Development and Functional Analysis of Lithocarpus polystachyus (wall.) Rehd Black Tea

Abstract

This study examined the development conditions and functional properties of a novel compound tea Lithocarpus polystachyus (wall.) Rehd (L. polystachyus, LPR) black tea (LPRBT). The compound tea was developed by fermentation using fresh leaves (Camellia sinensis cv. Qianmei 601) as the main raw material with LPR powder as an additive. Based on the single factor and orthogonal tests with sensory scores as indicators, a withered leaves–LPR powder mass ratio of 9:1 with a 6 h fermentation time was determined to be the production condition of LPRBT with a sensory score of 89.09. In addition, phlorizin content, anti-oxidation function, hypoglycemic function, and tumor suppressor effect of LPRBT were measured. The results demonstrated that LPRBT phlorizin content was significantly higher than apple. It also showed that the equivalent 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical clearance rate with Vitamin C (Vc) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical clearance rate was 81% of Vc. Both hydroxyl and superoxide anion radical clearance increased with the increase in LPRBT amount. LPRBT also showed a good inhibitory effect on α-glucosidase and α-amylase, indicating certain hypoglycemic activity. Moreover, it inhibited the growth of HeLa and A549 cancer cells showing tumor suppressor activity. This study provides a reference for the development and application of LPR food products.

1. Introduction

Lithocarpus polystachyus (wall.) Rehd (L. polystachyus, LPR), an evergreen plant of the Fagaceae family, also known as sweet tea, sweet vegetable, and wild golden firewood. LPR is a medicinal edible plant with a long history of use in China. People often consume the young leaves of LPR and use them in tea. Soaked leaves are sweet, refreshing, and have a strong aroma with a lasting aftertaste [1,2]. Dihydrochalcone phlorizin is the main sweet component of LPR, which is 300 times sweeter than sucrose [3]. Dihydrochalcone as an oxidant can clear free radicals and delay aging [4,5]. Phlorizin mainly participates in blood glucose homeostasis, pentose degradation, calcium ion homeostasis, etc., and has shown a positive effect on diabetics [6,7]. Phlorizin can specifically and competitively inhibit the transport of glucose molecules via SGLT1 and SGLT2 reducing blood glucose levels [5,8,9]. Phlorizin is metabolized into phloretin by lactase-phloretin hydrolase, β-glucosidase, and intestinal flora [10]. In addition, phloretin has an inhibitory effect on cancer cells, inducing cancer cell apoptosis in vitro [11]. Another study showed that phloretin effectively inhibits the migration and invasion of cancer cells [12].

Traditional black tea (CBT), with strong fusion properties, can be compounded with a variety of raw materials to improve aroma, taste, and consumer acceptance [13]. Currently, iced black tea, milk tea, pecan black tea compound beverage, citrus black tea, rose black tea, black tea biscuits, and black tea cake are the major food products that use black tea as the main ingredient [14,15]. However, a study on adding LRP as a natural sweetener to withered tea leaves for fermentation to produce black tea has not been reported yet. This study mixed withered tea leaves with LPR powder to develop the compound tea (LPRBT) with improved taste and health effects that can also be consumed by diabetics. This paper provides a reference for the development of tea healthcare functional products of LPR.

2. Materials and Methods

2.1. Materials

LPR fresh leaves and fresh tea leaves (C. sinensis cv. Qianmei 601) were collected from Meitan County, Guizhou Province. Non-small cell lung cancer A549 cells and cervical cancer Hela cells were maintained in our laboratory (The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)).

2.2. Preparation of LPRBT

LPR fresh leaves were steam-finished using a vertical biomass-fired steam boiler. The boiler was heated, and LPR fresh leaves were put into the steam for 5 s at a pressure of 0.4 MPa. Then, the leaves were taken out and dried at 70 °C using a tea roaster, and ground using a ball mill matcha machine (Hq-20) at 52 Hz and 1 kg/h. Lastly, the impurities were filtered out with an 80-mesh stainless steel sieve and the final product was stored at −20 °C for further use.

LPR powder was added to the withered tea leaves for kneading and fermentation. The withered tea leaves were firstly pressed lightly and kneaded for 10 min. After pressured and kneaded for 30 min, the LPR powder was added, and the complete mixture was pressured and kneaded for 10 min. The drying temperature was 120 °C, and the fragrance induction was performed at 85 °C for 2 h.

The fermentation was carried out at 26 °C for 6 h. The sensory scores of different combination products (withered leaves: LPR; 10:1, 6:1, and 2:1) were investigated. Based on the univariate test, withered leaves–LPR ratio and fermentation time were used as the factors for the orthogonal test. The optimal proportion of LPRBT was determined based on sensory evaluation (

Table 1. Orthogonal test factors.

Level	Factors
	Withered Leaves: LPR	Fermentation Time (h)
1	9:1	5
2	8:1	6
3	7:1	7

).

2.3. Physicochemical Indices of LPRBT

Sensory evaluation of LPRBT was determined following the GB/T 23776-2018 Methodology for sensory evaluation of tea. The total soluble sugar content was determined by anthrone sulfate colorimetry. The content of tea polyphenols was determined following the GB/T 8313-2018 Determination of total polyphenols and catechins content in tea. The amino acid content was determined following the GB/T 8314-2013 Tea-Determination of free amino acids content; the caffeine content was determined following the GB/T 8312-2013 Tea-Determination of caffeine content. The phlorizin content was determined by high-performance liquid chromatography. Meanwhile, apple flesh and apple peel were used as controls [16].

2.4. Preparation of LPRBT Extracts

First, 150 mL of boiled water (100 °C) was added with 3 g of LPRBT, and the solution was filtered with a 0.22 μm nylon filter membrane. One part of the collected filtrate was used for anti-oxidation and hypoglycemic function assays, and the other part was freeze-dried for tumor suppressor function assays.

2.5. Determination of Anti-Oxidation Function of LPRBT

2.5.1. Measurement of 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) Diammonium Salt (ABTS) Radical Clearance Activity

First, 2.5 mL of ABTS (7 mmol/L) and 44 µL of K₂S₂O₈ (140 mmol/L) were mixed thoroughly and kept overnight (12–16 h) in the dark at room temperature (25 °C) to obtain ABTS stock solution [17]. ABTS working solution was prepared by diluting ABTS stock solution with phosphate buffer (20 mmol/L, pH = 7.4) until OD₇₃₄ reached 0.700 ± 0.002. Then, 30 µL of LPR extract and 3.0 mL of ABTS working solution were mixed to react in the dark for 6 min. Then, the mixture absorbance was measured at 734 nm. Meanwhile, LPR powder and 1mg/mL Vitamin C (Vc) solution were used as controls. The ABTS radical clearance was calculated as follows:

$$\mathrm{ABTS} \mathrm{radical} \mathrm{clearance} (\%)=(1-\frac{A_1-A_2}{ A_3})\times 100$$

(1)

where A₁, A₂, and A₃ are the absorbances of sample, an equivalent amount of distilled water instead of ABTS working solution, and the blank control.

2.5.2. Measurement of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Clearance Activity

First, 0.5 mL of different samples was added with 1 mL of 0.2 mmol/L DPPH anhydrous ethanol and reacted at RT for 30 min in the dark after good mixing. The mixture was centrifuged at 6000 rpm/min for 10 min. The absorbance (517 nm) of the obtained supernatant was determined [18]. Meanwhile, LPR powder and 1mg/mL Vc solution were used as control. The DPPH radical clearance was calculated as follows:

$$\mathrm{DPPH} \mathrm{radical} \mathrm{clearance} (\%)=(1-\frac{A_1-A_2}{ A_3})\times 100$$

(2)

where A₁, A₂, and A₃ are the absorbances of sample, an equivalent amount of absolute ethanol instead of DPPH ethanol solution, and the blank control.

2.5.3. Measurement of Hydroxyl Radical Clearance Activity

First, 0.1, 0.2, 0.5, 0.8, 1.0 mL of sample was added into respective tubes, and volume was made up to 2 mL with distilled water. Then, 2 mL of 6 mmol/L FeSO₄, 2 mL of 6 mmol/L H₂O₂, and 2 mL of 6 mmol/L salicylic acid were added to each tube. The tubes were shaken well and kept for 30 min. Then, the mixtures were centrifuged at 5000 rpm/min for 5 min, and absorbance was measured at 510 nm [19]. CBT, LPR powder, and 1 mg/mL Vc solution were used as controls. The hydroxyl radical clearance was calculated as follows:

$$\mathrm{Hydroxyl} \mathrm{clearance} (\%)=(1-\frac{A_1-A_2}{ A_3})\times 100$$

(3)

where A₁, A₂, and A₃ are the absorbances of sample, the same volume of distilled water instead of the salicylic acid solution, and the blank control.

2.5.4. Measurement of Superoxide Anion Radical Clearance Activity

First, 0.1, 0.2, 0.5, 0.8, 1.0 mL of sample was accurately added into respective tubes and volume was made up to 1 mL with distilled water. These were then added with 4.5 mL of Tris-HCl buffer solution (pH = 8.2) preheated at 25 °C for 20 min and 0.4 mL of 25 mmol/L pyrogallol solution. After good mixing and heating at 25 °C for 5 min, the mixtures were added with 1 mL of 8 mol/L HCl solution to terminate the reaction, and the mixture absorbance was measured at 299 nm [20]. CBT, LPR powder, and 1 mg/mL Vc solution were used as controls. The superoxide anion radical clearance was calculated as follows:

$$\mathrm{Superoxide} \mathrm{anion} \mathrm{clearance} (\%)=(1-\frac{A_1-A_2}{ A_3})\times 100$$

(4)

where A₁, A₂, and A₃ are the absorbances of sample, an equivalent volume of distilled water instead of the pyrogallol solution, and the blank control.

2.6. Determination of Hypoglycemic Function of LPRBT

2.6.1. α-Glucosidase Activity Inhibition Assay

The groups were divided into the sample, positive (acarbose solution), and blank control groups (phosphate-buffered solution; pH = 6.8). Then, 0.3 mL of α-glucosidase was added, and 0.4 mL of 4-nitrophenyl beta-D-glucopyranoside (PNPG) solution was used as the substrate. The reaction was terminated after 30 min at 37 °C using 0.8 mL of Na₂CO₃ solution. The sample absorbance was measured at 400 nm, and the inhibition rate was calculated [21]. The concentrations of α-glucosidase, PNPG, acarbose, and Na₂CO₃ in the reaction system were 2 U/mL, 2 mg/mL, 5 mg/mL, and 25 mg/mL, respectively. The inhibition rate of α-glucosidase was calculated as follows:

$$\mathrm{Inhibition} \mathrm{rate} \mathrm{of} \alpha -\mathrm{glucosidase} (\%)= \frac{A_0-A_1}{A_0}\times 100$$

(5)

where A₀ and A₁ refer to the absorbance of blank control and sample group, respectively.

2.6.2. α-Amylase Activity Inhibition Assay

First, 500 μL of α-amylase solution (1 U/mL) was added to 500 μL of sample solution and reacted at 25 °C for 10 min. Then, 500 μL of soluble starch solution (1% w/v) was added and the reaction was carried out for another 10 min. Finally, the reaction was terminated with 1 mL of DNS reagent. The mixture was diluted to 10 mL with deionized water, and the absorbance was measured at 520 nm. Acarbose used as the positive control [22]. The α-amylase inhibition rate was calculated as follows:

$$\mathrm{Inhibition} \mathrm{rate} \mathrm{of} \alpha -\mathrm{amylase}/\%= 1-\frac{A_1-A_2}{A_3-A_4}\times 100$$

(6)

where A₁, A₂, A₃, and A₄ are the absorbances of sample group, sample blank group, control group and the blank group.

2.7. Determination of Tumor Suppressor Function of LPRBT

Hela and A549 cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 °C and 5% CO₂. The proliferation rate of Hela and A549 cells was measured by the Cell Counting Kit-8 (CCK8) method. The cells in the logarithmic growth phase were used to prepare a single-cell suspension, which was inoculated in a 96-well culture plate (100 μL/well). After culturing for 24 h at 37 °C, 10 μL of LPRBT extracts with different concentrations (50, 100, 200, 500 μg/g) were added and the culture was continued for another 24 h; each concentration treatment had three repeats. Next, 10 μL of CCK8 solution was added to each well, and the culture was continued for 0.5 h at 37 °C. The plate absorbance (450 nm) was read with a microplate reader and the cell viability was calculated as follows:

$$\mathrm{Cell} \mathrm{viability} (\%)= \frac{A_1-A_0}{A_2-A_0}\times 100$$

(7)

where A₀ and A₁ are the absorbances of the medium and CCK8 solution but without cells, the absorbances of the cells, CCK8 solution, and tea extract, and A₂ is the absorbances of the cells and CCK8 solution but without tea extract.

2.8. Statistical Analysis

All experiments were performed in triplicate, and the results were displayed as mean ± standard deviation. Statistical analyses were conducted with the IBM SPSS Statistics 19.0 software (SPSS Inc., Chicago, IL, USA). The statistical significance was investigated by one-way ANOVA with Duncan’s test at a significance level of p < 0.05.

3. Results

3.1. Optimization of BT–LPR Ratio

The results showed that LPR addition to withered leaves increased the sweetness of LPRBT and agglomeration. The sensory score is highest when the ratio of withered leaves and LPR is 6:1. At the withered leaves–LPR ratio of 10:1, LPRBT exhibited a licorice flavor (

Table 2. Univariate test results.

Sample Number	Appearance	Soup Color	Fragrance	Taste	Leaf Base	Overall Rating
10:1	Still tight and thin, yellowish-brown, moist, covered with gold cents	Bright yellow	Pure, slightly sweet, basically free of precipitation	Slightly astringent, slightly sweet	Tan, brighter	91.8
6:1	Tight and thin, curved, yellowish-brown, more moist, golden centimeters, lumps	Yellow red still bright	The sweet tea fragrance is obvious, lasting, and with slight precipitation	Mellow, sweet, refreshing	Tan, bright	94.3
2:1	Long block, brown, smooth, with gold cents	Yellow, brighter	Obvious sweet aroma, light tea aroma, with precipitation	Too sweet, obvious licorice flavor, weak tea flavor	Unexpanded, tan, bright	85.25

). Hence, for the subsequent orthogonal test, the withered leaves–LPR ratios of 9:1, 8:1, and 7:1 and the fermentation time of 5, 6, and 7 h were selected.

3.2. Orthogonal Test of LPRBT

Based on the analysis results of different production process parameters, the highest range value (R) was considered as the most important factor. The ratios of withered leaves–LPR as the vital impact factor was employed to affect the sensory score of the samples. The time of fermentation had the lowest impact on the orthogonal experiment. A higher K value indicated that the level had a more important effect on the sensory data of the samples for each factor. The best scheme could be determined according to the highest K value of each level. For withered leaves–LPR ratios 7:1 and 8:1, the sensory score decreased with the increase in fermentation time. The withered leaves–LPR ratio of 9:1 produced the highest (89.09) sensory score with a fermentation time of 6 h. Therefore, the optimum parameters for LPRBT production were determined as follows: withered leaves–LPR ratio of 9:1 and fermentation time of 6 h (

Table 3. Orthogonal test results.

Test Serial Number	Factors and Levels		Index
	(A) Withered Leaves: LPR	(B) Fermentation Time (h)	Sensory Score
1	1 (7:1)	1 (5)	88.29
2	1	2 (6)	87.83
3	1	3 (7)	87.14
4	2 (8:1)	1	87.62
5	2	2	87.61
6	2	3	87.28
7	3 (9:1)	1	88.69
8	3	2	89.09
9	3	3	88.33
K1	87.75	88.20
K2	87.50	88.18
K3	88.70	87.58
R	1.2	0.62

Note: Analysis of the results of the orthogonal experiment adopted the range analysis. K_1–3 was the sums’ mean of the indicators at each level of 1–3 for each factor of A and B. R = extreme difference, i.e., the difference between the highest and lowest value at each level of K_1–3 for each factor of A and B (to indicate the influence of each factor on the result).

).

3.3. Physiochemical Indices

Compared with the control (CK, 6 h) group, the optimized group (9:1, 6 h) exhibited significantly enhanced contents of tea polyphenols and soluble sugar, equivalent contents of caffeine and flavone, and significantly reduced contents of amino acids. Compared with LPR powder, the optimized group exhibited significantly enhanced contents of tea polyphenols and amino acids, and significantly reduced contents of caffeine, flavone, and soluble sugar. This indicated a substantial change in contents during fermentation (

Table 4. Compositions of LPRBT.

Sample (Withered Leaves: LPR, Fermentation Time)	Content (%)
	Tea Polyphenols	Amino Acid	Caffeine	Flavone	Soluble Sugar
7:1, 5 h	10.34 ± 0.39 d,e	0.26 ± 0.02 d,e	7.49 ± 0.57 b,c	17.34 ± 0.32 b	2.78 ± 0.06 b
7:1, 6 h	10.85 ± 0.54 c,d	0.25 ± ND e	6.33 ± 0.07 c,d	21.07 ± 0.89 a	2.40 ± 0.06 d,e
7:1, 7 h	12.05 ± 0.13 b,c	0.19 ± 0.01 f	6.86 ± 0.23 c,d,e	12.75 ± 0.28 c	2.60 ± 0.05 c
8:1, 5 h	9.42 ± 0.26 e	0.25 ± 0.02 d,e	7.26 ± 0.35 c,d	12.96 ± 1.34 c	2.15 ± 0.02 f
8:1, 6 h	11.3 ± 0.85 c,d	0.31 ± 0.01 b	8.19 ± 0.67 b	20.73 ± 1.91 a	2.45 ± 0.01 c,d,e
8:1, 7 h	10.34 ± 0.74 d,e	0.28 ± ND c,d	7.49 ± 0.41 b,c	12.20 ± 1.06 c	2.62 ± 0.07 b,c
9:1, 5 h	11.44 ± 0.89 c,d	0.30 ± ND b,c	7.26 ± 0.35 c,d	13.79 ± 1.09 c	2.62 ± 0.02 b,c
9:1, 6 h	13.02 ± 0.29 a,b	0.28 ± 0.01 b,c	6.16 ± 0.18 e,f	14.19 ± 0.99 c	2.35 ± 0.01 e
9:1, 7 h	12.89 ± 0.59 a,b	0.25 ± 0.01 e	5.94 ± 0.40 f	12.05 ± 1.57 c	2.56 ± 0.28 c,d
CK, 5 h	13.89 ± 1.60 a	0.31 ± 0.04 a,b	6.20 ± 0.50 e,f	12.01 ± 0.16 c	1.71 ± 0.07 g
CK, 6 h	10.93 ± 0.45 c,d	0.33 ± 0.02 a	6.52 ± 0.18 d,e,f	11.32 ± 0.89 c	2.15 ± 0.03 f
CK, 7 h	10.74 ± 0.30 c,d,e	0.30 ± 0.01 b,c	5.99 ± 0.12 f	11.93 ± 0.51 c	2.48 ± 0.06 c,d,e
LPR powder	10.85 ± 1.16 c,d	0.13 ± 0.01 g	21.15 ± 0.81 a	20.73 ± 4.16 a	4.88 ± 0.09 a

Note: ND refers to undetected standard deviation, and different letters indicate a significant difference (p < 0.05). CK refers to traditional black tea.

).

The phlorizin content in LPRBT, apple flesh, apple peel, and CBT was 4.11 ± 0.39, 0.042 ± 0.014, 0.338 ± 0.029, and 0 mg/g, respectively (Figure 1). Hence, the LPR addition significantly enhanced the phlorizin content of black tea compared with apple flesh (>10 times) and apple peel (>90 times).

3.4. Anti-Oxidation Function of LPRBT

The ABTS radical clearance activity of LPRBT (96.68%) was almost close to Vc (97.22%) and LPR raw powder (97.20%). The DPPH radical clearance activity of LPRBT (72.77%) was 81% of the positive control Vc (89.59%). The hydroxyl radical clearance activity of CBT was higher than that of LPRBT for the loading volume 0.1–0.2 mL. For the sample size >0.5 mL, the hydroxyl radical clearance activity of LPRBT was higher than that of CBT, indicating that LPRBT can have a higher ability to scavenge hydroxyl radical at a certain concentration. The superoxide anion clearance activity of LPRBT was higher than that of CBT, indicating that LPR improved the superoxide anion clearance property of CBT (Figure 2).

3.5. Hypoglycemic Function of LPRBT

LPRBT inhibition rate of α-glucosidase was lower (76.96%) than the commonly used hypoglycemic drug acarbose (80.23%) but higher than CBT (23.52%). Additionally, the LPRBT inhibition rate of α-amylase was lower (62.78%) than the positive control acarbose (79.10%) but higher than CBT (20.39%). This indicated the blood sugar-reducing property of LPRBT via inhibition of enzymes that convert macromolecular substances into glucose. However, the effect was lower than that of acarbose. Overall, the addition of LPR improved the α-glucosidase and α-amylase inhibition rate of black tea (Figure 3).

3.6. Tumor Suppressor Function of LPRBT

LPRBT inhibited the growth of Hela cells in a concentration-dependent manner. The IC₅₀ of CBT was about 283.1 μg/g indicating a better inhibitory effect of LPRBT (IC₅₀ 104.9 μg/g) against the Hela cells. For A549 cells, 100 μg/g LPRBT showed the highest inhibition effect; however, a further increase in concentration did not improve the inhibition effect. The IC₅₀ of LPRBT for A549 cells was 43.83 μg/g compared to the IC₅₀ of CBT 223.7 μg/g. This again indicated better cell inhibitory activity of LPRBT (Figure 4).

4. Discussion

LPR is sweet and has many health benefits, and it can be added to traditional tea beverages as a natural sweetener to enhance the taste and health value. Thus, in this study, a compound tea containing the LPR was developed. Dihydrochalcone phlorizin is the main sweet component of LPR, and apple is one of the main sources of natural phlorizin [23,24]. In this study, traditional black tea does not contain phlorizin, yet the content of phlorizin in LPRBT was 4.11 ± 0.29 mg/g, significantly higher than apple flesh (0.042 ± 0.014 mg/g) and apple peel (0.338 ± 0.029 mg/g). This indicates that LPRBT can retain this component of phlorizin very well. Phlorizin is the predominant dihydrochalcone and the most abundant polyphenolic compound in LPR, which can be metabolized to phloretin by lactase-phloretin hydrolase, β-glucosidase, and intestinal flora [10,25]. Phlorizin, trilobatin and phloretin were isolated from LPR leaves and they were all found to have good in vitro antioxidant activity [4,5]. Phloretin has synergistic antioxidant activity with caffeic acid and glutathione [26]. Rubio et al. [27] designed a new beverage combining extracts of green tea and apple and found it with high antioxidant power. Here, the scavenging rate of ABTS and DPPH free radical of LPRBT were similar to the effect of Vc, and LPR could improve the hydroxyl radical and superoxide anion clearance property of traditional black tea. It was shown that LPRBT has good antioxidant activity and the addition of LPR can improve the antioxidant capacity of traditional black tea, which may be caused by the addition of phlorizin, a functional component, to traditional black tea by LPR. In addition, phlorizin was believed to have similar properties to insulin [8,9,28] and to be able to improve to a great extent the complications associated with the diabetic state [6,29]. Our results indicated that LPRBT exhibited a good inhibitory effect against α-glucosidase and α-amylase, and its hypoglycemic effect was not as good as that of acarbose. While LPRBT had the efficacy of inhibiting enzymes that convert macromolecules into glucose, which played a role in lowering blood sugar. The addition of LPR can improve the inhibition of a-glucosidase and a-amylase by traditional black tea. Furthermore, trilobatin and phloretin from LPR also showed significant inhibitory activities against α-glucosidase and α-amylase [30,31]. However, it is not clear that compounds, phlorizin, trilobatin, or phloretin play a major role in the antidiabetic process of LPR, which will be analyzed for further studies [32,33,34,35]. The previous studies have found that phloretin could enhance the anticancer effects of cisplatin on non-small cell lung cancer cell lines by suppressing proliferation, inducing apoptosis, and inhibiting the invasion and migration of the cells [36]. Phloretin could also enhance the inhibition of cervical cancer cells by nanoencapsulation [37]. In our studies, LPRBT showed a good inhibitory effect against the cancer cells, which was better than traditional black tea. It indicates that the addition of LPR powder can improve the inhibitory effect of traditional black tea on cancer cells, which may be related to the rich phloretin content of LPRBT.

In summary, the results clearly suggest that LPRBT has good antioxidant, hypoglycemic, and tumor suppressor functions in vitro; however, direct evidence is lacking, and further in vivo studies might be needed to evaluate its potential beneficial effects for human health.