Effects of Macronutrients on Primary and Secondary Metabolism in Scutellaria baicalensis Georgi

Jinhua LIU Yanjing BIAN Zhixia LI Yongqing ZHANG

Abstract [Objectives]This study was conducted to clarify the roles of macronutrients in regulating plant primary and secondary metabolism of Scutellaria baicalensis Georgi.[Methods]The contents of chlorophyll （Chl） and soluble sugar were detected with ultraviolet spectrophotometry. The activities of phenylalanine ammonialyase （PAL）， cinnamate 4hydroxylase （C4H） and chalcone synthase （CHS） were determined with ultraviolet spectrophotometry. Secondary metabolites were detected by high performance liquid chromatography （HPLC）.[Results]The content of chlorophyll in treatments N， P and K was increased， showing significant difference from that of the control （P<0.05）. Among them， Treatment N has the highest content of chlorophyll. Soluble sugar content in treatments N， P and K （K>N>P>CK） was increased considerably and had significant difference from that of the control （P<0.05）. Compared with the control， PAL and C4H activity in treatments N， P and K were increased dramatically by 186.57%， 134.09%， 306.91% and 73.21%， 28.91%， 247.57%， respectively. CHS activity was also increased sharply in treatments N， P and K. Scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A contents in all treatments N， P and K （K>N>P） were increased， and had significant difference from that of the control （P<0.05）.[Conclusions]The results showed that macronutrients can increase leaf chlorophyll content， soluble sugar content， PAL activity， C4H activity and secondary metabolites of S. baicalensis.

Key words Macronutrients; PAL; C4H; CHS; Secondary metabolites; Scutellaria baicalensis

Nitrogen （N）， phosphorus （P） and potassium （K） are referred to as macronutrients， as they are required in large quantities for growth and development of plants. Macronutrients can influcne the quality of herbal medicines. Several hypotheses have suggested that there is a tradeoff between primary and secondary metabolism because of competition for common substrates， but nothing is known about regulation at the enzyme level. Scutellaria baicalensis is an important herbal medicine in common use. S. baicalensis is more widely planted in China， because of increasing demand for it. In this study， a set of experiments was performed to elucidate the effects of N， P and K on the activities of the key enzymes involved in flavonoid biosynthesis， such as phenylalanine ammonialyase （PAL）， cinnamate 4hydroxylase （C4H） and chalcone synthase （CHS）.

Materials and Methods

Materials

S. baicalensis was identified by Professor ZHANG Yongqing （Shandong University of Traditional Chinese Medicine）， and the pot experiment was carried out in the medicinal plants nursery in Shandong University of Traditional Chinese Medicine. The seeds of S. baicalensis were sown in cylindrical pots （21.00 cm high， 27.00 cm in diameter at the top， and 14.00 cm in diameter at the bottom）. Sixty days later， the seedlings were grown in Hoagland solution （control）， or Hoagland medium supplemented with 1.6 g/L N （Treatment N）， 136 mg/L P （Treatment P） or 642 mg/L K （Treatment K） or for 7 d. Six pots were prepared for each treatment.

Determination of the contents of chlorophyll （Chl） and soluble sugar

The contents of Chl and soluble sugar were detected with ultraviolet spectrophotometry[1].

Determination of PAL activity

PAL activity was assayed as previously described[2]. In detail， 0.2 g of S. baicalensis root was ground to a fine powder with a mortar and pestle in liquid N2. The powder was then extracted with 5 ml of 0.1 mol/L borate buffer （pH=8.8） containing 0.02 g PVP， 5 mmol/Lmercaptoethanol and 1 mmol/L EDTA， and then centrifuged at 10 000 r/min for 15 min. Subsequently， 100 l of the supernatant was collected and mixed with 2 ml of borate buffer （0.1 mol/L， pH= 8.8）. After 800 l of Lphenylalanine was added， the mixture was incubated at 30 for 30 min， and the reaction was terminated by adding 200 l of 6 mmol/L HCl. Finally， the absorbance of the mixture as read at 290 nm to calculate the yield of cinnamic acid. Each extract was assayed in triplicate[3].

Determination of C4H activity

C4H activity was assayed as previously described[2]. In detail， 0.2 g of S. baicalensis root was ground to a fine powder with a mortar and pestle in liquid N2. The powder was then extracted with 3 ml of 0.05 mol/L TrisHCl buffer （pH=8.9） containing 15 mmol/Lmercaptoethanol， 4 mmol/L MgCl2， 5 mmol/L Vc， 10 mol/L leupetin， 1 mmol/L PMSF， 0.15% polyvinylpolypyrrolidone （PVP， m/V）， 10% glycerol， and centrifuged at 10 000 r/min for 20 min. Subsequently， 100 l of the supernatant was collected and mixed with 2.2 ml of reaction buffer containing 2 mol/L transcinnamic acid， 50 mmol/L TrisHCl （pH=8.9）， 2 mol/L NADPNa2， and 5 mol/L G6PNa2， incubated on a shaker at 25 for 30 min， and the reaction was stopped by the addition of 6 mmol/L HCl （100 l）. Finally， the absorbance of the mixture as read at 340 nm. Each extract was assayed in triplicate.

Determination of CHS activity

Protein extraction

All steps were carried out at 0-4. Frozen S. baicalensis root （10 g） was ground using a pestle and mortar in the presence of sea sand and 10% （w/w） PVP. The frozen powder was mixed with extraction buffer （0.1 mol/L KPi buffer of pH 6.8， 1.4 mmol/L 2mercaptoethanol， 40 mmol/L ascorbic acid， 3 mmol/L EDTA， 10 pmol/L leupeptin and 0.2 mmol/L phenylmethyl sulphonylfluoride， flushed with N2 before use）. After thawing， the homogenate was centrifuged at 8 000 r/min for 20 min. The protein was then salted out using 30% to 70% of （NH4）2SO4. The 70% （NH4）2SO4 pellet was dissolved in 2.5 ml of 0.1 mol/L KPi buffer （pH 6.8）， 1.4 mmol/L 2mercaptoethanol， 40 mmol/L ascorbic acid and 5% （w/v） trehalose （flushed with N2 before use）， and then desalted in the same buffer with the use of a PD 10 column （Sephadex G25M， Pharmacia） according to the manufacturerюs directions. The protein sample was then frozen in liquid N2 and stored at -80.

Chalcone synthase assay

S. baicalensis protein extract （500 l） and 500 l of assay buffer （0.5 mol/L KPi buffer of pH 6.8， 2.8 mmol/L 2mercaptoethanol and 2% （w/v） bovine serum albumin） were mixed. The reaction was started by adding 100 l of 0.4 mmol/L malonylCoA （2 nmol） and 100 l of 0.2 mmol/L pcoumaroylCoA （1 nmol）. The reaction mixture was incubated at 30 for 40 min. At the end of the incubation， 1 ml of EtOAc was added， mixed using a vortex and then centrifuged for at least 2 min. The EtOAc layer was transferred to a new cup and evaporated to dryness using a vacuum concentrator. The residue was then redissolved in 300 l of MeOH and analysed by HPLC. The injection volume was 20 l. The recovery of naringenin was determined in separate experiments （n=7）， in which 15 ng of naringenin per sample was added to above described mixture of extract and buffer. Afterwards the same extraction procedure was performed as described above. The recovery of naringenin was determined by HPLC according to the height of the naringenin peak at 290 nm.

HPLC

All HPLC measurements were performed with an Agilent 1100 series HPLCDAD system. The eluent consisted of MeOHH2O85% H3PO4， （280≥136≥1） （pH 2.6）， and a flow rate of 1.0 ml/min was used （pressure： 140 bar）. Detection was performed at 290 nm.

Determination of secondary metabolites

Secondary metabolites were measured by HPLC. All HPLC measurements were performed with an Agilent 1100 series HPLCDAD system. The mobile phase consisted of MEOH， H2O， H3PO4. The mobile phase gradient program was shown in Table 1. The flow rate was kept at 1.0 ml/min， and the detection wavelength was set at 276 nm.

Assessment of linearity

The reference substances scutellarin （6.1 mg）， baicalin （3.0 mg）， wogonoside （2.1 mg）， baicalein （10.3 mg）， wogonin （1.5 mg） and oroxylin A （2.9 mg） was accurately weighed， put into a 100 ml volumetric flask containing methanol， and the volume was made up to the mark with methanol. Then， 2， 4， 6， 8， 10， 12 and 14 l of the standard solution were injected into the HPLC The representative linear equations for scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A were Y=90.464x-182.57 （R=0.999 1）， Y=193.59x-311.68 （R=0.999 2）， Y=86.437x-37.023 （R=0.999 2）， Y=156.23x-207.14 （R=0.999 1）， Y=49.077x-15.829 （R=0.999 3） and Y=1203.84x-110.16 （R=0.999 2）， respectively.

Sample preparation

The tested samples were pulverized into powder and passed through a 40mesh sieve.Each sample powder （0.5 g） was weighed accurately and extracted with 20 ml of ethanolwater mixture （7≥3） by ultrasonication for 60 min. The extract was filtered through 0.45 m microporous filters.

Assessment of precision

The solution for test was made according to the method described above， injected five times repeatedly to record the peak areas of scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A content. Their RSD values were 0.23%， 0.32%， 0.41%， 0.29%， 0.82% and 0.61% respectively. The results showed that the method had high precision.

Assessment of reproducibility

Five copies of S. baicalensis root （0.5 g each） were weighed precisely， pretreated as described above， and injected into the HPLC system to record the peak areas of scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A. Their RSD values were 0.43%， 0.22%， 0.51%， 0.69%， 0.32% and 0.71% respectively， which proved good reproducibility of the method.

Assessment of stability

The sample solution was assayed by HPLC 0， 2， 4， 6， 12， 18， 24， 48， 72 h， respectively after it was prepared. The result indicated that the RSD values were 0.86%， 0.57%， 1.02%， 0.93%， 1.12% and 0.95%， respectively， indicating that the sample solution was stable within 72 h.

Recovery experiment

S. baicalensis root （0.5 g） was taken precisely for five times. The proper amounts of scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A were added respectively. The solution for test was prepared as described above， and the content was determined. The average recovery rates （n=5） were 101.22%， 99.87%， 102.45%， 103.21%， 98.82%， 100.67%， and the RSD values were 0.63%， 0.93%， 0.89%， 1.29%， 1.02% and 0.91% respectively， indicating high recovery rate of the method.

Statistical analysis

Each experiment was repeated three times at least. Values were expressed as means÷S.E.， and analyzed by Statistical Analysis System （version 8.0， SAS Institute Inc.， NC， USA）.

Results and Analysis

Effects of macronutrients on leaf chlorophyll content

Chlorophyll， an important photosynthetic pigment， plays an important role in plant photosynthesis. The changes in leaf photosynthetic pigment content in different macronutrient treatments are shown in Fig. 1. The results indicated that Chl content in treatments N， P and K was increased， showing significant difference （P<0.05） from that of the control （CK）， and that of Treatment N was the highest.

Effects of macronutrients on soluble sugar content

Soluble sugar is an important primary metabolite in the formation of flavonoids. The influence of macronutrients on soluble sugar content is shown in Fig. 2. The results indicated that soluble sugar content in treatments N， P and K （K>N>P>CK） was increased considerably， showing significant difference from that of the control （P<0.05）， and that of Treatment K was the highest.

Effects of macronutrients on PAL and C4H activity

The changes of PAL and C4H in the shoot of S. baicalensis exposed to different macronutrients are shown in Fig. 3. PAL and C4H activity in treatments N， P and K were greatly increased by 186.57%， 134.09%， 306.91% and 73.21%， 28.91%， 247.5 in comparison to the control， respectively. There is a positive correlation between PAL and C4H （r=0.952， P<0.05）. They both provide an entry point for the biosynthesis of flavonoids. This showed that PAL and C4H are sensitive to N， P and K.

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Effects of macronutrients on CHS activity

The changes of CHS activity in the shoot of S. baicalensis exposed to different macronutrients are shown in Fig. 4. CHS activity in all treatments N， P and K was increased sharply， which showed that CHS is sensitive to N， P and K.

Effects of macronutrients on secondary metabolites

The changes of secondary metabolites in the seedling of S. baicalensis exposed to different macronutrients are shown in Fig. 5， Fig. 6 and Fig. 7. The results indicated that， the contents of scutellarin， baicalin， wogonoside， baicalein， wogonin and oroxylin A in treatments N， P and K （K>N>P>CK） wee all increased， and had significant difference （P<0.05） from that of the control， indicating that macronutrients are effective to the formation of secondary metabolites of S. baicalensis.

Correlation analysis

In all treatments N. P and K， flavonoids had no significant positive correlation with CHL and C4H activity， but significant correlation with soluble sugar content （R=0.980*）， PAL activity （R=0.988*）， and CHS activity （R=0.983*）; C4H and CHS activity had significant correlation with soluble sugar content （R=0.974*， R=0.985*）; CHS activity had significant correlation with PAL （R=0.958*） and C4H （R=0.957*） activity. The results proved that flavonoids （secondary metabolites） were accumulated when soluble sugar， PAL and CHS were massively accumulated in S. baicalensis root.

Discussion

Plants synthesize organic substances （primary metabolites） by leaf photosynthesis. Then， the primary metabolites are converted into secondary metabolites through the canalization of a series of enzymes. The secondary metabolites are key components of Chinese herbal medicines. Environmental degradation or improper cultivation measures will affect the formation and activity of enzymes， thereby affecting various metabolic pathways， and the yield and quality of medicinal plants.

Macronutrients are the main factor affecting plant growth and physiological process. Physiological indices of S. baicalensis seedlings exposed to different macronutrients were analyzed in this study. The results showed that S. baicalensis is sensitive to macronutrients， and the uses of macronutrients can increase leaf chlorophyll， soluble sugar content， PAL activity， C4H activity and secondary metabolites.

The phenylpropanoid pathway is the main route for flavonoids biosynthesis. The first step towards the phenylpropanoid biosynthetic pathway is catalyzed by PAL， which converts Lphenylalanine to transcinnamic acid. The second step is catalyzed by C4H， which converts Lphenylalanine transcinnamic acid to 4coumarate. Our data revealed that the activities of PAL and C4H were significantly increased compared to the control， which was consistent with the changes in secondary metabolites.

In conclusion， macronutrients can promote the formation of leaf photosynthetic pigment， and increase primary metabolites， the activity of PAL， C4H and CHS， and secondary metabolites of medicinal plants. So the quality of S. baicalensis can be improved by proper use of macronutrients， especially K.

References

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[2]JIN LP. Effect of drought stress in different ecological conditions of the Prunus mongolica maxim almond leaves in PAL and C4H activity[J]. Acta Agriculture BorealiSinica， 2009， 24（5）： 118-122.

[3]LIU JH， LI J， CUI SL， ZHANG YQ. Germination characteristics and secondary metabolism of Scutellaria baicalensis Georgi under different illumination time[J]. Agricultural science Technolagy，2014，15（8）：1312-1316.