Biotransformation of 3,4-cyclocondensed coumarins by transgenic hairy roots of Polygonum multiflorum

Two new coumarin glycosides (5 and 6), together with one known compound (4), were biosynthesized by transgenic hairy roots of Polygonum multiflorum with three 3,4-cyclocondensed coumarins as substrates (1 to 3). Their structures were elucidated by physicochemical and extensive spectroscopic analyses. The results of this study demonstrated that the hairy roots of P. multiflorum have the abilities to glycosylate 3,4-cyclocondensed coumarins in a regioand stereo-selective manner. Furthermore, the effects of three elicitors (salicylic acid, yeast extract and HgCl2) on the accumulation of the products were evaluated.

# These authors contributed equally to this work.treatment of skin fungal infection, preventing the skin fungal infections or suspending its development (Musa et al., 2008).
The methods of coumarins glycosylation include chemical synthesis, enzyme synthesis and biological synthesis.There are some disadvantages in the method of chemical synthesis, like high cost, very complex reactions (Garazd et al., 2005), and the yields are always very low.The insufficiency of the method of enzyme synthesis showed strict conditions and relative high cost.Comparing the aforementioned methods, biological synthesis has a lot of advantages.For instance, many reactions might occur in the cells according to the variety of enzymes, such as reductions, oxidations, and particularly glycosylation (Li et al., 2003).The glycosy-lation reaction of phenylpropanoids could be observed as the biotransformation of cinnamic, caffeic, and ferulic acids occur in plant cell cultures of Eucalyptus perriniana (Katsuragi et al., 2010).In our previous work, the transgenic hairy roots of Polygonum multiflorum have been demonstrated to have the abilitilies of glycosylating simple hydroxycoumarins and some phenolic compounds (Yan et al., 2008;Yu et al., 2008).The hairy roots have the characters of fast growth, genetic stability, cultivation without growth regulators, etc (Wang et al., 2002).There are lots of studies on the biotransformation of coumarin compounds by hairy roots.The root cultures of Panax ginseng have been demonstrated to have high glycosylation ability to the 7-OH coumarin (Li et al., 2002).The glycosylation reactions also have substrate specificity and the position selectivity.For example, phenolic compounds could be easily glycosylated by Pharbitis nil hairy roots.The 7-OH group of coumarins might be particularly vulnerable to glycosylation (Kanho et al., 2004).Therefore, in order to produce the coumarin glycosides with the method of biosynthesis and investigate the regio-and stereo-selectivity of transgenic hairy roots of P. multiflorum to 3,4-cyclocondensed coumarins, three 3,4-cyclocondensed coumarins (1, 2 and 3) were chosen as substrates in this paper.Furthermore, the effects of three elicitors (salicylic acid (SA), yeast extract and HgCl 2 ) on the yields of biosynthesis products were also investigated primarily.

General procedures
Three substrates were synthesized by Pechmann condensation.The structures of the substrates were determined using 1 H NMR, and those of the biosynthesis products were elucidated using 1 H NMR, 13 C NMR and heteronuclear multiple bond correlation (HMBC).The spectra were recorded by a Bruker Advance 400 MHz spectrometer in DMSO-d6 solution and chemical shifts were expressed in δ (ppm) referring to tetramethylsilane (TMS).Electrospray ionization-mass spectra (ESI-MS) were measured by a DSQ spectrometer (Thermo Electron Co.).High performance Liquid chromatography (HPLC) analyses were carried out by Agilent 1200 series with a diode array detection and Phenomenex C18 column (5 µm, 4.6 × 250 mm).Separation was performed on a C18 column by isocratic elution with water (A) and MeOH (B) (45:55, v/v), and the products were detected at 320 nm.Detection temperature was 30°C.The products were eluted with mobile phase of a mixture of water-MeOH (45:55, v/v) in 20 min.
The three compounds (1 to 3) were finally obtained by filtration and recrystallization from methanol.All the reactions were monitored by TLC (developing agent: ethyl acetate/petroleum ether, 1/1), and ultraviolet (UV) detection at 365 nm.The structures were finally determined by 1 H NMR, and the purity was given to be over 95% by HPLC analysis.

Culture conditions
Hairy roots of P. multiflorum were induced in our laboratory.The cultures (7.5 g, fresh weight) have been sub-cultured 10 days at intervals on Murashige and Skoog medium (MS: 200 ml in a 500 ml conical flask, pH 5.75).The incubation was cultured on a rotary shaker (110 rpm) for 8 days at 25°C in the dark.Prior to use, the cultures were sub-cultured three times with dropculture method.All groups were cultured in the same conditions throughout the experiments.substrates 1, 2 and 3   Each substrate (1, 2 and 3, each at 600 mg) was administered to 20 flasks of suspension cultures of P. multiflorum hairy roots (30 mg/flask).The hairy roots and media were separated by filtration with suction after cultures were incubated for 7 days.The medium was extracted with EtOAc for three times, and then was further extracted with n-BuOH for three times.The cultures were dried at 55°C and extracted with MeOH for 2.5 h (0.5 h × 5, 50°C).All the extracts were condensed and passed through silica gel column using a gradient elution of petroleum ether-EtOAc-MeOH system.First, the substrate and other impurities were eluted using the petroleum ether.When the solvents were EtOAc-MeOH (1:9 v/v) and the amount were five column volumes, the products could be completely separated.Subsequently, products were further purified by recrystallization in methanol.

Optimization of the biotransformation
In order to determine the biotransformation capacity of the hairy roots on the three 3,4-cyclocondensed coumarins and evaluate the optimization condition of compound 1, the effects of DMSO to the yields of products were investigated.Furthermore, the time course of compounds 1, 2 and 3 were also established.All experiments were repeated 3 times, and the data were the mean values of three experiments.
The earlier experiment estimated the effect of DMSO to the hairy roots.Every concentration was administered to three flasks cultures, which dissolved 10 mg of compound 1.Final concentrations of the DMSO in the medium were 0.25, 1.00, 2.50, 5.00 and 7.50% (v/v), respectively.After incubation, the hairy roots and media were separated by suction filtration.The medium was condensed to 100 ml and the cultures were dried at 55°C.The yield of the product was determined on the basis of the peak area from HPLC and expressed as a relative percentage to the total amount of the whole reaction products.The extraction and analytic procedures of the following experiments were the same as described earlier.
Meanwhile, to test the relationship of substrate concentration and product yield, compound 1 was administrated to three flasks cultures.Final concentrations of compound 1 in the medium were 0.05, 0.10, 0.25, 0.50 and 1.0 mg/L.
The relationship between the time of substrate supply and glucoside formation was examined as the following procedure.Compound 1 (10 mg) was administered to each flask; then, at 0.5, 1, 2, 3, 4, 5, 6, and 7 days intervals, three flasks were harvested.The time courses of compounds 2 and 3 were investigated with the same procedure as mentioned earlier.

Preparation of the elicitor solutions and administration
SA, yeast extract and HgCl2 were dissolved in DMSO separately and prepared in three concentrations.The elicitor solutions were added into three suspension cultures.Final concentrations of each elicitor in the medium were 0.1, 1.0 and 10.0 mg/L.And each flask was added as the same concentration of compound 1 (20 mg/flask).

Analysis of the sample
All samples were detected under the following conditions: the samples obtained from media were detected after being filtrated by the 0.45 µm microporous membrane.The samples from cultures (0.2 g) were extracted with 10 ml methanol, cooled to room temperature, filtrated by the 0.45 µm microporous membrane, and then determined.The yield of the products was determined on the basis of the peak area from HPLC and expressed as a relative percentage to the total amount of whole reaction products.

Structures of the biotransformation products
Structures of compounds 1, 2, and 3 were verified by comparing their NMR spectral data with literature (Garazd et al., 2002).Two new compounds (5 and 6) along with one known compound (4) were biosynthesized by hairy roots of P. multiflorum when compounds 1, 2 and 3 were administrated as substrates.The spectral data of compound 4 and two new compounds (5 and 6) are as follows.
7-O-β-D-glucopyranosyl-2,3dihydrocyclopenta[c]chromen-4-one (4).Colorless needle crystals.+ at m/z 217.4 and the molecule weight were 162 more than the substrate 2, which showed the molecular formula C 18 H 20 O 9 , indicating that compound 5 might be a glucosylation product of substrate 2. 1 H NMR spectrum displayed anomeric proton signal (δ 4.94, H, d, J = 7.6 Hz).The coupling constant (7.6 Hz) indicated that the configuration of sugar was β type.The 13 C NMR spectrum showed anomeric carbon at δ 101.1.The sugar generated by acid hydrolysis was compared with the standard of β-D-glucose by paper chromatography, and found that they are both R f value were the same.In the HMBC spectrum H-1 (4.60) of the terminal glucose and C-6 (61.0) of the inner glucose gave the linkage of the sugar units to be D-glucose.Thus, the structure of the product was proposed to be β-D-glucose.The signals of δ 2.21 (2H, m), 2.63 (2H, m), 3.23 (2H, m) 6.41 (1H, d, J = 2 Hz), 6.50 (1H, d, J = 2.0 Hz), 10.45 (1H, s) showed that the positions of 2, 3, 8, and 9 were not replaced, while sugar group was connected in one of the phenolic hydroxyl group.HMBC spectrum showed that the signal of C-9 (δ 156.4) was related to the anomeric proton and the δ 6.50 (1H, d, J = 2.0 Hz) signal, indicating that the sugar group was attached to the 9-OH.The signal δ 6.50 (1H, d, J = 2.0 Hz) was H-8.However, the δ 6.41 (1H, d, J = 2 Hz) was the H-6.δ 156.44 was related with the δ H 6.41, 6.50, indicating that the signal is the C-7 signal.δ 156.2 was connected with δ 6.50, it is for the C-6a signal.There were connection between δ 156.1 and δ H 2.21 (2H, m), 2.63 (2H, m), 3.23 (2H, m), indicating the signal is C-1a.δ 121.95 and δ H 2.21 (2H, m), 2.63 (2H, m), 3.23 (2H, m) were related, δ 121.95 is C-3a signal.Correlations were observed between δ 102.94 and H-8, suggesting δ 102.94 is C-9a signal.δ 101.06 is sugar carbon.Thus, compound 5 was elucidated to be 9 were 162 more than substrate 3, which showed the molecular formula C 18 H 20 O 9 , indicating that compound 6 might be a glucosylation product of substrate 2. In the 1 H NMR spectra of compound 6, an anomeric proton signal was observed at δ 4.95 (1H, d, J = 7.6 Hz), indicating that the configuration of sugar was β -type.The sugar generated by acid hydrolysis was compared with the standard of β-D-glucose by paper chromatography (PC), and it found that the R f value of both were the same.In addition, the component sugar in compound 6 was indicated to be β-D-glucose on the basis of the patterns of the carbon and proton signals caused by the sugar moiety in NMR spectra.In the HMBC spectrum, the signal of δ 131.3 (C-6) was related to the anomeric proton and indicated that the sugar group was connected to C 6 -OH.The 13 C NMR spectrum showed anomeric carbon at δ 101.Other signals were confirmed the same as compound 5. Thus, compound 6 was elucidated to be 6-O-β-D-glucopyranosyl -7-hydroxyl-2,3dihydrocyclopenta[c]chromen-4-one, which is a new compound.The structures of compounds 1, 2, 3, 4, 5 and 6 are as shown in Figure 1.

Optimization of the biotransformation condition
The yields of compound 4 displayed a declining tendency as the addition of DMSO, with the highest yield (78.42%) at the concentration of 0.25%.It was also found that the product was only stored in the hairy roots and not detected in the medium.The product secretion rate was 36.96% at the highest concentration of DMSO (7.50%), while the yield of compound 4 was only 14.81%.The 0.25% of DMSO was chosen to dissolve the substrates as the optimal concentration.
The yields of compound 4 did not display a significant difference when the concentrations of compound 1 were between 0.05 and 0.25 mg/ml.The range of the yield was among 78 and 82%, but the yield was only 24.20% when the concentration was 0.5 mg/ml.Therefore, the concentrations were chosen among 0.05 and 0.25 mg/ml in the following experiments.
As shown in Figure 2, the concentration of compound 1 decreased rapidly in one day (10 to 3.44 mg).The yield of compound 4 increased sharply at the first day, indicating that biotransformation happened on the first day, and compound 4 reached the maximum transformation rate at the fourth day (81.35%).
The time course of compound 5 (Figure 3) showed that compound 5 was rapidly synthesized in a half-day.The accumulation of compound 5 reached the highest at the third day, and the yield was 93.40%.As shown in Figure 4, the biosynthesis reaction of compound 6 occurred at the first day.The highest yield was 18.14% at the second day.After that, the total of the substrate and product tends to a steady state.

Effects of the elicitor administration
The yield of compound 4 was 82.15% (the concentration of 1 was 0.1 mg/ml) without any elicitor and increased about 11% as the addition of SA in the concentration of 1 mg/L (Table 1).The yield was very low when the concentration of SA was 10 mg/L, and compound 4 are only 5.78 mg/flask.The yield was 94.24% which increased 12% as the yeast extract were added in the concentration of 1.0 mg/L, respectively.There was little inducing effect with HgCl 2 addition, and the accumulation of the product was lower than those of when the concentration was 1.0 and 10 mg/L.
The yield of compound 4 declined obviously when the concentration of the three elicitors reached the maximum (10 mg/L).Over dosage of elicitors failed to increase the product accumulation.

Water solubility of the substrates and their products
The comparison of the water solubility of the substrates and their products was shown in Table 2.The water solubility of three biosynthesis products increased obviously, especially for compound 5, which has increased 75.6 folds.The result showed that conjugation of glucose residue to 3,4-cyclocondensed coumarins could increase the water solubility.Therefore, it is a useful way to improve the water solubility of the 3,4-cyclocondensed coumarins by glycosylation method.

DISCUSSION
The transgenic hairy roots of P. multiflorum showed strong ability to transform the 3,4-cyclocondensed coumarins, and the yield was much higher when compared with the chemical reaction (42.84%) (Garazd et al., 2005).The lowest conversion rate of 6,7-dihydroxy-2,3dihydrocyclopenta[c]chromen-4 coumarins (3) may be due to steric hindrance of the o-hydroxy.The sugar group connection positions indicated that the hairy roots were superior to glycosylate 9-OH or 6-OH when 7,9-dihydroxy or 6,7-dihydroxy 2,3-dihydrocyclopenta[c]chromen-4 coumarins were administrated.It shows that the hairy roots of P. multiflorum have the position selectivity.This result certifies that the biotransformation is indeed a useful way for the production of 3,4-cyclocondensed coumarin glycosides and new compounds.
The biotransformation was so rapid that it was an effective way to produce the 3,4-cyclocondensed coumarin glycosides.The major influence factors of  1) by hairy roots of P. multiflorum.At one day interval, three flask cultures were harvested and the yields of the substrate and product were analyzed.The result showed that the product yield reached the highest (81.35%) on the fourth day.The substrate was the lowest on the seventh day.Total quantity of compound 1 (■); fields of the compound 4 (product of the compound 1) (◆).
biotransformation occurring and the conversion rate (cell, organ, etc.) are enzyme system as well as the substrate structural features, which include co-culturing time, substrate concentration, etc (Michiko et al., 1995).The substrate concentration increasing within a certain range could effectively improve the yield of the product, but the appropriate maximum concentration should be studied.Yields and conversion rates will decline, because the high concentration will cause harm to the culture (Tripathi et al., 2002).The effects of the co-culturing time, concentrations of the DMSO and substrates were investigated, and the results have shown that the over dosage failed to increase the product accumulation.Therefore, the optimization conditions of the biotransformation were 0.25% of DMSO, concentrations of substrates among 0.05 and 0.25 mg/ml.The appropriate co-culturing time of the compound 4, 5, and 6 were four, three, two days, respectively.2), by hairy roots of P. multiflorum.At one day interval, three flask cultures were harvested and the yields of the substrate and product were analyzed.The result showed that the product yield reached the highest (93.40%) on the third day.The substrate was the lowest on the fourth day.Total quantity of compound 2 (■); fields of the compound 5 (product of the compound 2) (◆).,7-dihydroxy-2,3-dihydrocyclopenta[c]chromen-4one (3) by hairy roots of P. multiflorum.At one day interval, three flask cultures were harvested and the yields of the substrate and product were analyzed.The result showed that the product yield reached the highest (18.14%) on the second day.The substrate was the lowest on the seventh day.Total quantity of compound 3 (■); fields of the compound 6 of the compound 3) (◆).
According to the literatures, the plasma membrane could be stronger with the increase of elicitor concentration.Some studies showed umbelliferone could be stimulated in suspension cultures of plant tissues using 1.0 mg/L HgCl 2 (Smith et al., 2001).Over dosage of elicitors failed to increase the product accumulation.The yield of taxol increased 4 times as the concentration of SA was 0.10 mg/L, but when the concentrations of SA

Figure 1 .Figure 2 .
Figure 1.Biotransformation of the three 3,4-cyclocondensed coumarins by hairy roots of P. multiflorum showing the structures of the substrates (1, 2 and 3) and their products (5, 6 and 7) were as above.The structures of the products are all glycosides.

Figure 3 .
Figure3.chromen-4one (2), by hairy roots of P. multiflorum.At one day interval, three flask cultures were harvested and the yields of the substrate and product were analyzed.The result showed that the product yield reached the highest (93.40%) on the third day.The substrate was the lowest on the fourth day.Total quantity of compound 2 (■); fields of the compound 5 (product of the compound 2) (◆).

Figure 4 .
Figure 4.chromen-4one (3) by hairy roots of P. multiflorum.At one day interval, three flask cultures were harvested and the yields of the substrate and product were analyzed.The result showed that the product yield reached the highest (18.14%) on the second day.The substrate was the lowest on the seventh day.Total quantity of compound 3 (■); fields of the compound 6 of the compound 3) (◆).