Ultrasound-assisted extraction and antioxidant activity of polyphenolic compounds from three different fruit form of Lonicera caerulea L.

Abstract

This study compared different forms of Lonicera caerulea L. fruit (fresh berries, dried berries, freeze-dried powder) to assess their suitability for storage and transport by analyzing their polyphenolic compounds content via ultrasound-assisted extraction. Optimal conditions were identified for extracting phenolics, flavonoids, and anthocyanins with orthogonal experiment, and antioxidant activities of the polyphenolic compounds were detected by free radical scavenging activity against DPPH and ABTS. The optimal extraction conditions were identified as follows: for total phenolics, 60% ethanol, 30 min, and 300 W; for flavonoids, 80% ethanol, a 60:1 liquid to solid ratio, and 500 W; for anthocyanins, 40 min at 40 °C with 80% acidified ethanol. The polyphenolic compounds in dried berries showed stronger free radical scavenging abilities with the increase of compounds concentration, which was close to or more than that of ascorbic acid. Above all, dried berries were the most suitable form for guiding the harvest, storage, processing and high value-added product development of its Fruit, which provides practical basis for industrial application and functional product development of Lonicera caerulea L.

Introduction

Lonicera caerulea L. (Blue honeysuckle) is a deciduous shrubby plant of the family Lonicera. It is suitable for growth in a humid environment and has ability of excellent cold tolerance¹. Blue honeysuckle is widely distributed in Northeast China, particularly in the Chang-bai Mountain region and Heilongjiang Province^2,3. The fruits are rich in a diverse array of bioactive compounds. More than 180 compounds have been identified, including amino acids and their derivatives, vitamins, minerals, sugars, phenolic acids, flavonoids, terpenoids, fatty acids, organic acids, and anthocyanins⁴. Regular consumption of blue honeysuckle berries has been associated with health benefits, including the prevention of chronic diseases⁵. Various high-value bioactive compounds were extracted from its fresh berries, which has the effect of lowering blood pressure, improving myocardial function and ischemic disease, improving adolescent vision, and preventing skin aging⁶. Studies have demonstrated that the berries are rich in polyphenols such as tannins, flavonoids, anthocyanins/anthocyanidins, and phenolic acids which exhibit antioxidant and anti-inflammatory activities. Among these phenolic compounds, anthocyanins, the most abundant class in nature, have been identified as the predominant constituents. They contribute to reactive oxygen species (ROS) homeostasis through the activation of antioxidant pathways⁷.

Its berries exhibit a soft to firm texture and a bluish to dark purple color, with dimensions of approximately 1–2 cm in length and 1 cm in width⁸. To preserve heat-sensitive compounds such as phenols and vitamins, fresh berries are commonly processed into freeze-dried powder or dried berries. Freeze-dried powder offers advantages in storage and transportation and represents a mainstream form for deep processing⁹. Alternatively, traditionally dried berries are valued for their unique flavor and extended shelf life¹⁰. Dry and freeze-dried forms provide unique advantages for industrial processing and long-term storage. Dehydration effectively inhibits microbial the reproduction and enzymatic degradation of microorganisms, which extends product shelf life compared to perishable fresh berries and improving the economic feasibility of large-scale operational processes¹¹. Despite these advances, most existing studies have focused primarily on a single physical form of blue honeysuckle fruit, typically fresh or frozen samples, while a systematic comparison of extraction efficiency across different physical forms—such as fresh fruit, dried fruit, and freeze-dried powder — remains unexplored.

Multiple extraction methods can be employed to effectively isolate polyphenolic compounds from blue honeysuckle. For example, the traditional solvent extraction as follow, supercritical fluid extraction, enzyme-assisted extraction, and ultrasound-assisted extraction¹². Ultrasound-assisted extraction generated high-speed and intense cavitation effect, disrupted the cellular structure of the material, allowing the solvent penetrate into the material cells, thus shortened the extraction time and increased the yield^13,14. Compared with solvent extraction, ultrasound-assisted extraction shortens the treatment time while enhanced extraction efficiency^15,16. In contrast to supercritical fluid extraction, ultrasound-assisted extraction has lower operational costs. Furthermore, ultrasound-assisted extraction outperforms enzyme-assisted approaches by better preserving the structural integrity of polyphenolic compounds, primarily attributed to its controlled cavitation mechanism that minimizes molecular degradation during extraction¹⁷. Currently, researchers used ultrasound-assisted method to extract total phenolics and flavonoids from fresh berries of blue honeysuckle and evaluated their antioxidant activities¹⁸. The findings showed that ultrasound-treated methods increased the release of bioactive components from blue honeysuckle’s fruit puree and conducted in vitro antioxidant experiments to evaluate the antioxidant activity of blue honeysuckle¹⁹. Dadan et al. reported that ultrasonic treatment improved the extraction efficiency and anthocyanins content in blue honeysuckle²⁰. Fan et al. conducted qualitative and quantitative analyses of anthocyanins in frozen berries across 61 different genotypes sourced from the northeastern China of blue honeysuckle, highlighting the genetic diversity and chemical variability of this species²¹.

Therefore, this study aims to systematically evaluate the extraction efficiency and antioxidant activity of polyphenolic compounds from three physical forms of blue honeysuckle—fresh berries, dried berries, and freeze-dried powders. The findings are expected to facilitate comprehensive resource utilization, support the development of high-value functional products, and provide valuable insights into optimal storage, transportation, and processing strategies, thereby contributing to the sustainable development of the blue honeysuckle industry.

Materials and methods

Materials

Plant materials

Blue honeysuckle was obtained from Yichun Xinye Industry Co. (Yichun China) (Fig. 1a), dried berries and freeze-dried powder were obtained from Heilongjiang Greater Khingan Mountains Tahe County Greenfield Mountain Product Development Co. (Greater Khingan Mountains China) (Fig. 1b, c). The tested cultivar of blue honeysuckle was ‘Wulan’, which was stored at − 20 °C before preparation and subsequent analysis.

Fig. 1.

3 Fruit forms of blue honeysuckle: a fresh berries, b dried berries, c freeze-dried powder.

Reagents and instruments

Anhydrous ethanol, hydrochloric acid, potassium persulfate, vitamin C (analytical purity), DPPH, ABTS, were purchased from Beijing Boao Toda Technology Co., Ltd. (Beijing, China). Gallic acid (analytical purity), rutin (analytical purity), Folin-Ciocalteu reagent and anhydrous sodium carbonate were purchased from Macklin Biochemical Co. Ltd. (Shanghai, China).

KQ-250E Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, China), UV-2600 Ultraviolet-visible spectrophotometer (Shimadzu Instruments Co., Ltd., Suzhou, China), FA1004B electronic balance (Shanghai Youke Scientific Instrument Co., Ltd., Shanghai, China), AQ-180E pulverizer (Cixi Naio Trading Co., Ltd., Ningbo, China).

Experimental methods

Pre-treatment of different forms of blue honeysuckle berries

Fresh berries were washed, dried in an air oven at 25 ℃ for 2 h and sealed in volumetric flasks. freeze-dried powder was stored directly into volumetric flasks. After the fresh berries and dried berries were ground and sieved for 80 mesh screens, all berries powder samples were stored in a refrigerator at − 20 °C for use.

Single-factor experiment design

Weighed 1.00 g of fresh berries, dried berries and freeze-dried powder, respectively. Single factor experiment was carried out according to the set conditions (Table 1). The experiment was carried out to investigate the effects of various factors on the extraction yield.

Table 1.

Ultrasonic extraction parameters of total phenolics, flavonoids, anthocyanins.

Target compounds	Total phenolics	Flavonoids	Anthocyanins
Varied Factor
Ethanol (%)	40, 50, 60, 70, 80	50, 60, 70, 80, 90	40, 65, 80, 95, 100
Time (min)	10, 20, 30, 40, 50	-	0,10, 20, 30, 40
Power (W)	200, 300, 400, 500	300, 350, 400, 450, 500	-
Liquid to solid ratio	-	20:1, 30:1, 40:1, 50:1, 60:1	-
Temperature (℃)	-	-	20, 25, 30, 35, 40
Fixed Factor
Time (min)	-	30	-
Power (W)	-	-	300
Liquid to solid ratio	20:1	-	20:1
Temperature (℃)	25	25	-

Orthogonal experiment design

Based on the results of single-factor experiments, four-factor, three-level orthogonal experiment designs were employed to optimize the extraction conditions and identify the optimal processes for total phenolics, total flavonoids, and anthocyanins, respectively.

Table 2.

Design of orthogonal experiment.

Target compounds	Response factor	Level 1	Level 2	Level 3
Total phenolics	Fruit form	Fresh berries	Dried berries	Freeze-dried powder
	Ethanol (%)	60	70	80
	Time (min)	20	30	40
	Power (W)	300	400	500
Flavonoids	Fruit form	Fresh berries	Dried berries	Freeze-dried powder
	Ethanol (%)	60	70	80
	Liquid to Solid ratio	40	50	60
	Power (W)	400	450	500
Anthocyanins	Fruit form	Fresh berries	Dried berries	Freeze-dried powder
	Ethanol (%)	80	95	100
	Time (min)	20	30	40
	Temperature (℃)	30	35	40

Comparison of polyphenolic compounds from different forms of blue honeysuckle

1. The content of total phenolics

The content of polyphenolic compounds was determined by using modified version of the Folin-Ciocalteu method²². Briefly, the extract solution obtained by ultrasound-assisted extraction under optimized conditions was centrifuged at 1000 r/min and collected. Subsequently, 2 mL of the extract solution was mixed with 5.0 mL ultrapure water, 1 mL Folin–Ciocalteu reagent, and 2 mL of 7.5% Na₂CO₃ solution. After 15 min of reaction, the absorbance was measured at 765 nm. Calculation of total phenolics content based on gallic acid concentration. The formula was as follows:

Where C₁ is the standard solution concentration of gallic acid (mg/mL), N is the multiple of dilution, V is the sample solution volume (mL), M is the fruit powder weight (g), 98.2 is the extinction coefficient of 1% polyphenolic compounds solution. The total amount of phenolics was calculated from the gallic acid curve and expressed as mg/g gallic acid equivalent per one gram of dry weight (DW).

• (2)The content of flavonoids

Modified sodium nitrite-aluminum nitrate colorimetry method was used for determination²³. To each extract solution (2.0 mL), 5% sodium nitrite (0.6 mL) was added and vortexed. After 5 min, 10% aluminum nitrate (0.6 mL), 4% sodium hydroxide (3.0 mL), and 70% ethanol solution was added to 10 mL. The absorbance was measured at 510 nm after 15 min of reaction. Calculation of flavonoids content based on rutin concentration, using the following formula:

Where C₂ is the standard solution concentration of rutin (mg/mL), N is the multiple of dilution, V is the sample solution volume (mL), M is the fruit powder weight (g), 98.2 is the extinction coefficient of 1% polyphenolic compounds solution. The total amount of phenolics was calculated from the rutin curve and expressed as mg/g rutin equivalent per one gram of dry weight (DW).

• (3)The content of anthocyanins

The anthocyanins content in the extract solution was determined by using modified version of the method described in Zhai et al.’s study²⁴. 2 mL of the prepared extract solution in three different forms were taken and ultrapure water was used as a blank control, then the absorbances was measured at 530 nm. The content of anthocyanins was calculated as follow:

Where C₃ is the standard solution concentration of cyanidin-3-glucoside (mg/mL), N is the multiple of dilution, V is the sample solution volume (mL), M is the fruit powder weight (g), 98.2 is the extinction coefficient of 1% anthocyanins solution.

Antioxidant activity analysis of polyphenolic compounds from dried blue honeysuckle

1. DPPH free radical scavenging activity

DPPH free radical scavenging activity experiments were referred to Brand-Williams et al.’s method with slightly modifications²⁵. 2 mL of different concentrations of extracts from blue honeysuckle dried fruit were mixed with 2 mL of DPPH working solution (0.1 mmol/mL). The mixture of liquid was incubated at room temperature away from light for 30 min, and then the absorbances at 517 nm was measured and recorded as A₁. Replace the series of extracts and DPPH working solution with 2 mL of ethanol solution, then repeat the above procedure to measure the absorbance values, recorded as A₂ and A₃, respectively. Vitamin C as a positive control and conduct three parallel measurements. DPPH clearance was calculated as follow:

Where A₃ is the negative control (DPPH + ethanol solution), A₁ is the absorbances of the reaction solution (sample + DPPH) and A₂ is the blank control (ethanol solution + sample).

• (2)ABTS⁺ free radical scavenging activity

Determination of free radical scavenging activity of ABTS⁺ by a slightly modified of Re’s method²⁶. 140 mmol/L potassium persulfate was mixed with ABTS working solution (7 mmol/L) in the dark at room temperature and then stored overnight. Subsequently, this working solution (3.9 mL) was mixed with the sample solution (0.1 mL). The mixture of liquid was incubated at room temperature away from light for 10 min, and then the absorbances at 734 nm was measured and recorded. Vitamin C as a positive control and conduct three parallel measurements. ABTS clearance was calculated as follow:

where A₁ is the absorbances of the sample reaction solution (sample + ABTS⁺), A₂ is the blank control (sample + ethanol solution) and A₃ is the negative control (ABTS⁺ + ethanol solution).

Statistical analysis

The SPSS version 27.0 was used to perform statistical analysis. Each experiment was reported as the mean ± SD for at least three replicates for each sample. Differences between groups were compared using one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparison test. Significance of differences was established at P-value < 0.05.

Results and discussion

Single-factor experiment results

Effect of ethanol concentration, ultrasonic power, ultrasonic time on total phenolics yield

The total phenolics contents were quantified. The regression equation of standard curve of gallic acid was y = 248.7x + 0.0805 with R² = 0.9981. Figure 2 (a) shown that when the ethanol concentration increased from 40 to 70%, the yield of total phenolics extracted from blue honeysuckle’s fruit increased. However, the yield gradually decreased when the concentration of ethanol was further increased to 80%. This phenomenon may be attributed to the saturation of the extraction yield of total phenolics²⁷. Phenolic compounds in plants are usually bound to protein through hydrogen bonds. These bonds are not effectively disrupted by low-concentration ethanol, leading to a reduced yield during extraction. Concurrently, the extraction yield of certain alcohol-soluble impurities and pigments increased with the elevation of ethanol concentration. This interaction with total phenolics in ethanol subsequently influences the extraction of total phenolics. The finding aligned with previous research²⁸, optimal conditions for total phenolics extraction with 70% ethanol were determined.

Fig. 2.

The effects of the ethanol concentration (a), ultrasonic power (b), ultrasonic time (c) on the extraction yield of total phenolics in blue honeysuckle’s fruit.

As depicted in Fig. 2 (b), when the ultrasonic power increased from 200 to 400 W, the yield of total phenolics extracted from freeze-dried powder shown a significant increase trend, with a gradual rise observed in the yield from both fresh and dried berries. Conversely, further increases of ultrasonic power resulted in a progressive decline in yield. Due to the enhanced cavitation effects, the structural components of polyphenolic compounds were destroyed and the molecular structure was changed. Additionally, excessive power likely induced a thermal effect, contributing to the thermal degradation of polyphenolic compounds within the plant cells²⁹. The optimal ultrasonic power for total phenolics extraction with 400 W was determined.

The results were shown in Fig. 2 (c), when the extraction time was 30 min, the yield of total phenolics obtained a maximum value. The extraction yield decreased when the extraction time exceeded 30 min. When the ultrasonic time reached a certain threshold, the yield of total phenolics approached saturation. At this point, some impurities and pigments that easily combined with ethanol were extracted, which affected the yield of total phenolics^30,31. Therefore, the optimal ultrasonic time for extraction with 30 min was determined.

Effect of ethanol concentration, ratio of liquid to solid, ultrasonic power on flavonoids yield

The flavonoids contents were quantified. The regression equation of standard curve of rutin was y = 11.3x + 0.0092 with R² = 0.9998. According to Fig. 3 (a), it was observed that the extraction yield of flavonoids from fresh, dried, and freeze-dried powder blue honeysuckle initially increased and then decreased with varying ethanol concentrations, reaching the highest yield at an ethanol concentration of 70%. When the ethanol concentration exceeded 70%, the flavonoid extraction yield began to decline. This decline may have been attributed to the increase in ethanol concentration leading to the saturation of flavonoids, while simultaneously increasing the solubility of certain lipophilic components³². These components competed with flavonoids for binding with ethanol, resulting in a decreased extraction rate of flavonoids³³. Therefore, an ethanol concentration of approximately 70% was found to be optimal for extraction.

Fig. 3.

The effects of ethanol concentration (a), the ratio of liquid to solid (b), ultrasonic power (c) on the extraction yield of flavonoids in blue honeysuckle’s fruit.

According to Fig. 3 (b), it was observed that the extraction yield of flavonoids from fresh, dried, and freeze-dried blue honeysuckle gradually increased with the increase in the liquid to solid ratio. However, after reaching a certain ratio (50: 1), the flavonoid extraction yield from all three forms of blue honeysuckle showed little variation with further increases in the liquid to solid ratio. Although a larger solvent volume generally resulted in higher extraction rates, excessively high liquid to solid ratio could lead to waste of both solvent and energy. Additionally, increasing the solvent volume might extract some poorly soluble components, which could negatively affect the extraction rate of flavonoids^34,35. Therefore, a liquid to solid ratio of 50: 1 was determined to be optimal for extraction.

According to Fig. 3 (c), it was observed that the extraction yield of flavonoids from fresh, dried, and freeze-dried blue honeysuckle gradually increased with the increase in ultrasonic power. The maximum flavonoid extraction yield for all three forms of blue honeysuckle was achieved at an ultrasonic power of 450 W. However, as the ultrasonic power continued to increase, the flavonoid extraction rate exhibited a downward trend. This decline was likely due to the fact that increased ultrasonic power accelerated the cavitation effect, which disrupted cell walls and membranes, leading to changes in the molecular structure of flavonoids. Additionally, further increases in power generated thermal effects that could cause thermal decomposition of flavonoids within the cells^36,37. Therefore, an ultrasonic power of 450 W was determined to be the optimal experimental condition for extraction.

Effect of ethanol concentration, ultrasonic time, ultrasonic temperature on anthocyanins yield

As shown in Fig. 4 (a), for both fresh and blue honeysuckle dried berries, the extraction yield of anthocyanins increased with the rising ethanol concentration, reaching a maximum at an ethanol volume fraction of 95%. This increase may have been attributed to the low solubility of anthocyanins at lower ethanol concentrations; as the ethanol concentration increased, the extraction yields also improved. However, for freeze-dried powder, the maximum extraction yield of anthocyanins occurred at an ethanol concentration of 65%. When the ethanol volume fraction was further increased, the extraction yield of anthocyanins from the freeze-dried powder decreased. This decrease could be due to the higher hydrophilicity of anthocyanins in freeze-dried powder, which made it difficult for them to be extracted when the ethanol concentration was excessively high, resulting in a reduced extraction yield^38,39.

Fig. 4.

The effects of ethanol concentration (a), ultrasonic time (b), ultrasonic temperature (c) on the extraction yield of anthocyanins in blue honeysuckle’s fruit.

As illustrated in Fig. 4 (b), the extraction yield of anthocyanins from blue honeysuckle exhibited an increase with prolonged ultrasonic time. The optimal extraction yield was observed at an ultrasonic time of 30 min. Nevertheless, extending the ultrasonic time beyond this point resulted in a decline in the extraction yield. This reduction may be attributed to the fact, while ultrasound assistance initially promoted the liberation of anthocyanins into the solvent, facilitating their complete extraction, excessive ultrasound exposure could lead to the degradation of anthocyanin structures and inadvertent extraction of unwanted impurities⁴⁰. Consequently, ultrasonic time of 30 min was determined to be optimal for extraction.

The relationship between ultrasonic temperature and the extraction yield of anthocyanins from blue honeysuckle was illustrated in Fig. 4 (c). As shown, when the ultrasonic temperature ranged from 20 to 35 ℃, the extraction yield of anthocyanins increased with rising temperature, reaching a maximum at 35 ℃. However, as the temperature continued to increase beyond this point, the extraction yield of anthocyanins slightly decreased. The initial temperature was rise that reduced the viscosity of the sample, accelerated molecular movement and facilitating the penetration of the extraction solvent into the material, thereby the solubility of anthocyanins was enhanced⁴¹. Once the temperature further increased, excessive ultrasound heating may lead to structural degradation of the anthocyanins and a decrease in extraction yield⁴². Therefore, an ultrasonic temperature of 35 ℃ was determined to be optimal for extraction.

Orthogonal experiment results

Orthogonal experiment analysis of total phenolics extraction

As shown in Table 3, the extraction yield of total phenolics ranged from 0.993 ± 0.023 to 5.253 ± 0.095 mg/g. Analysis of variance revealed that the order of influencing factors was A > B > D > C, the primary and secondary factors affecting the total phenolic yield were fruit form, ethanol concentration, extraction time, and ultrasonic power (Table 4). The optimal conditions were determined as A₂B₁C₂D₁, corresponding to dried fruit, 60% ethanol, 30 min extraction time, and 300 W ultrasonic power. Validation experiments under these optimized conditions showed a total phenolic yield of 5.573 mg/g, which exceeded the highest value observed in the orthogonal experiments (Table 5).

Table 3.

Results of the orthogonal experiments for total phenolics extraction.

Number	Response factor					Extraction yield (mg/g)
	A	B		C	D
1	1	1		1	1	4.533 ± 0.334
2	1	2		3	2	4.367 ± 0.320
3	1	3		2	3	4.030 ± 0.762
4	2	1		3	3	5.190 ± 0.300
5	2	2		2	1	5.253 ± 0.095
6	2	3		1	2	4.913 ± 0.117
7	3	1		2	2	1.067 ± 0.112
8	3	2		1	3	1.020 ± 0.115
9	3	3		3	1	0.993 ± 0.023
K1	12.93	10.79		10.466	10.779
K2	15.356	10.64		10.55	10.347
K3	3.08	9.936		10.35	10.24
R	12.276	0.854		0.2	0.539

Table 4.

Analysis of variance (ANOVA) for total phenolics extraction.

Source of Variation	Sum of Squares (SS)	Degrees of Freedom (DF)	Mean Square (MS)	F-value	P-value
A	84.543	2	42.271	407.369	< 0.001
B	0.415	2	0.208	2.000	0.164
C	0.02	2	0.01	0.097	0.908
D	0.164	2	0.082	0.788	0.470
Error	1.868	18	0.104

Table 5.

Verification test result of total phenolics extraction.

Yield (mg/g)	Average (mg/g)	RSD (%)
5.514	5.573	1.5
5.537
5.669

Orthogonal experiment analysis of flavonoids extraction

The extraction yield of total flavonoids ranged from 20.820 ± 2.288 to 37.800 ± 3.223 mg/g (Table 6). The order of factor influence was determined as A > D > B > C (Table 7). The factors (Fruit form and ultrasonic power) exhibited significant effects on the extraction yield (P < 0.05), whereas ethanol concentration and liquid to solid ratio demonstrated no significant impact. The optimal conditions were identified as A₂B₃C₃D₃, corresponding to dried fruit, 80% ethanol concentration, 60:1 liquid to solid ratio, and 500 W ultrasonic power. Under these optimized conditions, the extraction yield was 37.907 mg/g (Table 8).

Table 6.

Results of the orthogonal experiments for flavonoids extraction.

Number	Response factor					Extraction yield (mg/g)
	A	B		C	D
1	1	1		1	1	24.270 ± 2.540
2	1	2		3	2	28.290 ± 2.299
3	1	3		2	3	28.360 ± 1.446
4	2	1		3	3	37.090 ± 3.639
5	2	2		2	1	32.653 ± 2.217
6	2	3		1	2	37.800 ± 3.223
7	3	1		2	2	20.820 ± 2.288
8	3	2		1	3	23.187 ± 2.686
9	3	3		3	1	21.293 ± 4.429
K1	80.92	82.18		85.257	78.216
K2	107.543	84.13		81.833	86.91
K3	65.3	87.453		86.673	88.637
R	42.243	5.273		4.84	10.421

Table 7.

Analysis of variance (ANOVA) for flavonoids extraction.

Source of Variation	Sum of Squares (SS)	Degrees of Freedom (DF)	Mean Square (MS)	F-value	P-value
A	912.428	2	456.214	55.142	< 0.001
B	14.218	2	7.109	0.859	0.440
C	12.384	2	6.192	0.748	0.487
D	62.377	2	31.189	3.770	0.043
Error	148.922	18	8.273

Table 8.

Verification test result of flavonoids extraction.

Yield (mg/g)	Average (mg/g)	RSD (%)
37.657	37.907	0.6
38.102
37.961

Orthogonal experiment analysis of anthocyanins extraction

The extraction yield of anthocyanins ranged from 2.08 ± 0.036 to 25.440 ± 0.884 mg/g (Table 9). All four factors exhibited significant effects on anthocyanin extraction (P < 0.05), with the order as A > D > B > C, corresponding to fruit form, temperature, ethanol concentration, and extraction time (Table 10). The optimal conditions were identified as A₂B₁C₃D₃, corresponding to dried fruit, 80% ethanol concentration, 40 min extraction time, and 40 °C extraction temperature. Validation experiments confirmed an anthocyanin extraction yield of 25.538 mg/g (Table 11).

Table 9.

Results of the orthogonal experiments for anthocyanins extraction.

Number	Response factor				Extraction yield (mg/g)
	A	B	C	D
1	1	1	1	1	4.43 ± 0.911
2	1	2	3	2	4.657 ± 0.464
3	1	3	2	3	4.866 ± 0.095
4	2	1	3	3	25.440 ± 0.884
5	2	2	2	1	22.033 ± 2.081
6	2	3	1	2	21.113 ± 0.411
7	3	1	2	2	2.626 ± 0.146
8	3	2	1	3	2.637 ± 0.151
9	3	3	3	1	2.08 ± 0.036
K1	13.953	32.496	28.18	28.543
K2	68.586	29.327	29.525	28.396
K3	7.343	28.059	32.177	32.943
R	61.243	4.437	3.997	4.547

Table 10.

Analysis of variance (ANOVA) for anthocyanins extraction.

Source of Variation	Sum of Squares (SS)	Degrees of Freedom (DF)	Mean Square (MS)	F-value	P-value
A	2259.746	2	1129.873	1593.715	<0.001
B	10.446	2	5.223	7.367	0.005
C	8.270	2	4.135	5.832	0.011
D	13.351	2	6.676	9.416	0.002
Error	12.761	18	0.709

Table 11.

Verification test result of anthocyanins extraction.

Yield (mg/g)	Average (mg/g)	RSD (%)
25.656	25.538	1.6
25.519
24.899

Comparison of the extraction yield of polyphenolic compounds

According to the orthogonal experiment results, the extraction yields of total phenolics, flavonoids and anthocyanins under their respective optimal conditions were determined. The highest levels of these polyphenolic compounds were obtained from dried berries, with yields of 5.573 ± 0.084 mg/g (total phenolics), 37.907 ± 0.227 mg/g (flavonoids), and 25.358 ± 0.403 mg/g (anthocyanins), respectively (Fig. 5). Its yields in fresh berries showed moderate, while the yields in freeze-dried powder showed the lowest. During the preparation of dried berries and freeze-dried powder, this result may be due to significant moisture evaporation occurred, such as drying and sun exposure, and result in weight loss. But polyphenolic compounds were remained intact and concentrated. The extraction yield of polyphenolic compounds in freeze-dried powder was significantly lower than that in other two forms. This discrepancy could be attributed to the complexity of the freeze-drying process, or improper drying methods that may lead to the deactivation of bioactive substances, thus polyphenolic yields in the freeze-dried powder were decreased⁴³. In a word, the extraction yields of total phenolics, flavonoids and anthocyanins in different physical forms exhibited significant difference.

Fig. 5.

The polyphenolic compounds content extracted from different forms of blue honeysuckle.

Table 12.

The optimal extraction conditions for total phenolics, flavonoids, anthocyanins.

Response factor	Total phenolics	Flavonoids	Anthocyanins
Ethanol (%)	60	80	80
Time (min)	30	30	40
Power (W)	300	500	300
Liquid to Solid ratio	20:1	60:1	20:1
Temperature (℃)	25	25	40

Antioxidant activity analysis

The DPPH and ABTS⁺ free radical scavenging activity of total phenolics

As shown in Fig. 6 (a) and (b), indicated that when the mass concentration ranged from 10 to 50 µg/mL, the radical scavenging activity of total phenolics from dried berries were significantly increased. At a mass concentration of 50 µg/mL total phenolics, the ability to scavenge DPPH and ABTS⁺ radicals reached its maximum, with scavenging rates of 94.38% and 87.54%, respectively. With the increase of concentration, the difference between total phenolic radical scavenging rate and ascorbic acid gradually decreases until it approached the equivalent value.

Fig. 6.

The free radical scavenging activity of total phenolics; DPPH (a), ABTS⁺ (b).

The DPPH and ABTS⁺ free radical scavenging activity of flavonoids

As shown in Fig. 7 (a) and (b), indicated that the radical scavenging capacity of dried berries flavonoids gradually enhanced with the increased of concentration within a mass concentration range of 0.5 to 2.5 mg/mL. Specifically, the DPPH radical scavenging rate increased from 32.72% to 78.03%, and the ABTS⁺ radical scavenging rate climbed from 23.37% to 82.05%. This results showed that the antioxidant effect of flavonoids increased with the increase in extract concentration⁴⁴.

Fig. 7.

The free radical scavenging activity of flavonoids; DPPH (a), ABTS⁺ (b).

The DPPH and ABTS⁺ free radical scavenging activity of anthocyanins

As illustrated in Fig. 8 (a) and (b), revealed that the radical scavenging capacity of dried berries anthocyanins increased with the increased of concentration within a mass concentration range of 20 to 120 µg/mL with the rise in their mass concentration, exhibiting a positive correlation. Specifically, the DPPH radical scavenging rate increased from 81.94% to 91.40%, and the ABTS⁺ radical scavenging rate climbed from 79.93% to 89.11%. At equivalent mass concentrations, the scavenging efficiencies of DPPH and ABTS⁺ radicals by blue honeysuckle anthocyanins were markedly superior to those observed with the vitamin C solution. These results indicate that anthocyanins are highly effective at scavenging radicals and demonstrate potent antioxidant activities⁴⁵.

Fig. 8.

The free radical scavenging activity of anthocyanins; DPPH (a), ABTS⁺ (b).

Conclusion

This study systematically optimized the ultrasound-assisted extraction conditions for polyphenolic compounds from three physical forms (fresh berries, dried berries, and freeze-dried powder) of Lonicera caerulea L. and evaluated their antioxidant activities. The optimal extraction parameters were determined as follows: for total phenolics, 60% ethanol, 30 min, and 500 W; for flavonoids, 80% ethanol, a liquid to solid ratio of 60:1, and 500 W; for anthocyanins, an extraction time of 40 min and a temperature of 40 °C, using 80% acidified ethanol. Orthogonal experiment analysis revealed that the fruit form had the most significant influence on extraction yield. Dried berries consistently provided the highest yields of total phenolics (5.573 mg/g), flavonoids (37.907 mg/g), and anthocyanins (25.538 mg/g). Subsequent Antioxidant activity analysis showed that polyphenolic compounds extracted from dried berries showed effective and concentration-dependent free radical scavenging activities against DPPH and ABTS⁺. It is the most suitable for polyphenolic compounds with strong antioxidant activities were extracted from dried fruit. This study provided critical insights and practical data for selecting optimal fruit forms, supporting the development of high-value functional products, and informing strategies for the storage, transportation, and industrial processing of blue honeysuckle berries.

Author contributions

Mei Yang : Writing - original draft, Methodology, Formal analysis. Yao Liu : Writing - original draft, Methodology, Data curation. Xinyu Huang : Methodology, Data curation. Shannan Zhou : Methodology. Xiaoxue Wei : Resources. Yuedong Lv : Funding acquisition. Chunjian Zhao : Resources, Funding acquisition. Dewen Li : Writing - review & editing, Resources, Methodology, Funding acquisition, Conceptualization. Ying Liu : Supervision, Resources, Funding acquisition, Conceptualization.

Funding

This work was financially supported by the Forestry Science and Technology Achievement Promotion Project (Hei[2024]TG30).

Data availability

The datasets used and/or analysed during the current study available from the corresponding authors on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.