Changes of bioactive composition and concentration in loquat flower extracted with water/Chinese Baijiu

Abstract

Loquat is a high-value fruit tree with medicine and fruit homology. Loquat flowers with special fragrance, strong cold resistance and rich in various bioactive components, are valuable agricultural auxiliary products and have been widely used for making floral teas and beverages in recent years. In this study, we found the concentration of active components increased from the floral buds to initial flowers along with flower development, the bioactives of initial flowers were the richest in four flowering stages, and loquat flowers contained major volatile components such as alcohols, aldehydes and esters, which are the source of fragrance. When extract with hot water, the most efficient method was 80 °C for 30 min or boiling water within 2 h. For Baijiu (56% Vol), the best solid-to-liquid ratio was 3:100 (Dry flower: Baijiu) in 6–12 h. Baijiu achieved higher bioactive content than water extraction, the amygdalin concentration in Baijiu reached 0.3 mg/mL.

1. Introduction

Loquat, belonging to Rosaceae, originated in China and has a cultivation history of 2200 years. There are two major production areas in the world for loquat cultivation: the East Asian production area and the Mediterranean coastal production area. The former mainly includes Japan and Korea in addition to China. As for the latter, the largest producer is Spain, with the second highest annual production in the world. China ranks first in terms of loquat area and production with a cultivation area about 170,000 hm² and an annual output about one million tons [1], which accounting for 80% of the world’s total production.

Loquat is a medicinal and edible evergreen fruit tree, current studies on loquat ethanol/water extracts and bioactivity provided scientific evidence and chemical mechanisms for the medical value of loquat [[2], [3], [4]]. Loquat blooms in winter, and has a flowering period of 3–4 months with 80–120 flowers in each inflorescence [5]. After fruit thinning, 2–4 fruits were reserved in each inflorescence. However, a large number of loquat flowers are abandoned in the field. Loquat flowers contain various components (e. g. flavonoids, phenolics, triterpenoids, amygdalin, volatile oils and oligosaccharides) that are effective in refreshing the mind, clearing lungs, moistening the throat, resolving phlegm, relieving cough, and pleasing the soul [4,6,7]. In recent years, loquat by-product utilization has become more common in China, mainly for beverage processing, such as loquat leaf-flower mixed beverage, and the flower-fruit tea [8]. However, there are few reports on how to extract the maximum of the bioactive efficacy, and most products were not fully extracted the active ingredients in loquat flowers. There is little information about the bioactive components at different developing stages of loquat flowers. The brewing method is monotonous and lacks diversity [2].

In this study, UV–Vis, HPLC, SPME-GC, FT-IR and conventional chemical analysis were used to investigate the effects of different soaking methods on the extraction of active ingredients and antioxidant capacity of active ingredients in loquat flower soaks, each component among different flowers developing stages were compared with the purposes to find a more suitable time of flower thinning. Moreover, two types of liquors with high and low alcohol degrees respectively were used in the extraction, aiming to excavate the utilization value of loquat flowers, provide reference for the development of functional Chinese Baijiu.

2. Materials and methods

The design of this experiment was briefly summarized in Fig. 1.

Fig. 1.

Experimental design roadmap.

2.1. Collection and pretreatment of loquat flowers

Inflorescence were picked from Ninghai Bai and Dahongpao loquat fruit trees grown in the Yangdu experimental base of Zhejiang Academy of Agricultural Sciences in December, 2021. The loquat flowers on the inflorescence were classified by the four flowering stages: floral bud, initial flower, full bloom and petal abscission. 100 flowers were picked for each stage and oven-dried at 40 °C to constant weights, then taken out and crushed into powders of the four flowering stages, which were refrigerated for use.

2.2. Flower extraction with water/Chinese Baijiu

2.2.1. Extraction of flower active components from water

With the samples at the same flowering stage, first the soaking time, and soaking temperature were set as two variables. At the solid-liquid ratio of 1%, a sample (1.000 g) was weighed, and distilled water was used as the extraction solution. After extraction, the filtrate was collected and the residues were retained. Then 50 mL of a 75% ethanol solution was added, and the new filtrate was collected after 30 min of ultrasonic treatment. The concentration of active ingredients in the two filtrates was determined, and the average extraction rate was calculated. On this basis, the soaking time and temperature were preliminary screened.

According to the preliminary screening results, the suitable soaking time and temperature were selected. Then single-factor experiments were set up with the four flowering stages and the aging time (no filtration after heating and soaking, and the soaking time continued at room temperature) of 0, 2, 6, 12 and 24 h to determine the suitable drinking time and the best flower picking stage for flower extraction.

2.2.2. Concentration and extraction of flower active components in Chinese Baijiu

The variable of the soaking solvents was set to investigate the effect of the solvents on the extraction of active ingredients. The samples were soaked with anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, water, high alcohol (56% Vol) or low alcohol (38% Vol) as solvents at a 1% ratio. The filtrates were collected after 60 min of ultrasonic treatment to determine the concentrations of the four active ingredients.

Two variables viz. Soaking time (3, 6, 12, 24, 48, 96 h) and the solid-liquid ratio (1, 2, 3, 4, 6, 8%) were set. Samples from the same flowering stage were used and soaked in two types of Chinese Baijiu viz. high alcohol (56% Vol) and low alcohol (38% Vol) as solvents, solvent volume was 50 mL. The filtrates were collected to determine the contents of the four active ingredients.

2.3. Concentration analysis of active components

2.3.1. Determination of total flavonoid concentration

The 200 μg/mL rutin-70% ethanol standard solution (0, 0.1, 0.5, 1, 2, 3, 4, or 5 mL) and the sample extract (0.5 mL) were transferred into a 10 mL colorimetric tube for reaction. Then the absorbance at 510 nm was measured with a UV-VIS Spectrophotometer (SHIMADZU UV-1800) using the aluminum nitrate chromogenic method [9]. With absorbance (Abs) as the y-axis and rutin standard solution concentration (mg/mL) as the x-axis, a standard curve equation y = 13.82x − 0.01180 was obtained, with the correlation coefficient R² = 0.9995. According to this equation, the total flavonoid concentration (mgRE)/L was calculated.

2.3.2. Determination of total phenol concentration

The 0.2 mg/mL gallic acid standard solution (0, 1.0, 2.0, 3.0, 4.0, 5.0, or 6.0 mL) and the sample extract (0.5 mL) were transferred into a 10 mL colorimetric tube for reaction. Then the absorbance at 760 nm was measured with the UV spectrophotometer according to Ref. [10]. With absorbance (Abs) as the y-axis and gallic acid standard solution concentration (mg/mL) as the x-axis, a standard curve equation y = 94.19x + 0.1403 was obtained, with R² = 0.9954. The total phenolic concentration (mgUA)/L of the extract was calculated according to this equation.

2.3.3. Determination of total triterpenoid concentration

The 0.2 mg/mL ursolic acid-ethanol standard solution (0, 0.1, 0.2, 0.3, 0.4, 0.5, or 0.6 mL) and the sample extract (50 μL) were transferred into a 10 mL colorimetric tube with a nitrogen blowing apparatus at 45 °C and 0.1 MPa for 5 min to make the standard solution evaporate quickly using the vanillin-perchloric acid color development method [11,12]. The absorbance at 548 nm was measured with the UV–Vis spectrophotometer. With absorbance (Abs) as the y-axis and the concentration of ursolic acid standard solution (mg/mL) as the x-axis, a standard curve equation y = 0.008567x − 0.01521 was obtained with R² = 0.9934. The concentration of total triterpenoids in the extract (mgGAE)/L was calculated according to this equation.

2.3.4. Determination of total amygdalin concentration

The 100 μg/mL amygdalin-ethanol standard solution (0, 1.0, 2.0, 4.0, 8.0, or 10.0 mL) was pipetted into a 10 mL colorimetric tube and fixed with anhydrous ethanol to 10 mL. The peak area was measured at 210 nm by HPLC [13]. The standard curve equation y = 16.47x + 0.01340 was obtained by taking the peak area as the y-axis and the concentration of bitter amygdalin standard solution (μg/mL) as the x-axis, with R² = 0.9939. Total amygdalin concentration was calculated based on this equation and the peak area of the sample extract.

2.4. Solid-phase micro-extraction-gas chromatography (SPME-GC)

Based on the method from Ref. [14] with revisions. SPME fibers, 65 μm Polydimethylsiloxane/Divinylbenzene and 100 μm Polydimethylsiloxane, were obtained from Supelco (Bellefonte, PA, USA). Hamilton Model 701 N 10 μL syringe (26s gauge, style bevel tip for autosampler) was purchased from Hamilton (Reno, NV, USA). Headspace 1 mL syringe was purchased from Gerstel (Gerstel GmbH, Mülheim an der Ruhr, Germany). The 20 mL vials with magnetic snap caps and PTFE coated silicone septa (Supelco, Bellefonte, PA, USA) were used for the automated analysis. In GC analysis, injector temperature was set at 250 °C. Helium was used as carrier gas at a constant flow rate of 1.2 mL/min. The oven temperature was set at 50 °C for 10 min. Each volatile aromatic substance was identified with the NIST08 standard library. The relative intensity of each volatile compound has been calculated as the ratio between the area of the specific molecule and the sum of the areas of all identified peaks (peak area normalization method) in the chromatogram [15].

2.5. Fourier transform infrared spectroscopy (FT-IR)

The tissues of loquat flowers (stamen, pistil, petal, receptacle and stalk) were dried, crushed and weighed (each sample 20 mg) with a 0.1 mg balance [16]. Spectrally pure potassium bromide was appropriately weighed and mixed at the flower tissue powder: potassium bromide ratio of 1:150. The mixture was ground to powder and then was put (700 mg) into a press mold. The mixed sample was pressed into a transparent sheet on a hydraulic press, and then scanned with an FT-IR meter from 400 to 4000 cm⁻¹ at a resolution of 4 cm⁻¹ and for totally 64 scans. The vibration intervals of the absorption peaks were identified using Origin Pro8.5, and the functional group region and fingerprint region were analyzed.

2.6. Determination of DPPH radical scavenging rate

Measure the DPPH radical scavenging rate with reference to the method of [9]. A 0.02 mL sample of the extract was added to 3 mL of a 0.1 mmol/L DPPH ethanol solution (anhydrous ethanol as the solvent). Then the mixture was shaken well and left to stand for 30 min in the dark. The absorbance at 517 nm was measured with the UV–Vis spectrophotometer using distilled water as a blank control.

The DPPH radical scavenging rate X% was determined as follows:

X% = [1 − (A_i/A₀)] × 100 (1)

where A_i is the absorbance of the solution after addition of the sample, and A₀ is the absorbance of the blank solution.

2.7. Statistical analysis

The results are expressed as mean ± standard deviation. Statistical analyses were done by Microsoft Excel software. Values of p < 0.05 were considered as significantly different, and p-value <0.01 as extremely different.

3. Results and analysis

3.1. Leaching analysis of active ingredients in water extraction of loquat flowers

The active ingredients in water leaching of loquat flowers at different time and temperature were compared in Fig. 2(a)–(c). The average extraction rate and antioxidant capacity were shown in Table 1. The antioxidant capacity were correlated with the concentrations of the four active ingredients. The antioxidant capacity were better at higher leaching concentration of the active ingredients. The highest correlation coefficient is 0.7947 in flavonoids. When the soaking time was identical, the active ingredient leaching concentration was higher and the extraction was more adequate under the conditions of 80 °C water soaking and boiling water boiling. The average extraction rate was above 86%, where the highest concentrations of flavonoids and phenolic components were achieved after leaching at 80 °C. The highest concentrations of phenols and triterpenoids were obtained after leaching in boiling water. Reportedly, the best extraction process of loquat flower flavonoids is in boiling water at 100 °C, and the temperature conditions [17] are consistent with our experimental results.

Fig. 2.

Study on changes of active components in water extract of loquat flower. (a) Changes of active components leached at different temperatures, (b) changes of active components at 80 °C for different time, (c) changes of active ingredients at different boiling time, (d) changes of active ingredients after steeping at 80 °C,Water, (e) changes of active components after boiling, (f) active components at different flowering stages at 80 °C water, (g) active components in boiling water at different flowering stages.

Table 1.

Average extraction rate and antioxidant capacity of active components.

Soaking temperature	Soaking time (min)	Average extraction rate (%)	DPPH· scavenging potential (%)
60 °C water	20	80.71	17.61
80 °C water		86.42	20.70
100 °C water		83.52	18.57
Boiling water		93.12	13.13
80 °C water	5	83.89	40.23
	10	86.13	29.88
	30	86.79	37.46
	40	88.33	35.43
	80	90.25	26.47
Boiling water	5	91.53	26.57
	10	89.81	26.15
	30	90.56	29.88
	40	91.40	25.83
	80	91.86	25.40

Note: Means within a column followed by the same letter are not significantly different at p-value <0.05.

Under the two conditions of 80 and 100 °C boiling water, the concentration of dissolved active ingredients both first increased and then decreased with the prolonging of brewing time, and the active ingredients dissolved and the extraction efficiency were both maximized at 30 min of steeping time. The results of 80 and 100 °C boiling water are constant with the 30 min of heating extraction [18]. After 80 °C soaking time exceeded 30 min, the overall concentration of active ingredients decreased, which may be because some active ingredients were oxidized when the extraction time was too long.

The changes of active ingredients with aging time are shown in Fig. 2(d) and (e). After 30 min of steeping at 80 °C, the leaching concentrations of the four active ingredients decreased with the prolonged aging time, among which the changes of amygdalin concentration were relatively stable. After treatment in the boiling water for 30 min, the total phenol, total flavonoid and total triterpene leaching concentrations increased slightly within 2 h and then gradually decreased. This was probably because the boiling water temperature was too high and the residual heat in the soaking solution made the active ingredients still dissolve slowly in short time.

3.2. Changes of active components at different flower developing stages

The evolution of the four floral active ingredients in loquat flowers is shown in Fig. 2(f) and (g). Under the two optimal temperature conditions of 80 °C water and boiling for soaking 30 min (p-value < 0.05, p-value < 0.05, respectively), the extracts from the flowers at the initial flowering stage contained the highest concentrations of the four active ingredients, and the flavonoid, total phenol, total triterpene, and total bitter amygdalin contents were 1250–1350 mg/L (125–135 mgRE/gDW), 920–1250 mg/L (92–125 mgGAE/gDW), 900–1550 mg/L and 170–230 mg/L respectively. The results at the full blooming stage were slightly behind, as the flavonoid, total phenol, total triterpene, and total amygdalin concentrations were 1090–1350, 670–900, 870–1060, and 160–170 mg/L respectively. The results were lower at the stages of floral buds and petal abscission. The above results indicate the initial flower have higher medicinal value compared to other flowering stages. As reported, the flavonoid content of loquat leaves can reach 192 mgRE/gDW, and the total phenolic content is 41–79 mgGAE/gDW [19], which are higher and lower respectively than our results in flower.

3.3. Leaching analysis of active components in floral liquor

The leaching active ingredients at different ratios of loquat flower to Chinese Baijiu were compared in Fig. 3(a) and (b). Between the two types of Chinese Baijiu at 12 h of steeping (when the changes in the concentration of active ingredients were more stable), the highest content (mg/gDW) of the four active ingredients was achieved in the test group with a solid-liquid ratio of 3% (p-value < 0.05, p-value < 0.05, respectively). Reportedly, the best process for its water extraction was based on a solid-liquid ratio of 1:20 [17], which was closer to our experimental results.

Fig. 3.

Study on Changes of Active Components in Chinese Baijiu Extract of Loquat Flower. (a) Extraction of high alcohol with different solid-liquid ratio for 12 h, (b) Extraction of low alcohol with different solid-liquid ratio for 12 h, (c) Optimum Solid - liquid Ratio of High Alcohol Extracted at Different Time, (d) Extraction of Optimum Solid-liquid Ratio Low alcohol at Different Time.

The Chinese Baijiu leaching active ingredients in loquat flowers compared at different development stages were shown in Fig. 3(c) and (d). After the leaching of active ingredients at a solid-liquid ratio of 3%, the extraction efficiency was the highest and leaching was more adequate after 6 h regardless the type of Chinese Baijiu, and the leaching content of active ingredients ceased to rise after 12 h and decreased.

3.4. Comparison of leaching effects of different solvents

The leaching concentrations of loquat flowers in different solvents are shown in Fig. 4. Results showed that loquat flower samples had the best leaching effect of active ingredients in high alcohol (56% Vol) (p-value <0.05), and demonstrated the highest overall leaching concentration, including amygdalin leaching concentration of 0.3 mg/mL. Reportedly, this concentration can induce apoptosis in cancer cells, and reduce Bcl-2 mRNA and protein expressions in DU145 and LNCaP prostate cancer cells as well as the viability of KB cells in oral squamous cell carcinoma cell lines, exhibiting cytotoxic, anti-proliferative and anti-cancer effects [4]. At the same time, it can inhibit TGF-β1 secretion in peripheral blood lymphocytes stimulated by phytohemagglutinin, and has an anti-renal interstitial fibrosis effect. In hematopoietic stem cells, 0.3 mg/mL bitter amygdalin reduced the mRNA and protein expressions of CTGF and TGF-β, and had an anti-liver fibrosis effect; it promoted the proliferation of T lymphocytes and had an immunomodulatory effect.

Fig. 4.

Comparison of solvent leaching effects on active components.

The leaching effect of the 50% ethanol solution was slightly behind; the leaching effect of the 100% ethanol solution was the worst. The effects of 0%, 25%, 75% ethanol solutions were comparable, probably because the various active ingredients have weaker polarity, while water is more polar and ethanol is less polar. According to the principle of similar solubility, it is more soluble in the 50% ethanol solution because of the closer polarity.

3.5. FT-IR analysis of loquat flower tissues

The peak-marked wave numbers in the infrared spectrum of each tissue are shown in Fig. 5(a), and the specific differences in the infrared spectra between tissues are shown in Fig. 5(b). Most of the spectral peaks appeared within 2000–600 cm⁻¹. Therefore, the corresponding functional groups and characteristics in this range were mainly analyzed. Firstly, the functional groups of amygdalin (primary alcohol, secondary alcohol, aromatic ethers), flavonoids (α-haloketones, benzene ring, methyl), phenols (phenol) and triterpenoids (non-cyclic ester, ester) were found in the tissues of loquat flower. And a high degree of similarity was found in the vibration interval of each organization in the infrared spectrum, but a certain difference was noticed in the peak level between tissues. The peak was the highest in receptacle, and the lowest in pistil. Fluctuation was obvious in stamen. The peaks at 1600 and 1060 cm ⁻¹ were higher, and the peak at 1700 cm ⁻¹ was lower, while the peaks of petal and stalk were the opposite. Between 3000 and 2000 cm⁻¹, the five loquat flower tissues showed obvious peaks near 2900 and 3300 cm⁻¹, indicating there may be a carbon-carbon triple bond in the five tissues. Stalk and stamen demonstrated peaks near 2300 cm⁻¹, and had some structural differences from the other three tissues. The results of this analysis can further prove the existence of various structures in the active components of loquat flowers [20].

Fig. 5.

FT-IR of loquat flower tissues. (a) Spectrum at 1800 and 500 cm⁻¹, (b) Spectrum in full wavelength range.

3.6. SPME-GC analysis of volatile components in loquat flower

The peak areas of SPME-GC in the full bloom stage are shown in Fig. 6, and the analysis results are shown in Table 2. A total of 15 volatile components were identified, mainly including alcohols, aldehydes and esters, with relative concentrations of 31.8%, 50.1% and 15.2%, respectively. The alcohols, esters and aldehydes were dominated by 3-hexene-1-ol (22.3%), methyl anisate (13.7%), and p-methoxybenzaldehyde (32.8%) and benzaldehyde (16.6%), respectively. Other components included trace o-methylbenzonitrile with a relative concentration of 0.2%.

Fig. 6.

Peak area map of volatile components in full bloom stage.

Table 2.

Volatile substances and their relative concentrations in full bloom stage.

Class	Name of substance	R.T.min	Relative concentration (%)	Molecular formula
Alcohols	3-Hexen-1-ol	7.229	22.3	C6H12O
	Trans-2-Hexenal	8.047	6.1	C6H12O
	Benzyl Alcohol	18.328	0.2	C7H8O
	1,2-Heptanediol	18.486	0.3	C7H16O2
	Phenylethyl alcohol	22.181	2.5	C8H10O
	4-Methoxybenzyl alcohol	31.647	0.4	C8H10O2
Aldehydes	Benzaldehyde	14.815	16.6	C7H6O
	Nonanal	17.646	0.1	C9H18O
	Phenylacetaldehyde	19.313	0.6	C8H8O
	4-Methoxybenzaldehyde	30.705	32.8	C8H8O2
Esters	Methyl cinnamate	34.793	0.3	C10H10O2
	Methyl anisate	34.998	13.7	C9H10O3
	Ethyl 4-methoxybenzoate	37.585	0.8	C10H12O3
Others	o-Tolunitrile	25.520	0.2	C8H7N
	2-Ethylfuran	11.549	0.5	C6H8O

Note: Relative concentration refers to the proportion of the substance in the total volatile components. Means within a column followed by the same letter are not significantly different at p-value <0.05.

4. Conclusions

The flower in the stage of initial flowers are the richest in active ingredients such as flavonoid, total phenol, total triterpene, and total aldehydes. The best way to drink the flower soak of water was steeping in 80 °C water for 30 min, or boiling for 30 min and drink within 2 h, the average extraction rate basically exceeded 86% and concentration of amygdalin reached 0.23 mg/mL. High concentration Chinese Baijiu (56% Vol) soaked the dried initial loquat flowers for 6–12 h with the optimal solid-liquid ratio of 3% was proved to be the most effective, in which the concentration of amygdalin reached 0.30 mg/mL. Chinese Baijiu extraction was better than the water soaking extraction under the same conditions, and the residues of loquat flowers can be repeatedly extracted and used after drinking. Aroma of loquat flowers came from volatile components, the main volatile components in the flowers were aldehydes (contributed for 50.1%), alcohols (contributed for 31.8%) and esters (contributed for 15.2%). The main components of alcohols, esters, and aldehydes were 3-hexene-1-ol (contributed for 22.3%), methyl anisole (contributed for 13.7%), and p-methoxybenzaldehyde (contributed for 32.8%) & benzaldehyde (contributed for 16.6%) respectively.

5. Suggestions

Loquat flower is a promising byproduct for functional beverage and food. It was suggested to thin loquat flowers before full bloom, dried and crushed to use for soak. The dried and crushed initial loquat flowers can also be used to make an effective medicinal usage by extracting in an appropriate volume of Chinese Baijiu, in which the concentration of amygdalin can effectively relief cough and anti-inflammatory, and even has an effect on preventing cancer, fibrotic and strengthening immunity. Besides, the volatile components in loquat can be used to produce functional food, flavors and spices. Further study are undertaking on the effects of flowers of different loquat varieties on active ingredients in our lab.

Author contribution statement

Zhebin Shen: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Junwei Chen: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

Jieli Zhu: Performed the experiments; Analyzed and interpreted the data.

Haixia Yu: Conceived and designed the experiments.

Funding statement

Junwei Chen was supported by Major science and technology projects “Breeding of New Loquat Varieties in Zhejiang Province” [2021C02066-3].

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.