In situ visual and content changes analysis of coumarins in Radix Angelicae dahuricae by LSCM combined with LC-MS technology

2.3.1 Screening of slice thickness

Fresh Radix Angelicae dahuricae was washed and cut into small segments of ∼ 0.5 cm to obtain a sample. Samples were poured into 15 % glycerin and vacuumized until the sample sank to the bottom of the bottle. Then the glycerin solution on the surface was sucked dry. The sample was immersed in the pre-cooled sample holder with OCT embedding agent, frozen on the freezing table until OCT turned white, and then moved to the head of the slicer for slicing. They were cut into 20 µm, 25 µm, 30 µm, 35 µm, 40 µm thick pieces, integrity and stretch were observed under the bright-field of the forward inverted integrated fluorescence microscope.

2.3.2 Screening of cryoprotectants

In the frozen sectioning process of plant tissue, cryoprotectants play an important role in the stability of active components in plant tissue and the integrity of sectioning. Currently glycerin and sucrose are commonly used as cryoprotectants (Bromfield and Rosa, 2013). Protectants can combine with free water in plant tissue and reduce damage to ice crystals to cells. Therefore, Radix Angelicae dahuricae samples were placed in distilled water, sucrose solution with mass fractions of 5 %, 10 %, 15 %, 20 %, 25 % and glycerin solution with the same volume fraction, and vacuumized until the materials were sunk to the bottom of the bottle. The best cryoprotectant was obtained by comparing the quality of slices treated with different cryoprotectants.

2.3.3 Establishing the evaluation equation for the frozen section

Traditional evaluation methods cannot judge the change in overall slice quality caused by the small concentration change of cryoprotectant, so we (Chen et al., 2022) improved it in a previous study. The evaluation equation of frozen section of medicinal plant Angelica sinensis was established, y = λ1a + λ2b + λ3c + λ4d + λ5e + λ6f + λ7g（λ1 ∼ λ7 is the grade coefficient, indicating the importance of each item, λ1 + λ2 + λ3 + λ4 + λ5 + λ6 + λ7 = 20). A represents the integrity of the observed object (the integrity of the secretory cavity and catheter), b refers to the retention degree of the test content (the retention degree of coumarin), c refers to the integrity of the cork layer and cork inner layer, d refers to the difficulty of freezing the material, e refers to the slice stretch, f refers to the integrity of tissue and cells, g refers to the reduction of ice crystal damage. The slices of the same concentration were repeated for 3 times, and the image quality of the 3 repetitions was scored according to items a to g, with a full score of 5 points for each item. The final total score is calculated after the average of the total scores of three repetitions. The evaluation equation is y = 5a + 4b + 3d + 2 (c + e + f + g). The higher the score, the better the slice quality.

2.4.1 Observation of coumarins autofluorescence

Coumarins can produce blue fluorescence under UV irradiation, and are widely used because of their high quantum yield and stability (Zhao et al., 2016). Therefore, imperatorin was selected for spontaneous fluorescence observation, which is the high content of coumarin in Radix Angelicae dahuricae. A small amount of imperatorin standard was placed on the concave glass slide, and 15 % glycerin was added to seal the slide. The slides were placed under the DAPI channel of the forward inverted integrated fluorescence microscope for observation.

2.4.2 Localization of coumarins in Radix Angelicae dahuricae root.

Samples obtained from frozen sections are fixed with 95 % ethanol for 30 min to remove coumarins, because coumarins are readily soluble in methanol, ethanol, chloroform and other organic solvents (Sun et al., 2020). After the film was sealed, it was observed under the DAPI channel of the forward inverted integrated fluorescence microscope.

2.5.1 Determination of coumarins fluorescence parameters in Radix Angelicae dahuricae

In this study, imperatorin and isoimperatorin standards were selected to determine coumarin fluorescence parameters, as they were high-content compounds in Radix Angelicae dahuricae. Imperatorin and isoimperatorin weighed 0.2 mg, dissolved in a 10 mL volumetric flask of methanol, and then ultrasonically dissolved for 2 min to obtain the test solution, respectively. The test solution was placed in the cuvette and fed into the F97 fluorescence spectrophotometer. Combined with the excitation wavelength of LSCM, the fluorescence value of each substance was detected to determine the best excitation wavelength of Radix Angelicae dahuricae in LSCM.

2.5.2 Relative quantitative analysis of coumarins in Radix Angelicae dahuricae at different periods

According to the research of Qiang Lu (Lu, 2009), the growth and development period of Radix Angelicae dahuricae can be divided into four stages. Seedling stage: mid-September to early March. Leaf growth period: early March to early May. Root growth period: from early May to mid-July. Harvest period. mid-July to mid-September. Therefore, fresh Radix Angelicae dahuricae from five different growth periods in early March, early May, mid-July, early September and early November were selected as materials. Fluorescence intensity refers to the intensity of the light emitting fluorescence. The fluorescence intensity is proportional to the concentration of the fluorescent substance, so the average fluorescence intensity is used to represent the coumarin content. Tissue sections of Radix Angelicae dahuricae at different stages were prepared under selected optimum frozen section conditions, and then fluorescence imaging was observed and photographed under LSCM with the selected excitation wavelength. Fluorescence images of 10 different visual fields were obtained for each tissue section, and the average fluorescence intensity was recorded.

2.6.1 Preparation of the control solution

Standards for imperatorin, isoimperatorin, bergapten, xanthotoxin and 7-hydroxycoumarine (Fig. 1), all at 1 mg, were accurately weighed and dissolved in methanol solution, respectively. Then, a control solution (100 µg/mL) was prepared. They are taken in an appropriate amount, mixed and diluted to obtain a mixed control solution with a concentration of 10 µg/ mL.

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Fig. 1

Structures of coumarins of Radix Angelicae dahuricae.

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2.6.2 Preparation of test solution

Radix Angelicae dahuricae in different periods was dried at 37 ℃ (Zhang, 2005) and then crushed and sieved through 40 mesh. The medicinal powder of Radix Angelicae dahuricae in different periods was weighed to 3 g, added 30 mL of methanol in a conical flask, soaked for 30 min, extracted in a 40 ℃ water bath by ultrasound (power: 360 W, frequency: 40KHz) for 30 min, cooled to room temperature, and then methanol was used to make up the lost weight. A certain amount of solution was taken into a centrifuge tube and centrifuged into a high-speed centrifuge (speed: 8000r/min, time: 10 min), and then the supernatant was taken for 0.22 µm microporous membrane filtration to obtain the test solution.

2.6.3 LC-MS conditions

The data were obtained by liquid chromatography mass spectrometry (Agilent 1290–6460, U.S.A.). Chromatographic column: Agilent Eclipse Plus-C18 (150 mm × 2.1 mm, 1.8-µm); mobile phase: 0.1 % formic acid (a)–methanol (b), gradient elution (0–0.5 min, 5 %-15 % B; 0.5–0.8 min, 15 %-28 % B; 0.8–1.0 min, 28 %–32 % B; 1.0–1.2 min, 32 %-38 % B; 1.2–1.5 min, 38 %-45 % B; 1.5–4.5 min, 45 %-65 % B; 4.5–5.5 min, 65 %-95 %B; 5.5–6.0 min, 95 %-5% B; 6.0–8.0 min, 95 %-5% B); Column temperature: 35 ℃; Flow rate: 0.3 mL/min; Injection volume: 10 µL.

Mass spectrometry conditions: the dry gas and atomizer are nitrogen; the temperature of dry gas is 295 ℃, the flow rate of dry gas is 11 L/min, the atomizer pressure is 15 psi, the electric spray ion source (ESI), the spray voltage is-4.0 KV, the positive and negative ion monitoring mode and multiple reaction scanning mode (MRM) are selected.

3.1.1 Investigation of optimum thickness of frozen sections

As shown in Fig. 2, when the slice thickness of Radix Angelicae dahuricae is less than 30 µm, the degree of tissue fragmentation and the difficulty of slicing increase. When the slice thickness exceeds 30 µm, the slice is too thick and the field of vision is dark, although the slice integrity and stretch are high, resulting in reduced cell tissue clarity. Therefore, 30 µm was chosen as the slice thickness of Radix Angelicae dahuricae.

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Fig. 2

Microscopic observation on frozen sections of Radix Angelicae dahuricae with different thickness Notes: A1:20 µm, A2:25 µm, A3:30 µm, B1:35 µm, B2:40 µm, B3:30 µm.

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3.1.2 The best condition of frozen section

It could be seen from Table 1 and Fig. 3 that different concentrations of glycerin and sucrose as protectants have a significant impact on the integrity of frozen sections of Radix Angelicae dahuricae. When the concentration of glycerin and sucrose is less than 15 %, slice integrity gradually increases with the increase of concentration. When the concentration exceeds 15 %, it is difficult to freeze and slice the tissue, and the slices are difficult to transfer to the slide. However, when sucrose solution of the same concentration is used as a cryoprotectant, the integrity of the observed object is lower than that of glycerin solution. It could be concluded that soaking in sucrose for a long time will lead to softening of tissue cells in Radix Angelicae dahuricae, and the degree of chip fragmentation is serious through the slicing process. The integrity of no cryoprotectant and distilled water as protectants is poor, as shown in Fig. 3 (C2, C3). Therefore, when 15 % glycerin is selected as the most cryoprotectant for the frozen section of Radix Angelicae dahuricae, the section quality is the best and the integrity is high, as shown in Fig. 3(A2). The use of protectants plays an important role in the stability of active ingredients in plant tissues and the integrity of sections in the freezing process of plant tissues.

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Fig. 3

Microscopic observation of frozen sections of Radix Angelicae dahuricae with different sectioning methods Notes: A1: 5% glycerin treatment, A2:15% glycerin treatment, A3:25%glycerin treatment, B1:5% sucrose treatment, B2:15% sucrose treatment, B3:20%sucrose treatment, C1:25% sucrose treatment, C2: distilled water treatment, C3: no cryoprotectant.

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3.3.1 Determination of fluorescence parameters of coumarins in Radix Angelicae dahuricae

Combined with the excitation wavelength of LSCM, 405 nm, 488 nm, 568 nm and 647 nm were used as excitation wavelengths, respectively. The fluorescence value of each standard was detected, as shown in Table 2 and Fig. 5. Imperatorin and isoimperatorin both excited fluorescence at different excitation wavelengths. Both have the highest fluorescence value at 405 nm excitation wavelength and the lowest fluorescence value at 647 nm excitation wavelength. Therefore, 405 nm was selected as the excitation wavelength for the subsequent test.

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Fig. 5

Fluorescence spectrum of standard (excitation wavelength = 405) Notes: A: imperatorin; B: isoimperatorin.

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3.3.2 Fluorescence imaging results of coumarins in Radix Angelicae dahuricae at different periods

The average fluorescence intensity of coumarin in Radix Angelicae dahuricae at different periods was analyzed using the coumarin fluorescence imaging method of coumarin combined with LSCM and ZEN 2 software, and it was found that the average fluorescence intensity at different periods was different (Table 3).

The average fluorescence intensity of coumarin in Radix Angelicae dahuricae showed a gradual increase from seedling stage to harvest stage. In early March, the average fluorescence intensity of coumarin in Radix Angelicae dahuricae at seedling stage was the lowest, only 165.170. In mid-July, the average fluorescence intensity of coumarin increased by 103.542, indicating that the root growth period is the key period for coumarin content accumulation. The average fluorescence intensity of coumarin in Radix Angelicae dahuricae during harvest time in early September was the highest, reaching 444.610. The nutrient growth period of Radix Angelicae dahuricae is from March to October, which is conducive to nutrient accumulation in the root. After November, Radix Angelicae dahuricae entered the reproductive growth period. In order to maintain life metabolism, the root system consumed some dry matter, so the average fluorescence intensity of coumarin began to decrease. In conclusion, the content of coumarin in Radix Angelicae dahuricae showed an “S” trend during the growth period (Fig. 6).

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Fig. 6

Variation trend of coumarin average fluorescence intensity in Radix Angelicae dahuricae in different periods.

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3.4.1 Methodological validation

The linear relationship, precision, repeatability, stability and sample recovery of the determination method were investigated. The correlation coefficient of the linear regression equation for imperatorin, isoimperatorin, bergapten, xanthotoxin and 7-hydroxycoumarine in their linear concentration ranges is 0.9985 ∼ 0.9996, and the linear range is 0.16 ∼ 6.00 μg/mL (Table 4). In the precision experiment, the RSD calculated by peak area is 0.59 %∼2.19 %. In the repeatability experiment, the RSD calculated by peak area is 0.49 %∼2.70 %. In the stability experiment, the test solution was injected and measured at 0, 2, 4, 8 and 12 h respectively, and the RSD of its peak area was 0.30 %∼2.45 %. The recovery rate of the sample is over 90.08 %, and the RSD is less than 3.94 %. Which met the requirements of quantitative analysis.

3.4.2 Sample content determination results

After fresh samples were harvested, they were dried at 37℃ to reduce volatilization of Radix Angelicae dahuricae during drying. As shown in Fig. 7 and Fig. 8, the coumarins content reached the highest in the early September, and increased most rapidly in the mid-July when the root was growing for a long time, indicating that this period is the accumulation period of coumarins content in Radix Angelicae dahuricae. The contents of imperatorin and isoimperatorin were high, which increased until the harvest period, and began to decrease during the reproductive growth period. The content of 7-hydroxycoumarin is less and tends to be stable. The content of bergapten lactone increased in the early stage of growth of Radix Angelicae dahuricae, and the content changed little in the late stage. Xanthotoxin content tended to be stable before reaching the harvest period, and began to decrease during the reproductive growth period. Total coumarin content in Radix Angelicae dahuricae in different periods increased in an “S” curve. In this work, the same batch of samples in the same period were analyzed both by LC-MS and LSCM. The average fluorescence intensity measured by LSCM represented the coumarin content. Therefore, the analysis results of total coumarin content change in different periods by LC-MS and LSCM were consistent, which verified the accuracy of the experiment.

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Fig. 7

LC-MS diagram of representative coumarins in Radix Angelicae dahuricae at different periods Notes: 1: 7-hydroxycoumarin, 2: xanthotoxin, 3: bergapten, 4: imperatorin, 5: isoimperatorin, A: mixed control solution, B1: early March, B2: early May, B3: mid-July, B4: early September, B5: early November.

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Fig. 8

Variation trend of coumarins content in Radix Angelicae dahuricae at different periods by LC-MS.

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