C3G quantified method verification and quantified in blue honeysuckle (Lonicera caerulea L.) using HPLC–DAD

Abstract

Blue honeysuckle is a source of anthocyanins with great potential as a food colorant, and a healthy and functional food material, and contains much cyanidin 3-glucoside (C3G), which has many benefits for human health. A rapid, reliable, accurate quantification method of anthocyanin content in different varieties of blue honeysuckle is critical to help in breeding and selecting excellent varieties which are used in the food processing industry and healthcare industry. Our objective was to verify the modified quantification method of C3G and quantified C3G content in three blue honeysuckle varieties of ‘Berel’, ‘Lanjingling’ and ‘Wulan’ using the modified HPLC method by Agilent 1200 system and CAPCELL PAK C18 column (150 mmⅹ4.6 mm, I. D., 5 μm, Japan), with detection at 530 nm, the solvent flow rate was 1 mL/min, the temperature of the column chamber is 35 °C. The results indicated that the modified method was validated in terms of linearity (R2 = 0.999), precision (RSD = 0.61%), stability (RSD = 5.23%), and recovery with a good level, and C3G can be quickly quantified in blue honeysuckle. In addition, ‘Wulan’ contains the highest C3G level compared with ‘Lanjingling’ and ‘Berel’.

Highlights

• •Blue honeysuckle contains cyanidin 3-glucoside.
• •Quantified cyanidin 3-glucoside in blue honeysuckle by HPLC-DVD is convenient and quick.
• •New blue honeysuckle variety ‘Wulan’ has high content of cyanidin 3-glucoside with 470.63 mg/100 g FW.

1. Introduction

Blue honeysuckle (Lonicera caerulea L.) is also named honeyberry and haskap which belongs to Caprifoliaceae, is a novel small blue fruit [1]. Blue honeysuckle is distributed in China, Russia, Europe, North America, and Japan [2]. The flavor and color of blue honeysuckle are similar to that of blueberry, It has some features that include extreme winter hardiness, and early ripe [3]. Blue honeysuckle does not require special care during cultivation, it likes neutral acidic soil, berries with a waxy coating, and the weight of berries from 0.3 to 2 g, and the yield of one bush is approximately 3–4 kg of fruit per season [4]. The fruits of some early varieties reach full maturity in mid-May, and some late varieties from Japan are in July [5,6]. Blue honeysuckle contains multiple anthocyanins [3,7], including cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, peonidin 3-glucoside, peonidin 3-rutinoside, the major anthocyanin in blue honeysuckle berry was identified as cyanidin 3-glucoside [8,9], accounting for 79–88% of total anthocyanins [10]. Blue honeysuckle has been developed into food products, in early 20th century, Russia used the blue honeysuckle berries to make jam and drink for astronauts because of high anthocyanins content [11], it also can be processed into many kinds of functional food, such as juice, dried fruit, fruit wine, fruit tea, yogurt, ice cream, freeze-dried powder, freeze-dried snacks, blue honeysuckle purees and canned blue honeysuckle, the color of most of blue honeysuckle foods are purple, because the color of anthocyanins is purple-red with high antioxidant ability [[12], [13], [14], [15], [16], [17]], the major bioactive anthocyanin of blue honeysuckle is cyanidin 3-glucoside (C3G), C3G derived from blue honeysuckle fruit has been reported that it has beneficial effects on whole body metabolism as a food Functional raw material, C3G enrichment in the diet attenuates weight gain [18,19].

C3G is as a main biological active substance in blue honeysuckle that has many functions, in the central nervous system (CNS), anthocyanins and its major component cyanidin 3-glucoside (C3G) have been reported to produce preventive and/or therapeutic activities in a wide range of disorders, such as cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, Multiple sclerosis, and glioblastoma [20], recent research indicated that C3G has biological effects and properties ranging from anti-inflammatory to anticancer [21,22]. The berry of blue honeysuckle also contains polyphenol compounds, flavonoid, Vitamin C, organic acids (citric acid, malic acid, quinic acid) and variety of minerals, it has the strong ability in antioxidant [11,23]. Recent research on blue honeysuckle approved that the biological activities of blue honeysuckle fruit with functions to protect the human health from aging resistance and obesity control, anti-cancer, lower heart diseases and diabetes, hypertension, gastrointestinal disorders, and bacterial infections, it indicated that the biological substances have a synergistic effect [24,25].

‘Lanjingling’ and ‘Wulan’ are two patent blue honeysuckle varieties from Northeast Agricultural University in China [26], derived from a cross between ‘Berel’ and ‘Blue bird’, The female parent ‘Berel’ is derived from a cross between the line ‘12–19’ (L. caerulea subsp. altaica) and mixed pollen of ‘Blue spindle’, ‘Blue bird’, and ‘Azure’. In 2003, Juniwe Huo introduced ‘Berel’ and ‘Blue bird’ to northeast agricultural university from M.A. Lisavenko Scientific Research Institute of Horticulture [27], ‘Berel’, ‘Lanjingling’, ‘Wulan’, these three varieties are major cultivars of blue honeysuckle in China.

The purposes of this study were to verify the method to quantify C3G by HPLC in blue honeysuckle and quantify the C3G in three varieties of blue honeysuckle ‘Berel’, ‘Lanjingling’ and ‘Wulan’ grown in China.

2. Materials and methods

2.1. Apparatus

The analytical HPLC equipment was an Agilent 1200 system consisting of a G1311A solvent delivery unit, a G1312A automatic sampler, a G1315B UV Vis photodiode array detector, a G1316A column oven, and an Agilent HPLC workstation (Agilent, China). The column applied in this work was a CAPCELL PAK C18 column (150 mm × 4.6 mm, I. D., 5 μm, Japan). SHZ-B Water bath thermostat oscillator, KEXI ultrasonic cleaner.

2.2. Reagents

All reagents used were chromatography grade. Acetonitrile, formic acid used for HPLC analysis were purchased from MACKLIN, China. The analytical standard of C3G, (purity ≥ 98%) was procured from Bomei, China. The water was used purified water, hydrochloric acid was AR grade purchased from Guangzhou Reagent Company, China.

2.3. Fruit sample

‘Berel’, ‘Lanjingling’ and ‘Wulan’ Blue honeysuckle fruits were provided by Xiangyang agricultural experiment station of Northeast Agricultural University, China, the blue honeysuckle seedings were transplanted in 2019, the latitude of 45.74°N and a longitude of 126.72°N, the altitude of 138.8 m above the sea level, the annual rainfall of 790.5 mm, the blue honeysuckle field had the drip irrigation system. The three samples of fully mature berry fruits were harvested by hand from several different plants of the same variety during the same day of July as shown in Fig. 1a–f, and respectively stored in 1 L plastic containers at −20 °C for chemical analysis. 30 g fruits were selected randomly from each variety, and ground into a sample pool by mortar for each variety waited for testing.

Fig. 1.

The samples of (a) ‘Berel’, (b) ‘Wulan’ and (c) ‘Lanjingling’ and cross-section of (d) ‘Berel’, (e) ‘Wulan’ and (f) ‘Lanjingling’.

2.4. Chromatographic condition

HPLC conditions were the same for all samples, fluid phase A was consisted of 1% (v/v) formic acid and purified water and fluid phase B was consisted of 1% (v/v) formic acid in acetonitrile. Gradient conditions: 0–2 min, 92% A in B; 2–5 min, 88–82% A in B; 5–10 min, 82–80% A in B; 10–12 min 80–75% A in B; 12–15 min, 75-70% A in B; 15–18 min, 70-55% A in B; 18–20 min, 55-20% A in B; 20–22 min, 20–92% A in B; 22–30 min, 92% A in B, re-equilibration was 8 min. The column oven temperature was set at 35 °C. The flow rate was 1 mL/min, and 20 μL treated samples were injected into the column, analytes were monitored using diode array detector (DAD) at a wavelength of 530 nm.

2.5. Standard curve

Cyanidin 3-glucoside standard substance was used as standard stock solution and diluted to seven concentration levels for generating calibration curve by HPLC, then filtered through 0.45 μm polypropylene syringe filter into the vials ready for HPLC analysis. The calibration curve was Y (peak area) = 52.154X + 55.597 (R² = 0.999). The content was expressed as mg of cyanidin 3-glucoside per 100 g of fresh weight.

2.6. Method validation

The reliability of the method mentioned was tested for method of precision, stability, and standard recovery rate. Method of precision was determined by analyzing constantly 5 times injection 20 μl of 50 mg/mL standard substance, then calculating relative standard deviation (RSD). The method of stability was determined by analyzing the same fruit sample at 0, 2, 4, 6, 8 h, calculating average C3G content and relative standard deviation (RSD), the blue honeysuckle extract samples were analyzed by HPLC using the conditions described in Section 2.7. The standard recovery rate verifies the accuracy of the data.

2.7. Sample content determination

The anthocyanin was exhaustively extracted from 1 g blue honeysuckle fruits, in triplicate, with methanol acidified with 50 mL 10% HCl (v/v), ultrasonic extraction for 30 min, then heated in boiling water 1 h for hydrolysis, after cooling then filtered through 0.45 μm polypropylene syringe filter into the vials ready for HPLC analysis.

2.8. Statistical analysis

All experiments were performed in triplicates and the average peak area was used for the accurate quantification and was represented as mean ± SD (P < 0.05).

3. Results

3.1. Method of precision and stability

The standard substance of C3G has the two significant peaks in the spectrum scan map as shown in Fig. 2c, the 530 nm was used as check wavelength to test standard substance and the content of C3G in blue honeysuckle in this study, generated the standard curve as shown in Fig. 2a. The response of C3G standard substance appeared in the spectrum scan map at 8.4min, the respond of one sample of blue honeysuckle fruit also appeared in the spectrum map at 8.4min under the same system shown in Fig. 2b and d, it indicated that under this system condition the C3G signal of the blue honeysuckle fruit responds at 8.4 min.

Fig. 2.

(a) The different concentrations of standard substance in 0.1 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L were measured by HPLC and generated the standard curve (b) HPLC chromatograms of 50 mg/mL standard substance cyanidin 3-glucoside, (c) spectrum scan map indicates that two range nm with strong absorption strength and (d) HPLC chromatograms of one of blue honeysuckle sample was used to compared to the standard substance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Quantification of cyanidin 3-glucoside (C3G) was continuously measured for five repeated standard substance 50 mg/L, the RSD was 0.61% (Table 1), it showed that the machine HPLC in good precision, but the area of peak in repeat 5 is lower than those repeat 1 to repeat 4, it revealed that the (C3G) area of peak of prepared samples that will reduce with the increase of time the samples were put in automatic sampling chamber of HPLC under environmental temperature.

Table 1.

The Area of peak of 50 mg/L standard substance of blue honeysuckle.

Samples	Area of peak mAU*s	SD	RSD%
Repeat 1	2639.33
Repeat 2	2615.18
Repeat 3	2615.18	15.99	0.61
Repeat 4	2615.18
Repeat 5	2598.81
Average	2612.75

Repeat 1 is the first time sample injection, repeat 2 is the second time sample injection, repeat 3 is the third time sample injection, repeat 4 is the fourth time sample injection, repeat 5 is the fifth time sample injection, the sample was the same.

The same blue honeysuckle fruit sample was tested every 2 h under the same system condition, the final measurement is in the eighth hour, RSD is 5.23 was shown in Table 2, the result revealed that the C3G content of the sample decreased with the increase of time.

Table 2.

The content of C3G of ‘Lanjingling’ blue honeysuckle every 2 h.

Samples	C3G content mg/100gFW	SD	RSD%
Repeat 0 h	364.31
Repeat 2 h	350.70
Repeat 4 h	336.43	17.80	5.23
Repeat 6 h	328.23
Repeat 8 h	319.74
Average	339.88

Repeat 0 h is the first sample injection after sample was prepared, repeat 2 h is 2 h removed from the last sample, repeat 4 h is the third time sample injection, repeat 6 h is the fourth time sample injection, repeat 8 h is the fifth time sample injection, the sample was the same.

3.2. Sample recovery test result

Nine blue honeysuckle samples were prepared and added the concentrations are 37.85 mg/L 45.93 mg/L, 56.78 mg/L standard substance, then these samples were measured the C3G concentration by HPLC,and calculated the recovery rate. RSD is 1.92%, 1.44% and 3.39% and data was summed in Table 3.

$$P=\frac{\mathrm{C}3-\mathrm{C}1}{\mathrm{C}2}\times 100\%$$

Table 3.

Recovery rate of standard addition of C3G.

C1 mg/L	C2 mg/L	C3 mg/L	Recovery rate %	Average Recovery rate %	RSD %
49.45	37.85	90.25	107.79	109.66	1.92
48.64	37.85	90.00	109.25
46.84	37.85	89.21	111.95
67.96	45.93	116.05	104.71	104.90	1.44
66.56	45.93	115.47	106.51
66.97	45.93	114.5	103.49
66.69	56.78	122.48	98.25	96.43	3.39
66.61	56.78	122.47	98.39
66.67	56.78	199.28	92.66

C1 is the concentration of samples, C2 is the concentration of addition standard substance, C3 is the concentration of after addition standard substance in samples, P represents recovery rate.

3.3. C3G content determination in three varieties of blue honeysuckle

‘Lanjingling’ and ‘Wulan’ are two new varieties of blue honeysuckle released in 2020 by Northeast Agricultural University, the three varieties of blue honeysuckle samples were treated and analyzed by HPLC using the conditions described in section 2.7, result revealed that the average content of C3G of ‘Wulan’ is 470.63 mg/100 FW, ‘Wulan’ contains the highest cyanidin 3- glucoside (C3G) compared to ‘Lanjingling’ and ‘Berel’, the data was shown in Table 4. Blue honeysuckle is a good resource of cyanidin 3-glucoside (C3G).

Table 4.

C3G content determination in three varieties of blue honeysuckle.

Three varieties of blue honeysuckle	C3G content mg/100 gFW
Berel	462.69 ± 10.37a
Lanjingling	306.34 ± 7.76b
Wulan	470.63 ± 10.55a

Data are means ± SD (n = 3). Different lowercase letters within rows indicate a significant difference at the 5% level (p < 0.05).

4. Discussion

4.1. C3G content determination in blue honeysuckle

Quantification of C3G in blue honeysuckle was investigated by HPLC. Two maximum absorption wavelengths appeared on C3G by HPLC, 280 nm and 530 nm as shown in Fig. 2c, the result is similar to that of Liang Chen [10,28]. In this study, 530 nm was used as determine wavelength to quantity the content of C3G in blue honeysuckle. The response of C3G standard substance and a fruit sample appeared in the spectrum scan map at 8.4 min under this system shown in Fig. 2b and d, it indicated that under this system condition the C3G signal of the blue honeysuckle fruit responds at 8.4 min. The respond time of C3G is dependent on the pressure of chromatographic column and the length of the column, the respond time of the C3G standard substance is the same as that of one of the substances in the sample, it indicates the substance at this time is C3G under same system condition [[29], [30], [31]]. The HPLC method would be verified by precision, stability and standard recovery rate, then the method would be as a responsible method [28,32]. In this study, precision of the current method for the determination of C3G was evaluated by calculating the RSD of the peak areas of five replicate injections of the standard solutions (50 mg/L), and was found RSD is 0.61% (Table 1). The samples were analyzed after 0, 2, 4, 6, 8 h respectively, the result showed that the RSD is 5.23% (Table 2). The range of recovery rate is 96.43%–109.66% (Table 3). The results showed that the current method is precise and stable.

Blue honeysuckle is rich in anthocyanins, the major bioactive anthocyanin of blue honeysuckle is C3G [19,33]. The C3G content reported for Canada-bred blue honeysuckle varieties range between 68 and 649 mg/100 g fresh weight (FW) [6,19,34]. C3G in blue honeysuckle constituted 221 mg/100 g FW (82%) and 170 mg/100 g FW in Polish bred ‘Zielona’ and Canada bred ‘Borealis’ cultivars, respectively [[35], [36], [37]]. In this study, the average C3G content in “Berel”, “Lanjingling”, “Wulan” is 462.69, 306.34, 470.63 mg/100 g FW, respectively. “Wulan” has the highest C3G content level among those varieties.

4.2. Application and expectation of blue honeysuckle

Historically, the blue honeysuckle (Lonicera caerulea L.) berry has been used in traditional medicine for thousands of years, people got blue honeysuckle berries from wet thicket or mountainous regions [38,39]. Blue honeysuckle is well known among the Japanese Ainu aboriginal people, recognizing the berry as the elixir of life [40]. Blue honeysuckle has been used in folk medicine, and it can reduce the risk of diabetes, hypertension, glaucoma, heart attack, anemia, malaria, osteoporosis, and gastrointestinal disease, some research provided evidence that C3G in blue honeysuckle plays a significant role in health promotion and disease prevention, flavonoids including quercetin and its glycosides, catechins, saponins, ascorbic acid, iridoids, and procyanidine are also found in blue honeysuckle berries [[41], [42], [43]].

The antioxidant capacity of blue honeysuckle has been known extensively, in addition, blue honeysuckle has the ability on anti-inflammatory, neuroprotective effects, cardiovascular benefits, anti-diabetic effects, anti-cancer effects [44,45]. Blue honeysuckle berries are becoming popular and used for making jams, wine, candies, jelly, puffed snacks, juice, juice concentrate, tea, canned and frozen fruit, and for medical products as an antioxidant and healthy food [13,14,17,46]. Further research should be carried at understanding the food processing, food matrix, extraction processes, and storage on the stability of C3G and other beneficial compounds. Validating the health benefits of blue honeysuckle berry food products using properly designed animal studies and human clinical trials, and providing more insight into the use of blue honeysuckle in wellness.

5. Conclusions

Blue honeysuckle contains cyanidin 3-glucoside and it can be as a good resource for extracting cyanidin 3-glucoside. The blue honeysuckle fruit samples treated by 1% hydrochloric acid methanol solution that the cyanidin 3-glucoside can be extracted from the samples, content determination of the cyanidin 3-glucoside in blue honeysuckle by HPLC is convenient and quick. The study indicates that the ‘Wulan’ blue honeysuckle has the highest content of cyanidin 3-glucoside rather than that ‘Berel’ and ‘Lanjingling’, it’s a good cyanidin 3-glucoside resource for extraction.

Author contribution statement

Liangchuan Guo: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Jinli Qiao, Chuntong Gong: Performed the experiments; Analyzed and interpreted the data.

Jia Wei, Jiacheng Li, Lei Zhang: Performed the experiments.

Dong Qin, Junwei Huo: Contributed reagents, materials, analysis tools or data.

Funding statement

This work was supported by the National key R＆D Program of China (2022YFD1600500), and Development Fund for Heilongjiang Provincial Undergraduate Universities supported by the central government (High-level Talents Project).

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.