Jinhua LIU, Qian LIU, Jia LI, Yongqing ZHANG

1. Jining Center for Food and Drug Control, Jining 272000, China; 2. Shandong University of Traditional Chinese Medicine, Jinan 250355, China

Abstract [Objectives] To explore the effects of temperature on the primary and secondary metabolism of Scutellaria baicalensis Georgi during the seed germination. [Methods] The superoxide dismutase (SOD) activity was determined using riboflavin-NBT; peroxidase (POD) activity was determined using guaiacol colorimetric method, catalase (CAT), ascorbate peroxidase (APX), phenylalanine ammonia lyase (PAL) and cinnamic acid-4-hydroxylase (C4H) activity were detected by ultraviolet spectrophotometry, and chalcone synthase (CHS) activity and the content of secondary metabolites were measured by high performance liquid chromatography (HPLC). [Results] The germination rate, germination potential and germination index of S. baicalensis seeds were significantly affected by temperature. The most suitable temperature for the germination of S. baicalensis seeds was 25 ℃. The activities of SOD, POD and CAT in S. baicalensis seeds treated at low and high temperature were higher than that treated at suitable temperature; the activities of PAL, C4H and CHS of S. baicalensis seeds treated at low and high temperature were lower than that treated at suitable temperature. There was a good positive correlation between flavonoids and soluble sugar, PAL activity and C4H activity, and the correlation coefficients were R=0.894*, R=0.956* and R=0.951*, respectively. [Conclusions] In adverse environment, S. baicalensis seeds have good defense capabilities. During the germination of seeds, the formation of secondary metabolites is significantly correlated to the activity of key enzymes. Therefore, high-quality medicinal materials can be obtained by taking measures to improve the activity of key enzymes.

Key words Germination, Phenylalanine ammonia lyase (PAL), Cinnamic acid-4-hydroxylase (C4H), Chalcone synthase (CHS), Secondary metabolites

1 Introduction

Scutellariae Radix (Huangqin) is the dried roots and rhizomes ofScutellariabaicalensisGeorgi, a perennial herbaceous plant in the Lamiaceae family.S.baicalensiscan clear away the heat and dry the dampness, purge fire and remove toxicity, cool the blood and stanch bleeding, and prevent abortion[1-3]. It is a commonly used Chinese medicinal material. Because of its outstanding efficacy and wide clinical applications, its market demand has increased substantially year by year, and the limited wild resources are far from being able to meet the market demands, which promote the gradual deepening of research on the artificial cultivation ofS.baicalensisin recent years[4-11].

Seed germination is a key stage in the life process of a plant and is the result of various physiological and biochemical metabolisms. Various metabolic activities in plants require enzyme catalysis. Temperature is an essential factor influencing the enzyme activity, and different enzymes have different catalytic temperature. Exploring the effects of temperature on the germination and metabolism ofS.baicalensisseeds is helpful for understanding the rules of seed germination and metabolism, so as to provide a reference for artificially regulating the synthesis and accumulation of active components.

2 Materials and methods

2.1 MaterialsTheS.baicalensisseeds used in this study were taken from theS.baicalensisplantation base in Kushan Township, Ju County, Rizhao City, Shandong Province. They were two-year-old plants that produced new seeds that year and were identified by Professor Zhang Yongqing from Shandong University of Traditional Chinese Medicine as seeds ofScutellariabaicalensisGeorgi.

AfterS.baicalensisseeds were selected and washed, disinfected with 75% ethanol, rinsed with distilled water, and placed in a petri dish with a diameter of 10 cm and covered with 3 layers of filter paper for germination. Placed the petri dish in a constant temperature incubator at 15, 20, 25, 30, and 35 ℃ and kept it moist. Made a record of the number of germination every day, calculated the germination rate of the first 6 d and the germination potential of the first 5 d. During the cotyledon expansion period, measured the activities of key enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and chalcone synthase (CHS).

2.2 Methods

2.2.1Determination of SOD activity. Weighed 0.2 g of fresh germinatedS.baicalensisseedlings, placed them in a pre-cooled mortar, added 1 mL of phosphate buffer (0.05 mol/L), ground them into a homogenate in an ice bath, added buffer to make the final volume 5 mL, centrifuged at 10 000 r/min for 10 min, and the supernatant was the crude SOD extract. Then took seven 5 mL test tubes, of which three were measuring tubes, and the other four were control tubes. Added the solutions according to Table 1.

After adding the test solution and mixing up, placed one control tube in a dark place, and kept the other tubes reacted under 4 000 lx fluorescent lamp for 30 min. After completion of the reaction, taking the non-illuminated control tube as the blank, measured the colorimetric values of the other tubes. The SOD activity was expressed as a unit of enzyme activity by inhibiting the photochemical reduction of NBT by 50%.

2.2.2Determination of POD activity. Weighed 0.2 g of fresh germinatedS.baicalensisseedlings, added 5 mL of enzyme extract (50 mmol/L of PBS, pH 7.8+0.1 mmol/L of EDTA) and ground with 0.05 g PVP ice-bath, then centrifuged at 4 ℃ and 10 000 r/min for 20 min, and took the supernatant as the enzyme solution, then took the test tube and added 2 mL of acetate buffer (0.1 mol/L), 1 mL of 0.25% guaiacol solution, 0.2 mL of enzyme solution, 0.1 mL of H2O2solution (0.75%), and quickly inverted and mixed. Poured the solution into a cuvette, adjustedA460to 0 and started timing, readA460value every 30 s (total reading 3 min).

2.2.3Determination of CAT activity. Weighed 0.2 g of fresh germinatedS.baicalensisseedlings and placed them in a pre-cooled mortar, added 1 mL of phosphate buffer (0.05 mol/L, pH 7.8), ground them into a homogenate in an ice bath, added buffer to make the final volume 5 mL, centrifuged at 4 ℃ and 10 000 r/min for 10 min, and the supernatant was the crude CAT extract. Added the test solutions according to Table 2.

After adding the enzyme solution, mixed quickly, and measured the absorbance at the wavelength of 240 nm, once every 30 s.

2.2.4Determination of APX activity. Weighed 0.2 g of fresh germinatedS.baicalensisseedlings and placed them in a pre-cooled mortar, added 1 mL of phosphate buffer (0.05 mol/L, pH 7.8), ground them into a homogenate in an ice bath, added buffer to make the final volume 5 mL, centrifuged at 4 ℃ and 10 000 r/min for 10 min, and the supernatant was the crude CAT extract. Added the test solutions according to Table 3.

After adding the enzyme solution, mixed quickly, and measured the absorbance at the wavelength of 240 nm, once every 30 s.

2.2.5Determination of PAL activity. Weighed 0.2 g of fresh germinatedS.baicalensisseedlings and placed them in a pre-cooled mortar, added 2 mL of boric acid buffer (0.1 mol/L, pH=8.8) and 0.02 g of polyvinyl pyrrolidone. The buffer contained 5 mmol/L of β-mercaptoethanol, 1 mmol/L EDTA, and ground the sample into a homogenate in an ice bath, centrifuged at 4 ℃ and 10 000 r/min for 15 min, and the supernatant was the crude enzyme extract. The reaction system includes 2 mL of boric acid buffer (0.1 mol/L, pH=8.8), 0.8 mL of L-phenylalanine (0.02 mol/L), and 0.1 mL of enzyme crude extract. For the control, no enzyme solution was added. Instead, 0.1 mL of distilled water was added. After 30 min in a water bath at 30 ℃, added 0.2 mL of hydrochloric acid (6 mol/L) to stop the reaction, and measured the absorbance at the wavelength of 290 nm. Calculated the specific activity of the enzyme using the following formula.

Specific activity of enzyme 0.01&A/(mg·h)=△A×D/0.01×M×T

where △Ais the change in absorbance during the reaction time,Mis the fresh mass of the sample,Tis the reaction time, andDis the dilution factor, namely, the total enzyme solution extracted is the multiple of the enzyme solution in the reaction system.

2.2.6Determination of C4H activity. The determination method was the same as that in Section2.2.5. Weighed 0.2 g of fresh sample, cooled with liquid nitrogen. Added 3 mL of extraction solvent for grinding. The extraction solvent contained 0.05 mol/L of Tris-HCl (pH=8.9), 1 mmol/L of PMSF, 15 mmol/L of β-mercaptoethanol, 10 μmol/L of Leupeptin, 5 mmol/L of Vc, 0.15% PVP (m/V), 4 mmol/Lof MgCl2, and 10% glycerol. The solution was centrifuged at 4 ℃ and 10 000 r/min for 20 min, and the supernatant was the crude C4H extract. The enzyme reaction solution contained 0.8 mL of enzyme extract, 2.2 mL of buffer [2 μmol/L of trans-cinnamic acid, 5 μmol/L G6-PNa2, 0.05 mol/L of Tris-HCl (pH=8.9), and 2 μmol/L of NADPNa2]. After oscillating reaction at 25 ℃ for 30 min, added 100 μL of 16 mol/L HCl to stop the reaction, centrifuged at 10 000 r/min for 15 min, and took the supernatant for colorimetric determination at 340 nm. The reference solution was the extraction solution not containing enzyme (but containing 0.8 mL of ddH2O).

2.2.7Determination of CHS activity. (i) Preparation of crude extract. All operations were carried out at 0-4 ℃. Put the frozen 10 g sample into a mortar, added a small amount of quartz sand and 10% (W/W) polyvinyl pyrrolidone. Used extraction buffer (0.1 mol/L of phosphate buffer with pH 6.8, 1.4 mmol/L of 2-mercaptoethanol, 40 mmol/L of ascorbic acid, 3 mmol/L of ethylenediaminetetraacetic acid (EDTA), 10 μmol/L of leupeptin and 0.2 mmol/L of phenylmethylsulfonyl chloride; cooled with liquid nitrogen before use) Mixed the frozen powder well. After unfreezing, centrifuged the homogenate at 8 000 r/min for 20 min, and then used 30%-70% (NH4)2SO4to salt out the extracted protein. Dissolved with 2.5 mL of phosphate buffer (0.1 mol/L, pH 6.8), 1.4 mmol/L of 2-mercaptoethanol, 40 mmol/L of ascorbic acid and 5% (W/V) trehalose (frozen with liquid nitrogen before use), Then used 70% (NH4)2SO4to salt out the precipitate, and finally used a Sephadex G-25M column (Pharmacia) to desalt to obtain the sample.

(ii) HPLC quantitative analysis. Took 500 μL of the extracted sample and 500 μL of buffer [0.5 mol/L of phosphate buffer, 2.8 mmol/L of 2-mercaptoethanol, 2% (W/V) bovine serum albumin] and mixed well. After adding 100 μL of malonyl-CoA (0.4 mmol/L) and 100 μL of cinnamoyl-CoA (0.2mmol/L) to the mixed solution, the reaction started. At this time, heated in a water bath at 30 ℃ for 40 min. After heating in a water bath, added 1 mL of ethyl acetate, mixed well with a vortex, and then centrifuged for 2 min. Transferred the ethyl acetate layer to a new container, and evaporated the ethyl acetate using a vacuum dryer. Then, used 300 μL of MeOH to dissolve the residue and performed the HPLC analysis at the injection volume of 20 μL.

(iii) HPLC conditions. Using Agilent 1100 high performance liquid chromatograph; Agilent DAD detector. The eluent was composed of MeOH, H2O, and 85% H3PO4(280∶136∶1) (pH 2.6), the flow rate was 1.0 mL/min, and the detection wavelength was 290 nm.

2.2.8Determination of secondary biomass content. (i) Instrument conditions. Agilent 1100 high performance liquid chromatograph; Agilent DAD detector; Agilent Chemstation workstation (Agilent Technologies, USA); used the gradient elution of water-methanol-phosphoric acid solvent system, the detection wavelength was 276 nm, the flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the injection volume was 20 μL. The gradient elution time program was listed in Table 4.

Table 4 Mobile phase gradient elution

(ii) Preparation of reference solution. Precisely weighed 6.1, 3.0, 2.1, 1.1, 10.3, 1.5 and 2.9 mg of baicalin, baicalein, wogonoside, wogonin, scutellarin, and oroxylin A reference substance, and dissolved with methanol to a volume of a 100-mL volumetric flask, and mixed well. The HPLC chromatogram was shown in Fig.1.

Note: A: scutellarin, B: baicalin, C: wogonoside, D: baicalein, E: wogonin, F: oroxylin A.

(iii) Linear relationship test. Took the above mixed reference solution of baicalin, baicalein, wogonoside, wogonin, scutellarin, and oroxylin A, injected 0, 2, 4, 8, 12, 16 and 20 μL respectively, and determined the peak area value according to the above chromatographic conditions. Taking the injection volume as the abscissa (X) and the peak area as the ordinate (Y), obtained the following regression equations:Y=90.464X-182.57 (R=0.999 1),Y=193.59X-311.68 (R=0.999 2) ,Y=86.437X-37.023 (R=0.992),Y=156.23X-207.14 (R=0.999 1),Y= 49.077X-15.829 (R=0.999 3) andY=1 203.84X-110.16 (R=0.999 2), indicating that the linear relationship is good.

(iv) Preparation of test solution. Precisely weighed 0.5 g of each sample ofS.baicalensis, added 25 mL of 70% ethanol, weighed, ultrasonically treated for 1 h, cooled, made up the weight with 70% ethanol, filtered the supernatant with a 0.45 m filter membrane, and took the subsequent filtrate as test solution.

(v) Precision test. Precisely absorbed 20 μL of the reference solution and injected 6 consecutive times to determine the peak areas of baicalin, baicalein, wogonin, wogonoside, scutellarin, and oroxylin A; theirRSDvalues were 0.23%, 0.32%, 0.41%, 0.29%, 0.82% and 0.61%, respectively, indicating that the precision was good and met the requirements.

(vi) Stability test. Precisely absorbed 20 μL of the reference solution and determined the peak area of baicalin, baicalein, wogonin, wogonoside, scutellarin, and oroxylin A at 0, 2, 4, 6, 12, 18, 24, 48, and 72 h, respectively; theirRSDvalues were 0.86%, 0.57%, 1.02%, 0.93%, 1.12% and 0.95% within 72 h, indicating that the test solution was stable within 72 h.

(vii) Recovery rate test. Precisely weighed 0.5 g of each sample with a known content and placed in a 25-mL volumetric flask, separately added the reference solution of baicalin, baicalein, wogonoside, wogonin, scutellarin, and oroxylin A, determined the recovery rate according to the above chromatographic conditions, the recovery rates of the six reference substances were 101.22%, 99.87%, 102.45%, 103.21%, 98.82% and 100.67%, respectively, and theRSDvalues were 0.63%, 0.93%, 0.89%, 1.29%, 1.02% and 0.91%, respectively, indicating that the recovery rates of the 6 reference substances met the requirements.

3 Results and analysis

3.1 Effects of temperature on seed germinationThere is a large difference in the suitable temperature for germination of different plant seeds. As indicated in Table 5,S.baicalensisseeds germinated very slowly at 15 ℃, and the germination rate, germination potential and germination index were all lower than other treatments. With the rise of temperature, at 15-25 ℃, the germination rate, germination potential and germination index showed a rising trend, and all indicators reached the highest value at 25 ℃, but at 25-35 ℃, all indicators showed a declining trend. At too high or too low temperature,S.baicalensisseedlings would not grow well, especially at high temperatures, rotting is likely to occur. These indicate that there are significant differences in germination rate, germination potential and germination index between different temperature treatments (P<0.05), and 25 ℃ is the optimum temperature for germination ofS.baicalensisseeds.

Table 5 Germination of Scutellaria baicalensis Georgi seeds at different temperatures

3.2 Effects of temperature on the antioxidant enzyme system of during seed germinationFig.2 shows that with the rise of the germination temperature, the activities of SOD, POD and CAT in the seeds first increased and then decreased. Specifically, the activities of SOD and CAT reached the highest value at 25 ℃, 366.97, 578.87 and 2 806.93 U/(h·g/Fw), respectively, but POD reached the highest value of 651.81 U/(h·g/Fw) at 20 ℃. By contrast, APX showed the opposite trend, reaching the highest value of 900.00 U/(h·g/Fw) at 35 ℃.

Fig.2 Effect of temperature on the antioxidant enzyme system during germination of Scutellaria baicalensis Georgi seeds

3.3 Effects of temperature on soluble sugar content during seed germinationFig.3 shows that the higher the temperature, the lower the soluble sugar content in the seeds, which is mainly related to the intensity of various metabolic activities in the seeds. Generally, the higher the temperature, the stronger the metabolism, and the more soluble sugars are consumed, resulting in a decrease in its content. There was no significant difference in the soluble sugar content between 25 and 20 ℃, but there were significant differences between 15, 30 and 35 ℃.

Fig.3 Effect of temperature on the soluble sugar content of Scutellaria baicalensis Georgi during germination

3.4 Effects of temperature on the activities of PAL and C4H during seed germinationAs shown in Fig.4, the activities of PAL and C4H inS.baicalensisseeds were different at different temperatures. The activity was highest at 20 ℃. When the temperature was lower than 20 ℃, the activities of PAL and C4H show an increasing trend as the temperature rises, and when the temperature was higher than 20 ℃, the activities of both showed a decreasing trend, and the decrease of PAL activity was larger. The results of statistical analysis showed that there was a good correlation between the activities of PAL and C4H (R=0.952,P<0.05), suggesting that their activities had the same trend with the temperature. There was no significant difference in PAL activity between 15 and 25 ℃ (P < 0.05). There were significant differences in PAL activity between 20, 25, 30, and 35 ℃ (P< 0.05). There was no significant difference in C4H activity between 30 and 35 ℃ (P<0.05), but there was a significant difference in C4H activity between 15, 20, 25, and 35 ℃ (P<0.05).

Fig.4 Effects of temperature on the activities of PAL and C4H during seed germination of Scutellaria baicalensis Georgi seeds

3.5 Effects of temperature on CHS activity during seed germinationAs shown in Fig.5, the CHS activity increased with the rise of temperature, reaching the highest value at 25 ℃. However, at 25-35 ℃, CHS activity sharply declined. Statistical analysis showed that there were significant differences in CHS activity between 15, 20, 25, and 30 ℃ (P<0.05), but there was no significant difference between 15 and 35 ℃ (P<0.05).

Fig.5 Effects of temperature on CHS activity during germination of Scutellaria baicalensis Georgi seeds

3.6 Effects of temperature on the secondary biomass content during seed germinationTable 6 shows that when the temperature rose from 15 ℃ to 20 ℃, the contents of scutellarin, baicalein and wogonin increased, and reached the highest value at 20 ℃. When the temperature rose from 20 ℃ to 35 ℃, the contents of scutellarin, baicalein and wogonin showed a declining trend. These suggest that both low temperature and high temperature are not conducive to the biosynthesis of scutellarin, baicalein and wogonin. At 15 ℃ and 20 ℃, the contents of baicalin and oroxylin A had similar changes, and the difference was not significant (P<0.05), but when the temperature rose from 25 ℃ to 35 ℃, both contents declined. When the temperature rose from 15 ℃ to 35 ℃, the content of wogonin showed a declining trend. When the temperature rose from 15 ℃ to 20 ℃, the total amount of these 6 components was increasing, but when the temperature rose from 20 ℃ to 35 ℃, their total amount dropped, which is the similar to the trend of changes in PAL and C4H. The statistical results show that the total amount of these 6 components has a good linear relationship with PAL and C4H (R=0.956,R=0.951,P<0.05), suggesting that both low temperature and high temperature are not conducive to the biosynthesis of flavonoids inS.baicalensis, and the most suitable temperature is 20 ℃.

Table 6 Effects of temperature on the secondary biomass content during germination of Scutellaria baicalensis Georgi seeds mg/g

3.7 Correlation analysisAt different temperatures, there was a good positive correlation between the content of flavonoids and the content of soluble sugars, PAL and C4H activities (Table 7), and the correlation coefficients wereR=0.894*,R=0.956*andR=0.951*, respectively. By contrast, the content of flavonoids and CHS did not show a good linear relationship, while there was a good positive correlation between the activities of PAL and C4H (R=0.952*). In addition, there was no good linear relationship between the soluble sugar content and the activities of PAL, C4H and CHS, and there is no linear correlation between the activities of CHS and PAL and C4H, indicating that different enzyme activities are greatly affected by temperature.

Table 7 Linear correlation analysis of soluble sugar content, key enzyme activity and secondary biomass content

4 Discussion and conclusions

Seed germination is a key stage in the entire life history of higher plants. Generally, the growth of seed plants begins with seed germination. Agricultural production also mostly begins with seeding (and seed germination). Therefore, quick seed germination and healthy growth can lay a solid foundation for high-quality and high-yield. In this study, we explored the germination ofS.baicalensisseeds at different temperatures, and found that the germination rate, germination potential and germination index ofS.baicalensisseeds are significantly affected by temperature, and the most suitable temperature for the germination ofS.baicalensisseeds is 25 ℃.

During seed germination, a series of physiological and biochemical changes occur in seeds. In order to reduce the destruction of active oxygen, the seeds themselves form a series of active oxygen scavenging systems. The antioxidant enzyme system plays an important role in the natural defense mechanism of plants. Through experiments on the effects of different temperatures on the germination ofS.baicalensisseeds, we found that the activities of SOD, POD and CAT inS.baicalensisseeds treated at low and high temperatures were higher than those inS.baicalensisseeds treated at moderate temperature, suggesting thatS.baicalensisseeds have a good ability to defend against the adverse environment when they encounter bad environment.

The main formation pathway of flavonoids is the phenylalanine pathway. In this pathway, the first key enzyme is phenylalanine ammonia lyase, and the second key enzyme is C4H, and chalcone synthase is a key enzyme that forms the branch of flavonoids. Through experiments on the effects of different temperatures on the germination ofS.baicalensisseeds, we found that the PAL, C4H, and CHS activities ofS.baicalensisseeds treated with low and high temperature were lower than those treated with moderate temperature, which was consistent with the change trend of flavonoids content. In addition, there was a good positive correlation between flavonoids and soluble sugar, PAL activity and C4H activity, and the correlation coefficients wereR=0.894*,R=0.956*andR=0.951*, respectively.