Effect of different drying degrees on content of various chemical components and in vitro bioactivity of Chrysanthemi Indici Flos

Abstract

Objective

Chrysanthemi Indici Flos (CIF), the inflorescence of Chrysanthemum indicum L., is widely used in traditional Chinese medicine. Traditionally, the content of active components in Chinese medicinal materials often changes during processing. However, the variation patterns of main active components in CIF during drying remain largely unknown. This study aims to investigate the contents of multiple chemical components and in vitro bioactivity of CIF with different drying degrees, so as to provide an optimized scheme for its origin processing.

Methods

Fresh CIF were dried at 60 °C to obtain samples with different water contents (80%WC, 60%WC, 40%WC, 20%WC and DS). HPLC was used to determine eight chemical components (chlorogenic acid, 3,5-O-dicaffeoylquinic acid, galuteolin, linarin, luteolin, naringenin, kaempferol and isorhamnetin). In vitro assays evaluated antioxidant activity (radical scavenging), anti-inflammatory activity (enzyme inhibition) and antibacterial activity (disk diffusion, MIC) against several strains.

Results

Among eight chemical components, chlorogenic acid and linarin decreased significantly with drying, luteolin and kaempferol first increased then decreased (peaking at 40%WC), and naringenin was highest in 80%WC. HPLC fingerprint similarity was lowest (0.559) between 80%WC and DS samples. Antioxidant (Total reducing capacity/DPPH/ABTS/·OH IC50: 80%WC > DS) and anti-inflammatory (XOD/LOX IC50: 80%WC > DS) activities strengthened with drying. Extracts inhibited Staphylococcus aureus (MIC 1.560–3.125 mg/mL), Listeria monocytogenes (DS group inhibition zone larger) and Escherichia coli (MIC 3.125 mg/mL), but not Pseudomonas aeruginosa or Salmonella paratyphoid.

Conclusion

Drying degree significantly affects CIF’s chemical profiles and in vitro bioactivities, with dry samples showing superior antioxidant/anti-inflammatory properties and specific antibacterial effects. These findings offer targeted guidance for optimizing primary processing parameters of CIF.

1. Introduction

Chrysanthemi Indici Flos (CIF, Yejuhua in Chinese), the capitulum of Chrysanthemum indicum L., has been used as an important traditional Chinese medicine (TCM) in China for 2 000 years. Chinese medical classics document that CIF is effective in clearing heat, detoxifying, reducing fire, and calming the liver. Nowadays, CIF is widely utilized in medicine, healthcare, detergents, and chemical products, reaching an annual demand of 10 000 tons (Li et al., 2023, Ren et al., 2024, Tian et al., 2020).

Modern pharmacological studies have demonstrated strong anti-diabetic antioxidant, anti-inflammatory, and antibacterial effects of CIF (Wang et al., 2022, Youssef et al., 2020, Yu et al., 2019, Wu et al., 2014). Previously reports have also highlighted the potential pharmacodynamic basis of CIF. Specifically, CIF is rich in various bioactive components, including flavonoids, organic acids, terpenoids, and volatile oils (Yang, Liu, & Shi, 2019). More than 40 flavonoids have been isolated and identified from CIF, including flavones, dihydroflavones, flavonols, and chalcones as the main types. (Dai and Sun, 2021, Shao et al., 2020). Regulating and increasing the content of these active components in CIF may enhance its pharmacological effects.

Obtaining high-quality CIF is one of the urgent challenges, with such quality defined by high levels of pharmacologically valuable active components and their strong bioactivity. The active components of medicinal plants, typically secondary metabolites, are closely linked to factors including origin, cultivation techniques, harvest timing, primary processing, deep processing, and storage (Juhaimi et al., 2020, Zhao et al., 2012). Noteworthily, previous studies have found that steaming, drying and other common processing methods have a significant effect on the content of chemical components in medicinal plants (Nozad et al., 2016). These studies offer valuable insights that examining quality changes in TCM during primary processing could be a promising approach to addressing the issues. Yet CIF often undergoes fixation and drying during origin processing. How the chemical content and extract activity of CIF change during drying remains insufficiently studied.

Here, CIF samples with different drying degrees were prepared. Contents of eight chemical components were quantified using high-performance liquid chromatograph (HPLC). Additionally, the basic bioactivities of CIF with different drying degrees were evaluated through in vitro antioxidant, anti-inflammatory, and antibacterial assays. This study explored changes in components and bioactivity of CIF during drying, aiming to provide theoretical references for the primary processing and quality control of CIF.

2. Materials and methods

2.1. Materials

Plant materials (Fresh CIF) were collected from Macheng City, Hubei Province, China (31°13′ N, 115°0′ E) in November 2020. After removing sediment and impurities, the CIF materials were prepared for further processing. Reference substances of chlorogenic acid (purity ≥98%, HPLC, J0120AS) and linarin (purity ≥ 98%, HPLC, N0901AS) were prepared from Meilun Biotechnology Co., Ltd (Dalian, China). Reference substances of galuteolin (purity ≥ 98%, HPLC, Y19A8H34394), 3,5-O-dicaffeoylquinic acid (purity ≥ 98%, HPLC, 79680010), luteolin (purity ≥ 98%, HPLC, Y01D656815), kaempferol (purity ≥ 98%, HPLC, G11A11L110978), isorhamnetin (purity ≥ 98%, HPLC, P17S9F68615), and naringenin (purity ≥ 98%, HPLC, YJ0603HA13) were prepared from Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

2.2. Processing methods of samples

In this study, an electric thermostatic with low temperature (60 °C) was used to obtain the CIF samples with different degrees of drying. Briefly, a certain amount of fresh CIF sample was taken and dried in the drying oven at 60 °C to a constant weight. The sample with constant weight is considered to be the usual dry sample state (water-free) under natural condition. During the drying process, the water content of the sample was determined by the weighing method, and the calculation formula is as follows: Water content = 1 − m₂/m₁. In the formula, m₁ represents the current weight of the sample, and m₂ represents the weight of the sample dried to constant weight (the weight of the dry sample). According to the above methods, five water content levels were set, and the water content of five groups of samples were controlled within a certain range: 80% ± 5% (80%WC, WC means water content), 60% ± 5% (60%WC), 40% ± 5% (40%WC), 20% ± 5% (20%WC) and dry sample (DS) (Fig. 1A). Although the weight of the sample is constantly changing during drying, the weight of the sample is converted to dry weight in order to analyze the chemical composition based on dry matter.

Fig. 1.

Comparison of the HPLC chromatograms of CIF with different degrees of drying. (A) Appearance of CIF sanples with different degrees of drying; HPLC chromatograms of standards (B) and CIF sample (C). 1-chlorogenic acid; 2-galuteolin; 3-3,5-O-dicaffeoylquinic acid; 4-linarin; 5-luteolin; 6-naringenin; 7-kaempferol; 8-isorhamnetin; (D) Comparison of the HPLC chromatograms of CIFs with different degrees of drying.

2.3. Measurement of the contents of multiple chemical components

To compare the differences of multiple chemical components of CIF with different degrees of drying, the contents of eight chemical components (chlorogenic acid, 3,5-O-dicaffeoylquinic acid, galuteolin, linarin, luteolin, naringenin, kaempferol and isorhamnetin) in CIF samples were measured. A previously reported method was referenced in the assay (Song et al., 2019). Briefly, 1 g CIF sample was accurately weighed (Weight was 80%WC as standard.), added with liquid nitrogen, grinded for 60 s, placed in a conical flask. Then the ground sample was added with 30 mL methanol and weighed. After 30 min of ultrasonic treatment, the sample was cooled to ambient temperature, re-weigh and make up the lost weight with methanol, and then filtered and the filtrate was collected. Then the sample was filtered through a 0.45 μm organic filter to obtain the sample solution. The eight active components of samples were determined using HPLC (Agilent Technologies Inc., CA, USA) (Fig. 1B, C). The determination condition was as below. Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6 mm × 100 mm, 3.5 μm). Mobile phase: acetonitrile to 0.1% phosphoric acid. Gradient elution: 0−6.5 min, 10%–18% acetonitrile; 6.5−10.5 min, 18%–20% acetonitrile; 10.5–18 min, 20%−60% acetonitrile; 18−26.5 min, 60%–80% acetonitrile. Flow rate: 1.0 mL/min. Column temperature: 30 °C. Detection wavelength: 326 nm. Injection volume: 5 μL. For the generation of standard curves, the standards of the eight active components were prepared according to Table S1. Then 1 mL of each standard solution was measured and diluted to 10 mL with methanol, filtered through a 0.45 μm organic filter to obtain the mixed standard solution. The mixed standard solution was diluted to 100%, 80%, 60%, 40%, 20%, 10%, 5% of the original solution, and then injected, respectively, and were detected at wavelength of 326 nm. The peak area values of each standard were recorded. The linear regression equations were then calculated using the sample volume of standards as abscissa ($x$) and the peak areas as ordinate ($Y$) (Table S2). For the quantity evaluation of estimated samples, the peak values of each active component were substituted to the linear regression equations to obtain the sample sizes. The method is used for methodological investigation (Table S3).

2.4. Measurement of antioxidant activities in vitro

2.4.1. Total reducing power assay

The activity of total reducing power was measured by the method of Prussian blue (Oyaizu, 1986). Briefly, 0.2 mL of varying concentration of extract solutions (10, 5, 2.5, 1.25, 0.625, 0.3125 mg/mL) were mixed with 2.5 mL of 0.2 mmol/L phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide (III) solution. The mixed solution was incubated at 50 °C for 20 min. After that, 2.5 mL of 10% trichloroacetic acid was added, and then the mixture was centrifuged substantially using a centrifugal apparatus (15 min, 3 000 r/min). Afterwards, 2.5 mL of upper layer was added to 2.5 mL of water and 0.5 mL of 0.1% ferric chloride, the absorbance was recorded at 700 nm by the Ultraviolet–Visible Spectrophotometer (UV-1100, MACY INSTRUMENT, Shanghai, China). The total reducing power was reflected by the absorbance value.

2.4.2. DPPH radical scavenging assay

The ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging was measured by a previously reported method and some adjustments were made in the method (Kim et al., 2018). Briefly, 1 mL of extract solutions at different concentrations (10, 5, 2.5 mg/mL) were added to 0.1 mL of 0.1 mmol/L DPPH solution. The mixed reaction solution was incubated for 30 min in the dark environment at 30 °C, and then the absorbance was measured at 517 nm by an enzyme-labeled instrument (Cytation 3, BioTek, USA). Methanol was used as control. The scavenging activity of DPPH radical (%) was calculated as follows: $\mathrm{Scavenging}\mathrm{activity}=(D_0-D_{\mathit{x}})/D_0\times 100\%$, where D_x denotes the absorbance of the sample with DPPH solution, and D₀ denotes the absorbance of control.

2.4.3. ABTS radical scavenging assay

The ability of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging was determined according to a previously reported method and some adjustments were made in the method (Huang et al., 2018). ABTS solution (7 mmol/L) was mixed with potassium persulfate solution (2.45 mmol/L) (v:v = 1:1). The mixture was placed at ambient temperature in dark for 12 h to obtain the stock solution of ABTS. To obtain the working solution whose absorbance reading of 0.70 ± 0.02 at 734 nm, the stock solution was diluted (v:v = 1:45). Then 1 mL of extract solutions at varying concentrations (10, 5, 2.5, 1.25, 0.625, 0.312 5 mg/mL) were added to 4 mL of working solution of ABTS, oscillate for 30 s and incubated for 30 min in the dark environment. And the absorbance was then measured at 734 nm by an enzyme-labeled instrument (Cytation 3, BioTek, USA). Methanol was used as control. The scavenging activity of ABTS radical (%) was calculated as follows: $\mathrm{Scavenging}\mathrm{activity}=(A_0-A_{\mathit{x}})/A_0\times 100\%$, where Ax denotes the absorbance of the sample with ABTS solution, and A₀ denotes the absorbance of control.

2.4.4. Hydroxyl radical scavenging assay

The ability of Hydroxyl radical scavenging was measured according to the method of salicylic acid (Smirnoff & Cumbes, 1989) and some adjustments were made in the method. Concisely, the reaction mixture generating hydroxyl radicals contained 0.1 mL of H₂O₂ (8.8 mmol/L), 0.1 mL of FeSO₄ (9 mmol/L), 0.1 mL of salicylic acid ethanolic solution (9 mmol/L), and 1 mL of the extract solutions of varying concentrations (10, 5, 2.5, 1.25, 0.625, 0.312 5 mg/mL). The mixed solution was incubated at 50 °C for 20 min. After that, the absorbance was measured at 734 nm by an enzyme-labeled instrument (Cytation 3, BioTek, USA). Methanol was used as control. The scavenging activity of Hydroxyl radical (%) was calculated as follows: $\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\left[H_0-\left(H_x-H_{x0}\right)\right]/H_0\times 100\%$, where H_x denotes the absorbance of the mixed solution, H₀ denotes the absorbance of control. For eliminating the influence of sample solution color on the assay, the background control was set (H₂O₂ is replaced by deionized water), H_x0 denotes the absorbance of control.

2.5. Measurement of anti-inflammation activities

2.5.1. Preparation of sample extract solution

For determining the in vitro anti-inflammatory activity of CIF with different degrees of drying, the solutions of sample extracts were prepared. First, 3 g sample was accurately weighed (Weight was 80%WC as standard.), added with liquid nitrogen, grinded for 60 s, placed in a conical flask. Then the ground sample was added with 100 mL methanol and weighed. After heating and refluxing in a water bath for 3 h, cooled the sample to ambient temperature, reweighed and compensated for the weight loss with methanol, then filtered and collected the filtrate.

Then the extract solution was concentrated into thick paste by rotary evaporator, and methanol was evaporated. And then the extract solids were dissolved in deionized water to obtain the sample extract solution (Tankeo et al., 2016).

2.5.2. Xanthine oxidase (XOD) inhibition assay

The ability of XOD inhibition was determined according to a previously reported method and some necessary adjustments were made in this study (Djouahri et al., 2014). Concisely, 50 μL of sample extract solutions at varying concentrations (10, 5, 2.5, 1.25, 0.625, 0.312 5 mg/mL) were added to 50 μL of XOD solution (0.4 U). The mixed reaction solution was incubated for 30 min in the dark at 37 °C. Then 50 μL of xanthine solution (0.24 mmol/L) was added to start the reaction. After 20 min of reacting at 25 °C, 50 μL of HCl (1 mol/L) was used to terminate the reaction. The absorbance at 290 nm was measured (Enzyme-labeled Instrument, Cytation 3, BioTek, USA). The inhibition activity of LOX (%) was calculated as follows: $\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\left[X_c-\left(X_a-X_b\right)\right]/X_c\times 100\%$, where X_a denotes the absorbance of the final reaction mixture, and X_b denotes the absorbance of background (The enzyme solution was replaced by 0.2 mol/L boric phosphate buffer with pH 7.5), X_c denotes the absorbance of control (the sample extract solution was replaced by deionized water). In calculation, all of the above absorbance subtracted the absorbance at 0 min of incubation.

2.5.3. Lipoxygenase (LOX) inhibition assay

The ability of LOX inhibition was determined according to a previously reported method and some adjustments were made in the method (Phan, Bucknall, & Arcot, 2018). Concisely, 20 μL of sample extract solutions at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.312 5 mg/mL) were added to 200 μL of LOX solution (About 5 000 U/mL). The mixed reaction solution was incubated for 30 min in the dark environment at 30 °C. Then 1.5 mL of linoleic acid solution was added to start the reaction. After 3 min of reacting at 30 °C, 3 mL of ethanol was used to terminate the reaction. And then 5 mL of distilled water was added to dilute the reaction mixture. The absorbance at 234 nm was measured (Ultraviolet–Visible Spectrophotometer, UV-1100, MACY INSTRUMENT, Shanghai, China). The inhibition activity of LOX (%) was calculated as follows: $\text{Inhibition activity}=\text{1}-(L_a-L_b)/(L_c-L_d)\times \text{1}00\%$, where L_a denotes the absorbance of the final reaction mixture, and L_b denotes the absorbance of background (The enzyme solution was replaced by 0.2 mol/L boric acid buffer with pH 9.0), L_c denotes the absorbance of reaction mixture without sample extract solution (replaced by deionized water), ‘L_d denotes the absorbance of control (Ethanol was added before adding the substrate to terminate the reaction).

2.6. Measurement of antibacterial activity

2.6.1. Bacterial strains

In this study, the following bacterial strains were obtained from microbial strains preservation center, College of Life Science, Nanjing Agricultural University (Nanjing, China): Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella paratyphoid, Pseudomonas aeruginosa. The activated strain was cultured in Luria Broth liquid (LB) medium for 6 h (200 r/min, 37 °C), and the bacterial solution was diluted with sterile normal saline to a concentration of about 1 × 10⁸ CFU/mL (0.5 michaelis turbidimetric tube was used as the bacterial solution turbidity standard) to obtain the test bacterial solution.

2.6.2. Measurement of antibacterial activity

The antibacterial activities of extracts from CIF with different degrees of drying were measured using the paper disk diffusion method (Bauer, Kirby, Sherris, & Turck, 1966), and some adjustments were made. Briefly, the aseptic filter paper was soaked in the sample extract solution (50 mg/mL), placed under the ultraviolet lamp for 8 h, and evaporated slightly. After that, the filter paper discs with different concentrations of extract were placed on the plate culture media (LB) coated with 0.15 mL bacterial solution (about 10⁸ CFU/mL), incubated at 37 °C for 24 h. Sterile normal saline was used as blank. The antibacterial activity was evaluated by measuring the diameter of the inhibition zones.

2.6.3. Measurement of minimum inhibition concentration (MIC)

The minimum inhibitory concentration was determined according to the double dilution method in a 96-well microplate (Sousa et al., 2015), and some necessary adjustments were made in this study. In short, the mixture consisted of 100 μL of sample extract solution (0.781 25−50 mg/mL), 100 μL of LB and 100 μL of bacterial solution (about 1 × 10⁸ CFU/mL). Sterile normal saline instead of sample solution was used as control. The MIC was defined as the lowest concentration that inhibited growth of bacteria.

2.7. Data analysis

Data were analyzed using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). All the results were expressed as the means ± standard deviations (n = 3). The comparison of means between groups was performed with one-way analysis of variance (ANOVA) followed by LSD test, and values of P < 0.05 were considered significant.

3. Results

3.1. Contents of multiple chemical components

There were significant differences in the contents of eight chemical components in CIF samples with different degrees of drying (Table 1). With the deepening of drying degree (water content decreases), the contents of chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid, and linarin decreased in general; the contents of luteolin, kaempferol, and isorhamnetin first increased and then decreased, with the final values in the DS group lower than those in the 80%WC group; the content of naringenin in the 80%WC group was significantly higher than that in other groups. According to the chromatogram of CIF samples (Fig. 2B), the compound with a retention time of approximately 21 min showed a relatively high relative content, but its identification was limited by the lack of a standard.

Table 1.

Contents of eight chemical components in CIF with different degrees of drying (mean ± SD, n = 3).

Groups	Eight chemical components detected in CIF samples with different degrees of drying
	Chlorogenic acid (µg/g)	Galuteolin (µg/g)	3,5-O-Dicaffeoylquinic acid (µg/g)	Linarin (µg/g)	Luteolin (µg/g)	Naringenin (µg/g)	Kaempferol (µg/g)	Isorhamnetin (µg/g)
80%WC	29.07 ± 1.72c	285.00 ± 33.77c	149.90 ± 2.00b	5.72 ± 2.47b	439.30 ± 0.49c	1 075.00 ± 70.59a	56.12 ± 4.05b	109.80 ± 2.44b
60%WC	51.51 ± 5.00c	362.40 ± 32.67b	288.70 ± 0.24b	5.32 ± 1.25b	566.20 ± 9.65b	706.00 ± 47.18b	73.71 ± 5.77a	119.80 ± 5.76a
40%WC	67.94 ± 13.49c	475.50 ± 26.72a	348.70 ± 18.28b	8.29 ± 0.39b	681.30 ± 21.74a	684.10 ± 123.30b	74.37 ± 1.74a	117.00 ± 3.54b
20%WC	591.80 ± 66.79b	502.50 ± 14.80a	1 715.00 ± 288.50a	8.55 ± 1.87b	458.80 ± 27.26c	653.70 ± 39.16b	34.73 ± 3.56c	90.99 ± 6.20c
DS	825.60 ± 20.64a	529.00 ± 1.49a	1 830.00 ± 166.30a	24.41 ± 3.51a	360.00 ± 18.78d	703.00 ± 79.24b	33.98 ± 6.33c	85.40 ± 4.54c

Note: Different lowercase letters indicate significant differences between groups, P < 0.05, the contents of chemical components are calculated based on dry weight.

Fig. 2.

Antioxidation ability of CIF with different degrees of drying in vitro. (A) Total reducing capacity, different letters indicate significant differences between groups (P < 0.05), while the same letter indicates no significant differences between groups; (B) DPPH radical scavenging ability; (C) ABTS radical scavenging ability; (D) Hydroxyl radical scavenging ability.

Using the Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A), the similarity of HPLC chromatograms among groups was analyzed (Fig. 1D, Table 2). The results showed that under the existing experimental conditions, the greater the difference in water content between samples, the lower the similarity of their HPLC chromatograms. The similarity between the 20%WC and DS groups was the highest (0.994), while that between the 80%WC and DS groups was the lowest (0.559).

Table 2.

Similarity of HPLC chromatograms of CIF with different degrees of drying.

Groups	CIF samples with different degrees of drying
	80%WC	60%WC	40%WC	20%WC	DS
80%WC	1	–	–	–	–
60%WC	0.968	1	–	–	–
40%WC	0.961	0.986	1	–	–
20%WC	0.612	0.746	0.792	1	–
DS	0.559	0.700	0.749	0.994	1

3.2. Antioxidant activity in vitro

In vitro antioxidant activity was evaluated using DPPH, ABTS, hydroxyl radical (·OH) scavenging assays, and total reducing power. At a sample concentration of 10 mg/mL, the absorbance values from the 80%WC to DS groups were 0.175, 0.210, 0.225, 0.240, and 0.356, respectively (Fig. 2A). Within a certain concentration range, the scavenging rates of the three radicals increased with sample concentration in a dose-dependent manner, eventually reaching an inflection point (Fig. 2B−D). There were significant differences in the IC₅₀ (half-inhibitory concentration) values of CIF with different drying degrees for the three radicals (Table 3). Overall, IC₅₀ values decreased with the deepening of drying degree: the 80%WC group had the highest IC₅₀, and the DS group had the lowest.

Table 3.

Radical IC₅₀ of extract from CIF with different degrees of drying (mean ± SD, n = 3).

Groups	Antioxidant activity in vitro (IC50, mg/mL)
	DPPH	ABTS	(·OH)
80%WC	3.827 ± 0.344a	7.773 ± 0.294a	4.024 ± 0.135a
60%WC	2.403 ± 0.033c	3.500 ± 0.137c	3.013 ± 0.107b
40%WC	2.926 ± 0.012b	4.946 ± 0.087b	2.506 ± 0.143c
20%WC	2.234 ± 0.007c	4.674 ± 0.091b	2.679 ± 0.073c
DS	1.347 ± 0.042d	2.837 ± 0.040d	2.279 ± 0.138d

Note: Different lowercase letters indicate significant differences between groups, P < 0.05, weight was extract as standard.

3.3. Anti-inflammatory activity in vitro

Anti-inflammatory activity was evaluated by the inhibition rates of xanthine oxidase (XOD) and lipoxygenase (LOX) (expressed as IC₅₀ values). Within a certain concentration range, the inhibition rates of XOD and LOX increased in a dose-dependent manner with the increase of sample concentration (Fig. 3). The IC₅₀ values of each group are listed in Table 4. Specifically, the IC₅₀ of the DS group for XOD and LOX inhibition was significantly lower than that of other groups, indicating that the DS group had the best in vitro anti-inflammatory effect compared to other groups. Furthermore, there were no significant differences in in vitro anti-inflammatory activity among the 40%WC, 60%WC, and 80%WC groups.

Fig. 3.

Anti-inflammation ability of CIF with different degrees of drying in vitro. (A) Xanthine oxidase (XOD) inhibition ability, (B) Lipoxygenase (LOX) inhibition ability.

Table 4.

Enzyme activity IC₅₀ of extract from CIF with different degrees of drying (mean ± SD, n = 3).

Groups	Methods for determining anti-inflammatory activity in vitro
	Xanthine oxidase (XOD) (mg/mL)	Lipoxygenase (LOX) (mg/mL)
80%WC	3.259 ± 0.142a	1.810 ± 0.067a
60%WC	3.542 ± 0.270a	1.879 ± 0.101a
40%WC	3.550 ± 0.188a	1.657 ± 0.144a
20%WC	2.921 ± 0.113b	1.403 ± 0.182b
DS	1.012 ± 0.036c	1.059 ± 0.119c

Note: Different lowercase letters indicate significant differences between groups, P < 0.05, weight was extract as standard.

3.4. Antibacterial activity in vitro

The inhibitory effects of CIF extract (50 mg/mL) on five strains differed. Extracts of CIF with different drying degrees showed obvious antibacterial effects on S. aureus (G⁺), L. monocytogenes (G⁺), and E. coli (G⁻), but no significant effect on P. aeruginosa (G⁻) and S. paratyphoid (G⁻) (Fig. 4). Measurements of the inhibition zone diameter of the first three bacteria (Table 5) showed: no significant difference in the inhibitory effect on S. aureus among groups; the DS group had a larger inhibition zone diameter for L. monocytogenes than other groups; for E. coli, the inhibition zone diameter first decreased and then increased with the deepening of drying degree. MIC values (Table 5) showed: for S. aureus, the MIC of the 80%WC and 60%WC groups was 3.1250 mg/mL, while that of the 40%WC, 20%WC, and DS groups was 1.562 5 mg/mL; for L. monocytogenes and E. coli, the MIC of all groups was 3.125 0 mg/mL.

Fig. 4.

Pathogenic bacteria inhibition of extract from CIF with different degrees of drying. (A) S. aureus, (B) L. monocytogenes, (C) E. coli, (D) P. aeruginosa, (E) S. paratyphoid.

Table 5.

Bacteriostatic diameter of extract from CIF with different degrees of drying (mean ± SD, n = 3).

Groups	Bacteriostatic diameter (mm)
	S. aureus	L. monocytogenes	E. coli
80%WC	12.10 ± 0.80a	15.91 ± 0.41b	11.37 ± 0.40b
60%WC	11.86 ± 0.65a	16.07 ± 0.95b	11.68 ± 0.39b
40%WC	12.08 ± 0.29a	15.51 ± 0.38b	9.93 ± 0.61c
20%WC	12.09 ± 0.57a	16.19 ± 0.31b	11.43 ± 0.04b
DS	12.96 ± 0.96a	17.06 ± 0.32a	16.40 ± 0.19a

Note: The diameter of the disc containing extract was 6 mm, the sample weight was extract as standard.

3.5. Relationship between the content of various chemical components and in vitro bioactivity of CIF

Correlation analysis of eight chemical components and in vitro bioactivities (antioxidant, anti-inflammatory, antibacterial) was performed based on component content and activity data (Fig. 5, Table 1). Among chemical components: chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid, and linarin showed positive correlations with each other; kaempferol and isorhamnetin showed a positive correlation. Between components and bioactivities: linarin, naringenin, kaempferol, and isorhamnetin contents were positively correlated with total reducing power, while chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid, and linarin contents were negatively correlated with total reducing power; in vitro anti-inflammatory activity showed potential correlations with chlorogenic acid, 3,5-O-dicaffeoylquinic acid, and linarin; the inhibition zone diameter of antibacterial activity was positively correlated with chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid, and linarin contents, and negatively correlated with luteolin, naringenin, kaempferol, and isorhamnetin contents.

Fig. 5.

Result of correlation analysis (pearson) between chemical components and in vitro bioactivity.

4. Discussion

4.1. Variations in chemical components with drying degree

The contents of eight chemical components in CIF showed distinct trends with decreasing water content, indicating that drying degree significantly affects the accumulation or degradation of specific components. Notably, the general decrease in chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid, and linarin with deeper drying contradicts the common cognition that fresh herbs retain higher active component contents, suggesting that drying may trigger specific metabolic processes (e.g., enzymatic degradation or chemical transformation) in CIF. The unidentifiable compound with a retention time of approximately 21 min (Fig. 1B, C) highlights a gap in current component characterization, warranting further identification with appropriate standards. The low similarity of HPLC chromatograms between groups with large water content differences (e.g., 80%WC vs. DS, similarity = 0.559) confirms that drying-driven component changes are sufficient to alter the overall chemical profile of CIF. This provides a chemical basis for distinguishing CIF samples with different drying degrees, supporting the potential of chromatographic fingerprinting in quality evaluation of dried CIF. To clarify the mechanisms underlying these variations, future studies should explore the effects of drying methods, temperatures, and durations on component dynamics, combined with analyses of key enzymes involved in secondary metabolism (e.g., phenylalanine ammonia-lyase, caffeoyl-CoA ligase), which may regulate flavonoid and phenolic acid biosynthesis or degradation during drying.

4.2. Drying enhances in vitro antioxidant and anti-inflammatory activities

The stronger antioxidant activity of dried CIF (lower IC₅₀ in DPPH, ABTS, ·OH scavenging, and higher total reducing power) is consistent with the biological properties of flavonoids and organic acids (Sato et al., 2011), which are major antioxidants in TCMs. Although some antioxidant-related components (e.g., chlorogenic acid) decreased with drying, the overall activity enhancement suggests that either the remaining components (e.g., kaempferol, isorhamnetin, which showed transient accumulation) play a dominant role, or drying promotes the formation of unmeasured antioxidants. Similarly, the improved in vitro anti-inflammatory activity (lower IC₅₀ for XOD and LOX inhibition) in dried samples may be linked to the dynamic changes in components. XOD and LOX are key targets in inflammation-related metabolic pathways (Younis et al., 2016, Indo et al., 2015), and the observed activity trends imply that drying may enrich components with higher XOD/LOX inhibitory potency. This is consistent with the correlation analysis suggesting potential contributions of chlorogenic acid, 3,5-O-dicaffeoylquinic acid, and linarin to anti-inflammatory activity, though their decreasing contents indicate a complex regulatory network (e.g., synergistic effects with other components).

4.3. Antibacterial activities of CIF with drying degree

The inhibitory effects of CIF extracts on S. aureus, L. monocytogenes, and E. coli are consistent with its traditional use in treating colds and diarrhea (Medicinal Flora of China), verifying the pharmacological relevance of these bacteria as targets. However, the lack of activity against P. aeruginosa and S. paratyphoid contrasts with previous reports (Zhu, Yang, Yu, Ying, & Zou, 2005), likely due to differences in extraction solvent polarity—methanol, used in this study, may not efficiently dissolve the lipophilic or high-polarity antibacterial components targeted by other solvents (e.g., ethanol or water). The minimal differences in antibacterial potency (inhibition zone diameter and MIC) between fresh and dried samples suggest that the key antibacterial components in CIF are relatively stable during drying, which may be attributed to their chemical stability (e.g., resistance to dehydration-induced degradation). However, this requires confirmation through targeted component quantification and activity assays.

4.4. Linking chemical components to bioactivities

Correlation analysis reveals that different components contribute differently to CIF’s bioactivities. For example, the positive correlation of kaempferol and isorhamnetin with total reducing power suggests their potential role as antioxidants, consistent with reports that flavonols exhibit strong free radical scavenging activity (Sato et al., 2011). In contrast, the negative correlation of chlorogenic acid (a known antioxidant) with total reducing power implies that its contribution may be masked by other components or that its activity is context-dependent (e.g., concentration thresholds). The association between anti-inflammatory activity and chlorogenic acid, 3,5-O-dicaffeoylquinic acid, and linarin is supported by prior studies: chlorogenic acid, for instance, inhibits lipopolysaccharide-induced inflammation in RAW_264.7 cells (Shan et al., 2009). For antibacterial activity, the positive correlation of phenolic acids (chlorogenic acid, 3,5-O-dicaffeoylquinic acid) with inhibition zone diameter are consistent with their broad-spectrum antibacterial properties, while the negative correlation with flavonoids may reflect antagonistic effects or tissue-specific roles. Notably, these correlations are preliminary and require validation through isolated component activity assays to confirm causal relationships. Additionally, the collective effects of multiple components (synergism or antagonism) should be considered, as TCM bioactivities often arise from multi-component interactions.

4.5. Limitations and future directions

This study is limited by the focus on non-volatile components (due to HPLC constraints); volatile components, which may contribute to bioactivities (e.g., anti-inflammatory or antibacterial), should be included using methods like GC–MS. Further, exploring the transformation mechanisms of active components via enzyme activity assays (e.g., phenylalanine ammonia-lyase for flavonoid biosynthesis) could clarify why drying enhances certain activities including antioxidant and anti-inflammatory. Drying degree significantly modulates CIF’s chemical composition and bioactivities, with dried samples showing stronger antioxidant and anti-inflammatory properties. These findings provide insights into optimizing CIF drying processes for maximal bioactivity and support the development of quality control standards based on key components and chromatographic fingerprints. Another study on CIF from different regions also confirmed that integrating HPLC fingerprints, similarity analysis, and fingerprint-bioactivity relationship modeling with antioxidant and enzyme inhibitory activities can identify efficacy-associated markers such as neochlorogenic acid, isochlorogenic acid A, and linarin, offering a referable methodological framework for monitoring quality consistency in related Asteraceae medicinal materials (Liu et al., 2023).

5. Conclusion

In summary, we investigated the contents of various chemical components and in vitro bioactivities of CIF with different drying degrees. The results of this study showed that the contents of chlorogenic acid, galuteolin, 3,5-O-dicaffeoylquinic acid and linarin in CIF increased significantly with the deepening of drying processing. The contents of luteolin, kaempferol and isorhamnetin increased at first and then decreased. While the content of naringenin decreased gradually. As for the bioactivity, the antioxidant and anti-inflammatory activity in vitro of CIF enhanced with the increasing of drying degree. The extracts of CIF with different degrees of drying had significant inhibitory effect on S. aureus, L. monocytogenes and E. coli, while had no obvious inhibitory effect on P. aeruginosa and S. paratyphoid. However, the in vitro antibacterial activity of CIF did not significantly change with the degree of drying. This study may provide some theoretical reference for the primary processing of CIF, especially the drying of crude drugs. And it will be an important foundation for further research and development of medicinal plants.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Tao Wang: Project administration, Conceptualization, Supervision, Writing – review & editing. Shuting Dong: Methodology, Investigation, Formal analysis, Data curation, Writing – original draft. Qiaosheng Guo: Supervision, Writing – review & editing. Qingjun Zou: Methodology, Writing – review & editing. Feng Yang: Methodology, Writing – review & editing.

Appendix A. Supplementary material

The following are the Supplementary data to this article: