GC-MS.

3.2 Bioactivities of different hydroquinone concentrations

Because hydroquinone was the first time identified,the bioactivities of different hydroquinone concentrations were conducted on wheat by measuring the RL,SL after culturing 4 days. The results (Table 2) showed that the growth of wheat seedling could be promoted with the concentration increasing by 1.0 mg/L and then inhibited with the concentration increasing and the treatment of 1×10^-1 mg/L hydroquinone had no difference with 1.0 mg/L. The RL,SL under the 1.0 mg/L treatment could reach 4.43cm,4.53cm respectively by the 6th days and increased by 2.42,4.16 times compared to control,which indicated that hydroquinone possessed bioactivities to promote plant growth and development and had similarity to plant growth regulators.

3.3 Comparison bioactivities between hydroquinone and other hormones

Further study on the bioactivity of hydroquinone was conducted by comparing to NAA,IAA,GA3 and BAP with 1×10^-1mg/L,1.0mg/L,respectively. The results (Figure 2 and Table 3) showed that the growth and development of wheat seedling was significantly promoted from 4th days to 8th days compared to other hormones and the hydroquinone of the RDW and SDW at the concentration of 1×10^-1 mg/L could increase by 18.18%,44.44% and 52.17%,75.00% compared to both NAA and BAP at the concentration of 1.0 mg/L. All of the data and statistical analysis (Table 3) indicated that the hydroquinone could stimulus the wheat seedling growth and development and hasten the nutrient substances absorption and accumulation,which further demonstrated hydroquinone had the similarity biology characteristics to plant regulators.

Figure 2: Compare bioactivities between hydroquinone and other hormones on wheat (6th days). BAP, B2=1.0 mg/L; B1=1×10^-1 mg/L. IAA, I2=1.0 mg/L; I1=1×10^-1 mg/L. GA3, G2=1.0 mg/L; G1=1×10^-1 mg/L. NAA, N2=1.0 mg/L; N1=1×10^-1 mg/L. Hydroquinone, H2=1.0 mg/ L; H1=1×10^-1 mg/ L.

3.4 Effect of hydroquinone on vitro culture of embryo of P.hexandrum seed

Considering the hydroquinone was separated from the seed of P.hexandrum and since the relative content of it was low compared to other separated compounds. So the 0.5 mg/L high concentration of hydroquinone,IAA,GA3 was respectively selected at the basic of before study on the vitro culture of embryo of P.hexandrum seed [7,15]. The results (Figure 3 ) showed that after culturing 30 days,the embryo growth (Figure 3 ,M3) of P.hexandrum seed was significantly hastened compared to IAA (Figure 3 ,M1) and GA3 (Figure 3 ,M2),and the seedling (M3) was strong and healthy with the root developed. In addition; after culturing 40 days,the bulb of seedling (Figure 3 ,M3-1) began to rosette euphylls that needs 1 year to grow and develop at natural Condition that showed the growth and development of P.hexandrum was hastened and the cycle of the growth and development shortened by adding hydroquinone at a degree concentration.

Figure 3: Effect of hydroquinone on vitro culture of seed embryo of P.hexandrum. M1: Seedling cultured with 1.0 mg/L IAA after 30 days; M2: Seedling cultured with 1.0 mg/L GA3 after 30 days; M3: Seedling cultured with 1.0 mg/L Hydroquinone after 30 days; M3: Seedling developed from bulb cultured on M3 after 40 days.

4 Conclusions

• Majority of nineteen compounds including seven kinds fatty acid,three kinds paraffin hydrocarbon,three kinds alcohol aldehyde,etc separated and identified from P.hexandrum seed had been proved to have inhibition bioactive to majority of plants seed germination and the relative high content of (Z,Z)-9,12-Octadecadienoic acid,Hexadecanoic acid,Oleic acid and 1-Phenanthrenemethanol could be the primary reason of unhealthy seed germination and dormancy mechanism of P.hexandrum.

• Hydroquinone was the first time identified from P.hexandrum seed and the concentrations 1×10^-1~1.0 mg/L of hydroquinone possessed bioactivities and could stimulus the wheat seedling growth and development and hasten the nutrient substances absorption and accumulation that showed hydroquinone had the similarity biology characteristics to plant regulators.

• Adding 0.5 mg/L concentration of hydroquinone in the MS medium on the vitro culture of embryo of P.hexandrum seed could hasten and shorten the cycle of the growth and development of P.hexandrum,which will provide an important foundation for the use of biotechnology to obtain podophyllotoxin in the near future.

• The ratio of hydroquinone conspicuously lower compared to (Z,Z)-9,12-Octadecadienoic acid,hexadecanoic acid,oleic acid,dotriacontane,etc was the main reason that couldn’t promote the seed germination of P.hexandrum. The growth and development of seed embryo of P.hexandrum could be promoted when the concentration of hydroquinone increased by proper degree (1×10^-1~1.0 mg/L). In addition,the bioactivity mechanism of Hydroquinone still needs further studying.

Acknowledgements

We acknowledge with thanks to Dr. Zahoor Ahmed and Dr. Shahid Masood for extending lab facilities of Plant Genome Research Institute,NARC,Islamabad.

References

1. Chen Y.H. (1979) Research on resource of Podophyllum category in China. Acta Pharmaceutica Sinica,14(2): 101-107. (In Chinese)
2. Chattopadhyay S.,Srivastava A.K.,Bhojwani S.S.,et al. (2001) Development of suspension culture of Podophyllum hexandrum for the production of podophyllotoxin. Biotechnol Lett,23: 2063-2066.
3. Airi S.,Rawal R.S.,Dhar U.,Purohit A.N. (1997)Population studies on Podophyllum hexandrum Royle—a dwindling medicinal plant of the Himalaya. Plant Genet Resour Newsl,110: 20-34.
4. Archana G.,M.Lakshmi N.. (2000) Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs. Cytotechnology. 34: 17-26.
5. Chen S.W.,Wang Y.H.,Yan J.,et al. (2007)Synthesis and anti-HIV-1 activities of novel podophyllotoxin derivatives. Bioorganic & Medicinal Chemistry Letters,17: 2091-2095.
6. Nayar M.P.,Sastry A.P.K. (1990) Red data book of Indian plants. Botanical Survey of India,Calcutta.
7. Rekha K.,Subedar P.,Sanjoy C.,et al. (2007) GA3 induced changes in slow growing endangered Himalayan plant Podophyllum hexandrum and hastening of vegetative growth. Plant Growth Regul,51: 207-215.
8. Bush E.J.,Jones D.W. (1995) Asymmetric total synthesis of (-)-podophyllotoxin. J Chem Soc,Perkin Transaction,1: 1489-1492.
9. Berkowitz D.B,Choi S.,Maeng J.H. (2000) Enzyme-assisted asymmetric total synthesis of (−)-podophyllotoxin and (−)-picropodophylline. J Org Chem,65: 847-860.
10. Moraes R.M.,Lata H.,Bedir E.,et al. (2002) American May apple and its potential for podophyllotoxin production. Trends in new crops and new uses. pp: 527-532.
11. Chattopadhyay S.,Srivastava A.K.,Bhojwani S.S.,et al. (2002) Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor. J Ferment Bioeng,93: 215-220.
12. Nadeem M.,Palni L.M.S.,Purohit A.N.,et al. (2000) Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. Biol Conserv,92: 121-129.
13. Li C.D.,Li W.,Li M.F.,et al. (2008) Seed dormancy and germination in endangered plant Podophyllum hexandrum Royle. Bulletin of Botanical Research,28(5): 618-621. (In Chinese)
14. Yan Q.C. (2001) Seed Science. Published by China Agricultural Press,China,pp: 92-104.
15. Qi Y.L.,Li M.F.,Sun P.,et al. (2008) In vitro culture of maturated embryo of Podophyllum hexandrum Royle. J Biology,25(4): 39-41. (In Chinese)