Phytochemical composition and potential biological activities assessment of raspberry leaf extracts from nine different raspberry species and raspberry leaf tea

Abstract

BACKGROUND:

The scientific interest regarding raspberry leaf composition and potential benefits grow. Still, little is known about raspberry leaves.

OBJECTIVE:

Extracts from nine raspberry leaf species and Autumn Bliss raspberry leaf tea (LT) were evaluated for their phenolic characteristics along with potential biological activities.

METHODS:

Phenolic constituents in leaf extracts from LT and nine raspberry species, including European red raspberry (ERR), Australian red raspberry (ARR), U.S. Raspberry (USR), Autumn Bliss (AB), Heritage (H), Tulameen (T), Longyuan Qiufeng (LQ), Qiuping (Q), and Fengman Hong (FH) were evaluated by UPLC-MS. Regarding to the bioactive activities, antioxidant activities (DPPH and ABTS assays), antidiabetic properties (α-amylase inhibition and α-glucosidase inhibition assays), antibacterial activities (growth inhibition zone measurement and a minimum inhibitory concentration (MIC)), and anti-inflammatory properties (inhibition of hyaluronidase activity and inhibition of albumin denaturation), were investigated.

RESULTS:

The results showed that ERR extract exhibited the highest levels of total phenolic (5.56±0.058 mg/g), the highest chlorogenic acid (597.03±10.90 mg/kg) and rutin level (2771.9±41.52 mg/kg). Autumn Bliss LT possessed the lowest total phenolics, and lowest DPPH scavenging activity. A high correlation between phenolic content and antioxidant activity was found in these extracts.

CONCLUSIONS:

High phytochemical contents and biological activities of these raspberry leaves, including antioxidant, antidiabetic, antibacterial, and anti-inflammatory effect were shown, which indicated their consumption would be beneficial to health.

Keywords

Raspberry leaf phytochemicals antioxidant antibacterial antidiabetic anti-inflammatory

1 Introduction

Raspberry has been demonstrated to exert anti-inflammatory, antioxidant, anticancer, antimicrobial, and anti-Alzheimer activities [1 –4], however, little is known about the raspberry leaves. Berry leaves are byproducts of berry cultivation, and tons of leaves are wasted annually. Studies have shown that the main bioactive compounds in berry leaves are similar to that of berry fruits [5]. However, their traditional therapeutic use against several diseases, such as the common cold, inflammation, and diabetes, has been lost attention already. Nevertheless, the scientific interest regarding the leaf composition and beneficial properties grows, indicating that berry leaves may be considered as an alternative source of bioactives [5].

Tea made from the leaves of Rubus idaeus L. (raspberry) has been used for centuries as a folk medicine to treat wounds, diarrhoea, and colic pain [6]. It was reported that raspberry LT might facilitate labor. Two different clinical studies were performed to assess the efficacy of raspberry leaf preparations in pregnancy. However, no clinically significant differences were observed among the control and raspberry leaf treated groups on maternal blood loss, length of gestation, the likelihood of medical facilitating of labor, and need for pain relief during labor [7–8]. The bioactive potential of raspberry leaves in exhibiting cytoprotective activity on human laryngeal carcinoma and colon adenocarcinoma has been documented [9]. Antithrombotic activity of raspberry leaves was reported by Han et al. [10].

The phytochemicals contents and biological activities exerted by the closely related berry species were reported to be different [3]. Therefore, we hypothesized that leaves from different raspberry species possess different properties. In this current study, leaf extracts from Autumn Bliss raspberry LT and nine raspberry species, including European red raspberry (ERR), Australian red raspberry (ARR), U.S. Raspberry (USR), Autumn Bliss (AB), Heritage (H), Tulameen (T), Longyuan Qiufeng (LQ), Qiuping (Q), and Fengman Hong (FH) were evaluated for their phytochemical contents (total phenolics, total procyanidins, and total flavonoids), quantitative analysis of each phenolic compound, antioxidant properties (DPPH and ABTS assays), antidiabetic properties (α-amylase inhibition and α-glucosidase inhibition assays), antibacterial activities (growth inhibition zone measurement and a minimum inhibitory concentration (MIC)), and anti-inflammatory properties (inhibition of hyaluronidase activity and inhibition of albumin denaturation).

2 Materials and methods

2.1 Materials

Leaves from nine different raspberry species, including Autumn Bliss (AB), Australian red raspberry (ARR), European red raspberry (ERR), Fengman Hong (FH), Heritage (H), Longyuan Qiufeng (LQ), Qiuping (Q), Tulameen (T), and U.S. Raspberry (USR) were obtained from the experimental field at Department of Horticulture in Heilongjiang Academy of Agricultural Sciences (Heilongjiang, Harbin, China) in August, 2018, and then were homogenized and saved in a desiccator after air drying. LQ was selected and bred in Heilongjiang Academy of Agricultural Sciences. Q was selected and bred in Shenyang Agricultural University. FH was bred in Special Agricultural Products Institute of China Academy of Agricultural Sciences and Jilin Agricultural Bureau.

All the standards for UPLC, including rutin, quercetin, hyperin, chlorogenic acid, ellagic acid, isorhamnetin, epicatechin, catechinic acid, syringic acid, gallic acid, ferulic acid, caffeic acid, and luteolin (HPLC grade) were obtained from Shanghai YuanYe Biotechnology Co., Ltd. (Shanghai, China). α-Amylase (4000 U/g), α-glucosidase (50000 U/g), hyaluronidase (300000 U/g), and bovine serum albumin were obtained from Shanghai YuanYe Biotechnology Co., Ltd. (Shanghai, China). All the other reagents were obtained from Tianli Chemical Reagent Co., Ltd. (Tianjin, China).

2.2 Preparation of the raspberry leaf extracts

Homogenized dried raspberry leaves (5.0 g) were extracted with 50 mL methanol containing 0.1 % HCl in an ultrasonic bath at room temperature for 1 h [11]. Then the extracts were left for 24 h in the dark at 4°C, followed by filtration and clear supernatants collection (the extraction procedure was repeated for three times). Rotary evaporation under reduced pressure at 40°C was used to evaporate fractions to dryness. The fractions were then combined and methanol/water (60 : 40, v/v) was added to ca. 100 mL. The final extracts were stored at 4°C until further analysis [12].

2.3 Autumn Bliss raspberry leaf tea (LT) preparation

Autumn Bliss raspberry leaves were air-dried in the greenhouse with an intensity of 5 g/dm³ for 1 h, followed by streaming at induction cooker for 45 mins. After rolling for 25 mins, finally LT was obtained after dried in the oven at 50°C for 12 h.

The method for LT extracts was followed according to the 2.2.

2.4 Determination of total phenolic content

The amount of total phenolics in raspberry leaf extracts was determined using Folin-Ciocalteu method, described by Pantelidis et al. [13]. Briefly, 0.05 mL of raspberry leaf extracts and 0.45 mL water were mixed with 2.5 mL of 1 : 10 diluted Folin-Ciocalteu’s phenol reagent, followed by 2 mL of 7.5% (w/v) sodium carbonate. The mixture was then allowed to stand for 5 mins at 50°C. Finally, the absorbance was measured at 765 nm against a blank. Phenol content was estimated from a standard curve obtained from gallic acid. Total phenolic contents were expressed as mg gallic acid equivalent per gram dry weight (mg GAE/g). Samples were read in triplicate.

2.5 Total procyanidins content measurement

Vanillin-hydrochloric acid (V-HCl) method [14] was used to measure total procyanidins content. Briefly, methanol solution (1 mL) containing 0.8 mL of sample was placed in a tube to which 6 mL of methanol solution containing 4% vanillin was added. After stirring vigorously, 3 mL of concentrated hydrochloric acid was added to the solution. After stirring with a tube mixer every 5 min for 15 min, the absorbance at 500 nm of the red solution was measured by a spectrophotometer. Catechol was used to perform standard curve. Total procyanidins contents were expressed as mg equivalents of catechol per gram dry raspberry leaf (mg catechol/g).

2.6 Total flavonoid content determination

The total flavonoid content was evaluated using aluminium nitrate nonahydrate [15]. The total flavonoids concentrations were calculated from rutin calibration curve and expressed as mg/g dry raspberry leaf (mg rutin/g).

2.7 UPLC-MS quantitative analysis of each phenolic compounds

Phenolic compounds were detected using Agilent 6470A triple quadrupole LC/MS system (Agilent Technologies, USA). Samples were separated using a Agilent ZORBAX Eclipse Plus C18 column (3.0*50 mm 1.8μm). Volume injection was 5μL and the temperature was 40°C. The elution was conducted as follows:

0∼0.5 min, 0% A ∼90% A, 100% B∼10% B; 0.5∼2.0 min, 90% A ∼10% A, 10% B∼90% B; 2.0∼4.0 min, 10% A, 90% B; 4.0∼4.1 min, 10% A∼90% A, 90% B∼10% B; 4.1∼6.0 min, 90% A, 10% B.

Mobile phase was consisted of water containing 0.1 % formic acid (solvent A) and acetonitrile (solvent B). The rate for mobile phase was 0.3 mL/min. Identification of the compounds was done by comparison of their retention time with those of the standards. Stock solutions of standards were prepared in methanol:DMSO (90 : 10, v:v).

Mass spectrometry data were obtained by an electrospray ionization (ESI) source in positive and negative ion mode. Data acquisition was performed in multiple reaction monitoring (MRM) mode. Optimal condition was:

Highly purified nitrogen as nebulizing gas with flow of 11 L/min and nebulizer pressure of 20 psi; capillary voltage of 4 kV; desolvation temperature of 325°C; collision pressure of 138 kPa using highly purified nitrogen as collision gas.

Specific MRM mode parameters for the targeted compounds were optimized through Agilent optimizer software (Mass Hunter Optimizer).

2.8 Assessment of antioxidant effect

2.8.1 Determination of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

DPPH scavenging activity assay was described by Butsat et al. [16] with modifications. Briefly, raspberry leaf and LT extracts (100μL) were mixed with 4 mL of 0.1 mmol/L of DPPH in ethanol. The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm against a blank. IC50 values were calculated using linear regression analysis. The test was performed in triplicate.

2.8.2 [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] ABTS radical scavenging activity measurement

ABTS free radical scavenging activity was analyzed by following standard protocol [17]. The ABTS cation radical scavenging activity was produced by the reaction between 9 mL of ABTS solution and 100μL of raspberry leaf and LT extracts, followed by incubation in the dark at room temperature for 6 min. Ethanol was used as a blank and all absorbance was taken within 6 min. Concentrations of extracts resulting in 50 % clearance of ABTS (IC50) were determined graphically.

2.9 Antidiabetic assay in vitro

2.9.1 α-Amylase inhibitory assay

100μL of raspberry leaf and LT extracts in phosphate buffer and 50μL of α-amylase (10 U/mL) were combined in a 96-well plate. The mixture was incubated at 37°C for 15 min. After that, 50μL of 1 % (w/v) starch solution in 0.02 mol/L sodium phosphate buffer (pH 6.9) was added at timed intervals and then further incubated at 37°C for 10 min. The reaction was terminated by the addition of 5μL of dinitrosalicylic acid (DNS). The absorbance was measured at 540 nm against a blank and α-amylase inhibitory activities were shown as IC50.

2.9.2 α-Glucosidase inhibitory activity

100μL of the raspberry leaf and LT extracts were combined with 50μL of α-glucosidase enzyme solution (1 U/mL) in a 96-well plate. Then, 20μL of p-NPG (5 mmol/L) was added as a substrate and the mixture was incubated at 37°C for 15 min in the dark. The reaction was terminated by the addition of 10μL of Na₂CO₃ (1 mol/L). α-Glucosidase activity was determined spectrophotometrically at 405 nm by measuring the quantity of p-nitrophenol released from p-NPG, and α-glucosidase inhibitory activities were shown as IC50.

2.10 Antimicrobial effect assessment

2.10.1 Growth inhibition zone measurement

Antibacterial activities of raspberry leaf and LT extracts were determined by agar well diffusion method according to a previous publication [18]. Inoculum containing 10⁶ cfu/mL of each bacterial culture to be tested was spread on nutrient agar plates with a sterile swab moistened with the bacterial suspension. Subsequently, 8 mm diameter wells were punched into the agar medium and filled with 100μL (25 mg/mL) of leaf or LT extracts and allowed to diffuse at room temperature for 2 h. The plates were then incubated in the upright position at 37°C for 24 h. Wells containing the same volume of distilled water were served as negative controls. After incubation, the diameters of the growth inhibition zones were measured in mm. Three replicates were carried out for each extract against each of the test organism.

2.10.2 Determination of a minimum inhibitory concentration (MIC)

The MIC of the raspberry leaf and LT extracts was determined according to the micro broth dilution technique [19]. It was performed in 96-well plates for determining the minimum inhibitory concentration (MIC). Standardized suspensions of the test organisms (Escherichia coli, staphylococcus aureus, and bacillus subtilis) were inoculated into a series of 96-well plate, including one growth and one sterility control. Brain Heart Infusion (BHI) and sabouraud dextrose broth containing leaf or LT extracts in decreasing concentrations: 20,10, 5, 2.5, 1.25, and 0.625 mg/mL, was incubated at 37°C for 24 hours. After overnight incubation these tubes were observed for turbidity. The microtiter plate showing the minimum turbidity was noted for MIC.

2.11 Anti-inflammatory effect assessment

2.11.1 Inhibition of hyaluronidase activity

Hyaluronidase activity inhibition was determined by Elson Morgan assay [20] with slight modifications. 500μL of bovine testicular hyaluronidase (2.5 U/mL) dissolved in 0.2 mol/L acetate buffer (pH = 3.6) was mixed with 500μL of raspberry leaf or LT extracts. The mixture was incubated for 10 min at 37°C. After that, 100μL of calcium chloride (12.5 mmol/L) was added to the reaction mixture and incubated for 10 min at 37°C. This Ca^2 + activated hyaluronidase was treated with 500μL hyaluronic acid (0.6 mg/mL), and incubated at 37°C for 40 min. After incubation the enzyme reaction was terminated by the addition of alkaline borate solution (300μL) and subsequently heated for 5 minutes at 100°C. After cooling down, 3 mL of N,N-dimethylaminobenzaldehyde (67 mmol/L) was added to all the tubes and incubated in water bath at 37°C for 10 minutes. The absorbance was measured at 585 nm. Inhibition of hyaluronidase activities was shown as IC50.

2.11.2 Inhibition of albumin denaturation

The assay was studied according to a previous publication with slight modifications [21]. Raspberry leaf and LT extracts were dissolved in acetate-sodium acetate buffer solution. 2 mL of diluted raspberry leaf or LT extracts was mixed with 3 mL of 2 % albumin solution in acetate buffer solution (0.1 mol/L, pH = 5.5) and incubated at 37°C for 20 min. Denaturation was induced by keeping the reaction mixture at 75°C in water bath for 10 min. After cooling, the turbidity was measured at 660 nm. Percentage of inhibition of denaturation was calculated from control where no extracts were added. Each experiment was done in triplicate. Inhibition of albumin denaturation was shown as IC50.

2.12 Statistical analysis

All analyses were run in triplicate and were expressed as mean±standard deviation. Statistical analysis was done by using SPSS 24.0 Software (SPSS Inc., Chicago, IL, USA). Differences between means were analyzed by ANOVA test and then followed by LSD (p < 0.05).

Correlations were calculated between the contents of phenolic, or flavonoid, or proanthocyanidin vs. antioxidant, or antimicrobial, or anti-inflammatory, or antidiabetic activities using Spearman’s correlation coefficients.

3 Results and discussion

3.1 Total phenolic, flavonoid, and proanthocyanidin content measurements

As shown in Fig. 1, ERR extract exhibited the highest levels of total phenolics (5.56±0.058 mg/g dry ERR) and flavonoids (3.77±0.063 mg/g dry ERR). Total proanthocyanidin content in H (3.06±0.049 mg/g dry H) was significantly higher than that in other raspberry leaves and raspberry LT (p < 0.05).

Fig. 1

Total phenolics, flavonoids and proanthocyanidins contents of 9 red raspberry leaves and leaf tea. The same phytochemical in different raspberry leaves were compared. The bars with the same letter are not significantly different from each other, p < 0.05 (mean±S.D., n = 3).

Compared to its raw material AB, LT showed significant lower contents of the total phenolics, flavonoids, and proanthocyanidins (p < 0.05), indicating a potential loss during processing.

3.2 Phenolic compounds quantification

As shown in Table 1, polyphenols including vanillic acid, arbutin, sinapic acid, naringin, hesperitin, isoliquiritigenin, quercetin, and coumarin were undetermined in all these nine raspberry leaves and LT.

Table 1
Polyphenol contents in 9 raspberry leaves and LT extracts

AB mg/kg ARR mg/kg ERR mg/kg FH mg/kg H mg/kg LQ mg/kg Q mg/kg T mg/kg USR mg/kg LT mg/kg

Vanillic acid – – – – – – – – – –

Arbutin – – – – – – – – – –

Gallic acid 20.34±1.01cd 55.58±4.27b 22.45±3.30cd 3.99±0.24d 32.33±1.11c 11.55±1.22d 5.52±0.75d 21.41±2.46cd 14.83±1.97cd 320.99±25.14a

Chlorogenic 442.94±18.79b 45.77±2.33e 597.03±10.90a 6.11±0.14e 202.69±4.32c 229.57±3.27c 116.38±11.39d 637.74±54.31a 23.39±1.48e 236.97±34.44c

acid

Caffeic acid 6.60±1.48b – 5.87±0.79bc – 7.23±0.26b 1.72±0.08d 4.21±0.08c 9.64±1.39a 0.84±0.21d 10.21±1.55a

Epicatechin 1.19±0.04d 0.82±0.01d 126.70±6.61b 1.55±0.10d 91.12±4.07c – – 216.52±11.06a 3.89±0.17d –

(+)-Catechin – – 234.10±8.13b – 171.44±7.24c – – 387.07±12.46a 6.07±0.25d –

Syringic – – – – 1.97±0.18 – – – – –

acid

Rutin 1385.50±33.39d 1095.06±11.57e 2771.9±41.52a – 1869.83±20.72b 583.87±11.85g 545.91±19.00g 130.9±40.65h 1730.77±2.62c 743.82±30.30f

Ellagic acid 53.88±10.76d 66.76±1.62d 89.35±1.61c – 139.47±8.85b 11.45±1.51e 5.77±0.35e 139.29±16.87b 21.3±1.13e 218.38±12.71a

Hyperoside 60.45±4.01c 31.55±2.78d 123.54±2.27b 5.55±0.16e 239.13±10.39a 7.17±0.15e 8.7±0.52e 6.55±0.08e 55.82±3.97c 32.22±4.23d

Ferulic acid – – – – – 1.33±0.15b – 3.79±0.25a – 3.47±0.19a

Sinapic acid – – – – – – – – – –

Naringin – – – – – – – – – –

Quercetin 0.47±0.06b – 0.27±.010c – – 0.076±0.004d 0.034±0.001d – 0.19±0.006c 0.62±0.06a

Luteolin – – – – 0.82±0.04a – – – – –

Isorhamnetin 1.26±0.09d 3.89±0.16b 3.43±0.25bc 0.36±0.05de 6.96±1.20a 2.59±0.21c 0.49±0.025de 3.58±0.15b 0.26±0.04e 0.90±0.05de

Hesperitin – – – – – – – – – –

Isoliquiritigenin – – – – – – – – – –

Quercetin – – – – – – – – – –

Coumarin – – – – – – – – – –

	AB mg/kg	ARR mg/kg	ERR mg/kg	FH mg/kg	H mg/kg	LQ mg/kg	Q mg/kg	T mg/kg	USR mg/kg	LT mg/kg
Vanillic acid	–	–	–	–	–	–	–	–	–	–
Arbutin	–	–	–	–	–	–	–	–	–	–
Gallic acid	20.34±1.01cd	55.58±4.27b	22.45±3.30cd	3.99±0.24d	32.33±1.11c	11.55±1.22d	5.52±0.75d	21.41±2.46cd	14.83±1.97cd	320.99±25.14a
Chlorogenic	442.94±18.79b	45.77±2.33e	597.03±10.90a	6.11±0.14e	202.69±4.32c	229.57±3.27c	116.38±11.39d	637.74±54.31a	23.39±1.48e	236.97±34.44c
acid
Caffeic acid	6.60±1.48b	–	5.87±0.79bc	–	7.23±0.26b	1.72±0.08d	4.21±0.08c	9.64±1.39a	0.84±0.21d	10.21±1.55a
Epicatechin	1.19±0.04d	0.82±0.01d	126.70±6.61b	1.55±0.10d	91.12±4.07c	–	–	216.52±11.06a	3.89±0.17d	–
(+)-Catechin	–	–	234.10±8.13b	–	171.44±7.24c	–	–	387.07±12.46a	6.07±0.25d	–
Syringic	–	–	–	–	1.97±0.18	–	–	–	–	–
acid
Rutin	1385.50±33.39d	1095.06±11.57e	2771.9±41.52a	–	1869.83±20.72b	583.87±11.85g	545.91±19.00g	130.9±40.65h	1730.77±2.62c	743.82±30.30f
Ellagic acid	53.88±10.76d	66.76±1.62d	89.35±1.61c	–	139.47±8.85b	11.45±1.51e	5.77±0.35e	139.29±16.87b	21.3±1.13e	218.38±12.71a
Hyperoside	60.45±4.01c	31.55±2.78d	123.54±2.27b	5.55±0.16e	239.13±10.39a	7.17±0.15e	8.7±0.52e	6.55±0.08e	55.82±3.97c	32.22±4.23d
Ferulic acid	–	–	–	–	–	1.33±0.15b	–	3.79±0.25a	–	3.47±0.19a
Sinapic acid	–	–	–	–	–	–	–	–	–	–
Naringin	–	–	–	–	–	–	–	–	–	–
Quercetin	0.47±0.06b	–	0.27±.010c	–	–	0.076±0.004d	0.034±0.001d	–	0.19±0.006c	0.62±0.06a
Luteolin	–	–	–	–	0.82±0.04a	–	–	–	–	–
Isorhamnetin	1.26±0.09d	3.89±0.16b	3.43±0.25bc	0.36±0.05de	6.96±1.20a	2.59±0.21c	0.49±0.025de	3.58±0.15b	0.26±0.04e	0.90±0.05de
Hesperitin	–	–	–	–	–	–	–	–	–	–
Isoliquiritigenin	–	–	–	–	–	–	–	–	–	–
Quercetin	–	–	–	–	–	–	–	–	–	–
Coumarin	–	–	–	–	–	–	–	–	–	–

“–” not detected. The same polyphenol content in different raspberry leaves was compared. Samples labeled with the same letter are not significantly different from each other, p < 0.05 (mean±S.D., n = 3).

Phenolic acids, including ellagic acid (6–218 mg/kg), caffeic acid (range from 1–10 mg/kg), which was lower than a previous publication, where 550 mg/kg caffeic acid of dried leaf was reported [5] and chlorogenic acid (range around 6–638 mg/kg) were determined in these raspberry leaves. Consistently, a publication showed ∼700 mg/kg dried leaf of chlorogenic acid in raspberry leaves [9]. Gallic acid, which ranged from 4–321 mg/kg was detected in raspberry leaves and LT. Other phenolic acids, including ferulic acid and syringic acid were low in raspberry leaves.

Chlorogenic acid levels in ERR (597.03±10.90 mg/kg) and T (637.74±54.31 mg/kg) were highest in all the leaves and LT. Catechin, epicatechin, ferulic acid levels were highest in T, compared to other raspberry leaves. ERR was dominantly rich in rutin and chlorogenic acid. H was high in hyperoside and isorhamnetin. H was the only raspberry leaf which luteolin was detected (0.82±0.04 mg/kg). LT possessed the highest concentration of caffeic acid, gallic acid, ellagic acid, ferulic acid, and quercetin, which were even higher than that in AB.

3.3 Antioxidant effect assessment

The antioxidant capacities of raspberry leaf and LT extracts were investigated using two different in vitro antioxidant assays: DPPH and ABTS assays. In DPPH assay, in the presence of antioxidant compound, purple DPPH solution turns into yellow color [22]. The antioxidant effect of raspberry leaf and LT extracts on DPPH free radical was due to its hydrogen-donating ability. Similar to DPPH assays, ABTS assay measures the scavenging activity of the free radicals.

3.3.1 DPPH free radical scavenging activity

The results of the DPPH scavenging activity of raspberry leaf and LT extracts were shown in Table 2.1. The IC50 values exhibited that the scavenging abilities of ARR (27.81±0.19 mg/mL), FH (29.55±1.47 mg/mL), LQ (28.66±1.46 mg/mL), Q (29.59±1.47 mg/mL), and T (29.31±0.18 mg/mL) were comparable to Vitamin C (28.07±0.22 mg/mL). The scavenging abilities of LT, AB, and H were significantly lower than that of Vitamin C. However, ERR and USR showed higher scavenging abilities than Vitamin C.

Table 2.1
Antioxidant activities of 9 raspberry leaves and LT extracts Table 2.1 IC50 values of DPPH·radical scavenging capacity of 9 raspberry leaves and LT extracts

Varieties AB ARR ERR FH H LQ Q T USR LT VC

IC50/(mg/mL) 32.10±0.22b 27.81±0.19d 23.74±0.22e 29.55±1.47cd 31.14±0.24bc 28.66±1.46d 29.59±1.47cd 29.31±0.18cd 18.16±0.11f 34.65±1.54a 28.07±0.22d

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	32.10±0.22b	27.81±0.19d	23.74±0.22e	29.55±1.47cd	31.14±0.24bc	28.66±1.46d	29.59±1.47cd	29.31±0.18cd	18.16±0.11f	34.65±1.54a	28.07±0.22d

Table 2.2

IC50 values of ABTS+·radical scavenging capacity of 9 raspberry leaves and LT extracts

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	17.20±0.12b	15.44±0.21bcd	13.25±0.11e	15.57±1.19bcd	17.13±0.12bc	15.26±1.18cd	14.25±1.15de	15.59±0.16bcd	11.18±0.15f	16.75±1.22bc	19.62±0.12a

The same radical scavenging capacity in different raspberry leaves was compared. Samples labeled with the same letter are not significantly different from each other, p < 0.05 (mean±S.D., n = 3).

3.3.2 ABTS radical scavenging activity measurement

The raspberry leaf and LT extracts exerted higher scavenging effects against ABTS radicals than DPPH radicals. As shown in Table 2.2, all the raspberry leaf and LT extracts exhibited significantly increased ABTS radical scavenging activities than Vitamin C (p < 0.05).

3.4 Assessment for antidiabetic in vitro

α-Amylase and α-glucosidase are the main enzymes involved in the hydrolysis of carbohydrates. Natural products which inhibit the activities of α-amylase and α-glucosidase could interfere with the hydrolysis and absorption of dietary carbohydrates and serve as an alternative therapy for the treatment of obesity [23, 24]. Raspberry leaf and LT extracts were evaluated for their inhibitory effects on α-amylase and α-glucosidase enzymes by in-vitro method.

These nine raspberry leaf and LT extracts exhibited concentration-dependent inhibitions on α-amylase and α-glucosidase activities. IC50 values of these extracts against α-amylase and α-glucosidase activities were calculated (Table 3). α-Amylase inhibition capacities of red raspberry leaves and LT extracts were found to be lower than that of Vitamin C. ARR, ERR, and USR showed the most potent capacities, however, AB showed the weakest capacity. In these 9 raspberry leaves and LT extracts, ARR and FH showed the highest inhibitory potential against α-glucosidase, followed by USR, and H.

Table 3
Antidiabetic activities of 9 raspberry leaves and LT extracts. Table 3.1 IC50 values of α-amylase inhibition capacity of 9 raspberry leaves and LT extracts

Varieties AB ARR ERR FH H LQ Q T USR LT VC

IC50/(mg/mL) 3.73±0.31a 0.70±0.03ef 0.81±0.13ef 1.10±0.26de 1.56±0.26d 1.38±0.09de 3.18±0.60b 2.27±0.28c 0.84±0.12ef 1.05±0.05e 0.52±0.12f

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	3.73±0.31a	0.70±0.03ef	0.81±0.13ef	1.10±0.26de	1.56±0.26d	1.38±0.09de	3.18±0.60b	2.27±0.28c	0.84±0.12ef	1.05±0.05e	0.52±0.12f

Table 3.2

IC50 values of α-glucosidase inhibition capacity of 9 raspberry leaves and LT extracts

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	1.82±0.13de	0.75±0.16h	1.66±0.19ef	0.83±0.06h	1.46±0.10fg	2.20±0.09bc	2.36±0.34b	2.00±0.10cd	1.32±0.04g	1.60±0.21efg	3.51±0.32a

Samples labeled with the same letter are not significantly different from each other, p < 0.05 (mean±S.D., n = 3).

Ellagic acid is reported to be one of the main bioactive compounds in the raspberry leaves [5]. Our previous publication has already demonstrated that ellagic acid played an important role on ameliorating the symptoms, such as decreasing blood glucose of high fat diet induced obesity in a mouse model [25]. Consumption of quercetin and quercetin-containing fruits was also reported to decrease blood glucose concentration, hepatic metabolism, and gene expression patterns in obese mice [26]. Tannins and vascular complications of diabetes have been reviewed recently [27]. Therefore, phytochemicals such as ellagic acid, quercetin, and tannins might account for the anti-obesity effect of raspberry leaf and LT.

3.5 Antimicrobial effect assessment

Markers of antibacterial activities of 9 raspberry leaves and LT, including growth inhibition zone and MIC, were shown in Table 4. The inhibition zones given by AB, ARR, ERR, H, LQ, T, USR, and LT against E.coli were more than 9 mm (diameter), which were significantly higher than FH and Q. The inhibition zones amongst all the raspberry leaf and LT extracts against S.aureus were not as obvious as against E.coli. To S.aureus, the inhibition zones of AB, ARR, H, USR, and LT were bigger than ERR, FH, LQ, Q, and T, which no obvious inhibition zones were observed. On the other hand, to B.subtilis, the inhibition zones produced by AB, ARR, H, and USR were biggest, which were bigger than ERR, FH, T, and LT. However, no obvious inhibition zones were observed after treatment by LQ or Q (Table 4).

Table 4
Antibacterial activities of 9 raspberry leaves and LT extracts

E. Coli S. aureus B. subtilis

Inhibition zone MIC (mg/mL) Inhibition zone MIC (mg/mL) Inhibition zone MIC (mg/mL)

AB ++ 5 + 5 ++ 5

ARR ++ 5 + 10 ++ 5

ERR ++ 5 +– 20 + 10

FH + 20 +– 20 + 10

H ++ 5 + 10 ++ 10

LQ ++ 5 +– 20 +– 20

Q + 10 +– 20 +– 20

T ++ 10 +– 20 + 10

USR ++ 5 + 10 ++ 5

LT ++ 10 + 5 + 10

	E. Coli	S. aureus	B. subtilis
AB	++	5	+	5	++	5
ARR	++	5	+	10	++	5
ERR	++	5	+–	20	+	10
FH	+	20	+–	20	+	10
H	++	5	+	10	++	10
LQ	++	5	+–	20	+–	20
Q	+	10	+–	20	+–	20
T	++	10	+–	20	+	10
USR	++	5	+	10	++	5
LT	++	10	+	5	+	10

+–Indicated no obvious inhibition zone was observed; + indicated the diameter of the inhibition zone ranged from 5–8 mm; ++ indicated the diameter of the inhibition zone was >9 mm.

FH showed highest MIC value (20 mg/mL), when it was used to inhibit E.coli growth. AB, and LT exhibited potent abilities to inhibit the growth of S. aureus. AB, ARR, and USR showed strong inhibitory abilities to the growth of B. subtilis.

3.6 Anti-inflammatory effect assessment

The hyaluronidase inhibition rate and albumin denaturation inhibition rate of food compounds are considered to be indicators of anti-inflammatory effect.

Hyaluronidase, an enzyme involved in normal tissue remodeling. Upregulation of hyaluronidase activity, which results in an increase of hyaluronan destruction, contributes to joint disease and other types of inflammatory conditions [28].

Denaturation of tissue proteins is a well-documented cause of inflammatory diseases. The in-vitro anti-inflammatory effect of individual extract was evaluated against denaturation of albumin [29].

Various phytochemicals such as tannins, kaempferol, myricetin, quercetin, catechin, and epicatechin were reported to inhibit hyaluronidase activity or albumin denaturation [28, 30].

The anti-inflammatory study was conducted by assessing the hyaluronidase inhibition rate and albumin denaturation inhibition rate of raspberry leaf and LT extracts. As shown in Table 5, hyaluronidase inhibition activities of AB and H were lowest amongst nine raspberry leaf and LT extracts. On the other hand, FH exhibited highest albumin denaturation inhibition activities (p < 0.05).

Table 5
Anti-inflammatory activities of 9 raspberry leaves and LT extracts.

Table 5.1. IC50 values of hyaluronidase inhibition capacity of 9 raspberry leaves and LT extracts

Varieties AB ARR ERR FH H LQ Q T USR LT VC

IC50/(mg/mL) 7.86±1.20b 0.62±0.18d 2.24±0.33d 2.30±0.44d 10.09±2.09b 1.35±0.44d 1.24±0.20d 5.55±1.10c 1.60±0.50d 1.61±0.38d 19.59±4.49a

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	7.86±1.20b	0.62±0.18d	2.24±0.33d	2.30±0.44d	10.09±2.09b	1.35±0.44d	1.24±0.20d	5.55±1.10c	1.60±0.50d	1.61±0.38d	19.59±4.49a

Table 5.2

IC50 values of albumin denaturation inhibition capacity of 9 raspberry leaves and leaf tea extracts

Varieties	AB	ARR	ERR	FH	H	LQ	Q	T	USR	LT	VC
IC50/(mg/mL)	6.51±0.41c	3.33±0.02g	4.04±0.27f	2.51±0.10h	5.01±0.28e	7.00±0.65b	3.13±0.05g	7.94±0.27a	4.84±0.10e	3.53±0.14g	5.95±0.08d

Samples labeled with the same letter are not significantly different from each other, p < 0.05 (mean±S.D., n = 3).

3.7 Correlation between phytochemical content and antioxidant activity

Phytochemicals, including phenolic, flavonoid, and proanthocyanidin, might contribute to the antioxidant activities of the raspberry leaves. Hence, correlation analyses was performed to investigate the correlation between the phytochemicals and antioxidant activities. Results showed a strong positive correlation between total phenolics and ABTS scavenging activity IC50 (r = –0.915, p < 0.05), and total phenolics and DPPH scavenging activity IC50 (r = –0.830, p < 0.05) (Table 6). In detail, ERR extract exhibited the highest levels of total phenolic (5.56±0.058 mg/g dry ERR), as well as the highest chlorogenic acid (597.03±10.90 mg/kg), rutin level (2771.9±41.52 mg/kg). The high level of rutin concentration was reported to be correlated with a good DPPH scavenging activity [31].

Table 6
Correlation analysis between antioxidant activities and contents of active components in raspberry leaves and LT extracts

Spearman’s Rho Correlation Polyphenols Flavonoids Proanthycidins

ABTS –0.915^ 0.0424 0.0545

DPPH –0.830^ –0.188 0.0182

Spearman’s Rho Correlation	Polyphenols	Flavonoids	Proanthycidins
ABTS	–0.915^**	0.0424	0.0545
DPPH	–0.830^**	–0.188	0.0182

^*Indicates p < 0.05, ^**indicates p < 0.01.

Previous reports by Milivojević et al. [32], and Abu et al. [33] supported a positive correlation between phenolics and antioxidant capacities in the berry extracts. Therefore, phenolics influence antioxidant activities of raspberry leaf considerably.

However, no strong correlation was observed between antimicrobial, anti-inflammatory, or antidiabetic activities vs. contents of active components (Table S1).

4 Conclusion

Based on the current study, raspberry leaf and LT extracts showed significant in vitro antioxidant, antibacterial, antidiabetic, and anti-inflammatory properties. The presence of phenolic compounds is particularly important for antioxidant properties. Raspberry leaf may be exploited in preparation of herbal drugs. However, further in vitro and in vivo trials at different doses and concentrations on antioxidant, antibacterial, anti-obesity, and anti-inflammatory effects are required. The active ingredients which are responsive to the antioxidant, antibacterial, antidiabetic, and anti-inflammatory properties require future study.

Conflict of interest

The authors have no conflict of interest to report.

Footnotes

Acknowledgments

This work was supported by “the Fundamental Research Funds for the Central Universities” (grant number: 2572018BA07), and “Applied Technology Research and Development Project of Harbin Science and Technology Bureau” (grant number: 2017RAYXJ012).

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