# Analysis of Chemical Constituents Changing in Physical Process and Nutritional Components ofMalus hallianaKoehne Tea

Authors: Zhenhua Yin; Yong Zhang; Juanjuan Zhang; Wenyi Kang
Journal: Journal of Food Quality
 (2017)

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## Abstract

This study aimed to establish a HPLC method for simultaneous determination of the changing of quercitrin, 3-hydroxyphloridzin, and phloridzin in physical process ofM. halliana tea. Meanwhile, the nutritional compositions were determined, using anthrone-sulfuric acid colorimetry and direct titration determination of total sugar and reducing sugar, respectively, in order to provide theoretical basis for quality control and tea production. The results showed that the regression equations for quercitrin, 3-hydroxyphloridzin, and phloridzin were linear in the range of 0.0972–12.15 μg (r=0.999 8), 0.0932~11.65 μg (r=0.999 1), and 0.9~112.5 μg (r=0.999 6), respectively. The average recoveries ranged from 98.19% to 99.35%. The contents of crude protein and the crude fat were measured by spectrophotometric detection and soxhlet extraction detection, respectively. The contents of total sugar, reducing sugar, the fat, and protein were 6.8 g/100 g, 8.5 mg/100 g, 2.399 g/100 g, and 4.362 g/100 g, respectively, inM. halliana tea.


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## Body

## 1. Introduction

Malus halliana (M. halliana)Koehne, belonging to the family Rosaceae, is abundant and widely distributed in Jiangsu, Zhejiang, Anhui, Shanxi, Sichuan, and Yunnan provinces in China, which grows in the jungle of slopes or stream sides, commonly cultivated for ornamental industry [1].Chinese Materia Medica records that its taste is light, bitter, and flat, and it can regulate the menstrual function and blood and treat metrorrhagia [2]. In folk medicine, it has been used as traditional Chinese herbal medicine in the treatment of traumatic injury, fractures, and hemorrhage [3]. In recent years, more studies have focused on gardening cuttings, cultivation, breeding, and so on [4, 5]. TheM. halliana-derived healthy drink has been developed [6].Drinking tea not only can add some of the necessary trace elements in the human body, but also has a pharmacological function and health effects on the human body. Modern medicine researches have shown that it not only can prevent thrombosis, reduce blood viscosity, increase high-density lipoprotein, and reduce the body’s capillary permeability and brittleness, but also possesses certain preventive effects on cardiovascular and cerebrovascular diseases and antiaging and increases immunity [7]. Drinking tea can make people excited, has diuretic impact, results in sterilization, has anti-inflammatory, cardiac, and other effects, enhances memory, and prevents diabetes [8, 9].Our group has conducted a series of related researches in early stage, including chemical composition [3, 10, 11], pharmacological activity [12–15], and content determination [15]. Flavonoids are characteristic constituents inM. halliana, and pharmacological investigations indicate that it possesses antioxidant [12], hepatoprotective [13],α-glucosidase inhibitory [14], tyrosinase-activating [15], and antimicrobial [16] activities.To date, onlyM. hupehensis is used as drinking tea known as Ning Qing tea, which is widely used as summer cool and refreshing drink in Three Gorges region of China [17]. The main component was phloridzin [18], and there has been a patent to disclose a preparation method and application as antidiabetic, hypolipidemic, antioxidant, and antitumor medicine ofM. hupehensis tea extract (ZL 200710053716.8).Our previous research has found that phloridzin is also the main ingredient ofM. hallianaleaves [11]. While the natural resource ofM. halliana was rich, no studies on the quality control and the analysis of the nutritional compositions ofM. hallianatea are available in the literatures. Our study was undertaken to adopt the traditional tea processing technology to develop theM. halliana tea, and the contents of quercitrin, 3-hydroxyphloridzin, and phloridzin inM. hallianatea were measured using high-performance liquid chromatography (HPLC). In the separation and purification of theM. hallianatea, we found that quercitrin, 3-hydroxyphloridzin, and phloridzin contained in the tea were more than other flavonoids, for example, juglanin, avicularin, afzelin, and 5,7-dihydroxychromone. In addition, the literature reviews found that the activities of these three compounds inMalus genus were more reported. In comprehensive analysis, we used these three compounds as the object of study. The contrast analysis of the changes in the same compositions ofM. hallianatea before and after processing was also carried out. Meanwhile, the main nutritive components ofM. hallianatea were studied. Further studies on quality control standards and nutritive components ofM. hallianatea are essential for providing theoretical basis for quality control and tea production.


## 2. Materials and Methods

### 2.1. Material and Reagents

Quercitrin, 3-hydroxyphloridzin, and phloridzin with purity greater than 98% were made by our laboratory. Acetonitrile (HPLC grade) was purchased from Avantor Performance Materials, Inc. (USA). Methanol (HPLC grade) was purchased from Tianjin Shield Fine Chemicals Co., Ltd. The water was Wahaha pure water.The series of glucose standard solutions (10~100μg/mL): the glucose (Xiangshui Tianyi Huagong Co., Ltd., Jiangsu) was weighed via drying at constant temperature using a constant temperature incubator (LZJS, Shenzhen in the South China Sea Masson Technology Industrial Co., Ltd.) at 105°C. Metered volume was filled with water to 1000 mL and then shaken well and 1, 2, 4, 6, 8, and 10 mL were sucked from it, respectively. Then they were transferred into six volumetric flasks (100 mL), diluted with water to volume, and shaken well, respectively. They were stored at 0–4°C refrigerator for two weeks.


### 2.2. Plant

Air-dried leaves were collected from Jinming district, Henan University (Kaifeng, Henan province of China). Among them, the first batch of samples was collected on March 11 in 2016, the second batch was collected on March 16 in 2016, the third batch was collected on March 21 in 2016. The plant was identified asM. hallianaof family Rosaceae by Professor Changqin Li (Henan University, Kaifeng, China). The voucher specimens were deposited in the Institute of Natural Products, Henan University (number 20160312).


### 2.3. Preparation ofM. halliana Tea

MarchM. hallianaleaves were used. TheM. hallianatea was made by de-enzyming, rolling, drying, and the same processes. Specific tea was processed as described in the patent [19].


### 2.4. Analysis of Chemical Components

#### 2.4.1. Preparation of Standard Solution and Test Sample Solution

The three standard solutions of quercitrin, 3-hydroxyphloridzin, and phloridzin were prepared in methanol at the concentrations of 486, 466, and 4500μg·mL−1 and stored at 4°C, respectively.M. hallianaleaves or tea (0.02 g, 50 mesh) were mixed with 1 mL of methanol solution in centrifuge tube. The mixture of solid and liquid was centrifuged at 5000 r·min−1 for 5 min after being extracted by ultrasonic (50 min, 500 W). Then, the extract was filtrated through a 0.22 μm microporous membrane. The subsequent filtrate was taken as the test sample solution.


#### 2.4.2. Development of the HPLC

LC-20AT high-performance liquid chromatography (Shimadzu) was used and the system was equipped with a degasser, a quaternary gradient low pressure pump, the CTO-20AC column oven, a SPD-M20A UV-detector, and a SIL-20AC autosampler. The data were acquired and processed using LC-solution chromatography data processing system.Two chromatographic separation steps were all performed on a Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) at a column temperature of 25°C. The flow rate of mobile phase was 0.8 mL·min−1. The UV detection wavelength was set at 270 nm. All the injection volumes were 10 μL. The mobile phase consisted of acetonitrile-1% phosphoric acid (20 : 80, v/v). The HPLC chromatograms of the standard solution and the extract of sample were shown in Figure 1.Figure 1
HPLC chromatograms of sample solution (A) and standard solution (B); quercitrin (1); 3-hydroxyphloridzin (2); and phloridzin (3).


### 2.5. Analysis of Nutritional Components

#### 2.5.1. Determination of Crude Protein Content

0.5010 g (40 mesh) ofM. halliana tea powder was accurately weighed. The extraction method of crude protein was determined according to the method as described in Chinese National Standard (GB 5009.5-2016) and ultraviolet spectrophotometric method was used to measure the protein content. At the same time, blank experiment was carried out with the same method to calculate the total nitrogen content. The protein content formula is as follows: protein content = total nitrogen × 6.25.


#### 2.5.2. Determination of Crude Fat Content

5.001 g ofM. hallianatea powder was accurately weighed. Crude fat was measured by the soxhlet extraction method as described in Chinese National Standard (GB 5009.6-2016) previously. The fat content formula is as follows: fat content = (m1-m0)/m2×100, where m1 refers to quality of absorption bottle and crude fat (g); m0 represents the quality of absorption bottle (g); m2 is the quality of sample (g).


#### 2.5.3. The Content Determination of Reducing Sugar

M. hallianatea powder was accurately weighed as 4.0036 g. The reducing sugar extraction method was determined according to the method described in Chinese National Standard (GB 5009.7-2016) and we used direct titration method for calibration. The reducing sugar formula is as follows: the reducing sugar content %=[m1/(m×V/250×1000)]×100.m
1 is quality of the reducing sugar (mg); V is consumption volume of sample solution in calibration (mL); m is quality of sample (g).


#### 2.5.4. The Content Determination of Total Soluble Sugar

0.1 g ofM. halliana tea powder was accurately weighed and extracted. The total soluble sugar content was determined according to the method described previously [20]. Meanwhile, the method for determining the total soluble sugar was the same as that used for determining the reducing sugar content. The total soluble sugar formula is as follows: the mass percentage of total sugar = (c×f×VSampletotal/106×m)×10  (c is sugar concentration of standard curve (μg/mL); f is dilution ratio; VSampletotal is total volume of sample (mL); m is quality of sample (g)).


## 2.1. Material and Reagents

Quercitrin, 3-hydroxyphloridzin, and phloridzin with purity greater than 98% were made by our laboratory. Acetonitrile (HPLC grade) was purchased from Avantor Performance Materials, Inc. (USA). Methanol (HPLC grade) was purchased from Tianjin Shield Fine Chemicals Co., Ltd. The water was Wahaha pure water.The series of glucose standard solutions (10~100μg/mL): the glucose (Xiangshui Tianyi Huagong Co., Ltd., Jiangsu) was weighed via drying at constant temperature using a constant temperature incubator (LZJS, Shenzhen in the South China Sea Masson Technology Industrial Co., Ltd.) at 105°C. Metered volume was filled with water to 1000 mL and then shaken well and 1, 2, 4, 6, 8, and 10 mL were sucked from it, respectively. Then they were transferred into six volumetric flasks (100 mL), diluted with water to volume, and shaken well, respectively. They were stored at 0–4°C refrigerator for two weeks.


## 2.2. Plant

Air-dried leaves were collected from Jinming district, Henan University (Kaifeng, Henan province of China). Among them, the first batch of samples was collected on March 11 in 2016, the second batch was collected on March 16 in 2016, the third batch was collected on March 21 in 2016. The plant was identified asM. hallianaof family Rosaceae by Professor Changqin Li (Henan University, Kaifeng, China). The voucher specimens were deposited in the Institute of Natural Products, Henan University (number 20160312).


## 2.3. Preparation ofM. halliana Tea

MarchM. hallianaleaves were used. TheM. hallianatea was made by de-enzyming, rolling, drying, and the same processes. Specific tea was processed as described in the patent [19].


## 2.4. Analysis of Chemical Components

### 2.4.1. Preparation of Standard Solution and Test Sample Solution

The three standard solutions of quercitrin, 3-hydroxyphloridzin, and phloridzin were prepared in methanol at the concentrations of 486, 466, and 4500μg·mL−1 and stored at 4°C, respectively.M. hallianaleaves or tea (0.02 g, 50 mesh) were mixed with 1 mL of methanol solution in centrifuge tube. The mixture of solid and liquid was centrifuged at 5000 r·min−1 for 5 min after being extracted by ultrasonic (50 min, 500 W). Then, the extract was filtrated through a 0.22 μm microporous membrane. The subsequent filtrate was taken as the test sample solution.


### 2.4.2. Development of the HPLC

LC-20AT high-performance liquid chromatography (Shimadzu) was used and the system was equipped with a degasser, a quaternary gradient low pressure pump, the CTO-20AC column oven, a SPD-M20A UV-detector, and a SIL-20AC autosampler. The data were acquired and processed using LC-solution chromatography data processing system.Two chromatographic separation steps were all performed on a Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) at a column temperature of 25°C. The flow rate of mobile phase was 0.8 mL·min−1. The UV detection wavelength was set at 270 nm. All the injection volumes were 10 μL. The mobile phase consisted of acetonitrile-1% phosphoric acid (20 : 80, v/v). The HPLC chromatograms of the standard solution and the extract of sample were shown in Figure 1.Figure 1
HPLC chromatograms of sample solution (A) and standard solution (B); quercitrin (1); 3-hydroxyphloridzin (2); and phloridzin (3).


## 2.4.1. Preparation of Standard Solution and Test Sample Solution

The three standard solutions of quercitrin, 3-hydroxyphloridzin, and phloridzin were prepared in methanol at the concentrations of 486, 466, and 4500μg·mL−1 and stored at 4°C, respectively.M. hallianaleaves or tea (0.02 g, 50 mesh) were mixed with 1 mL of methanol solution in centrifuge tube. The mixture of solid and liquid was centrifuged at 5000 r·min−1 for 5 min after being extracted by ultrasonic (50 min, 500 W). Then, the extract was filtrated through a 0.22 μm microporous membrane. The subsequent filtrate was taken as the test sample solution.


## 2.4.2. Development of the HPLC

LC-20AT high-performance liquid chromatography (Shimadzu) was used and the system was equipped with a degasser, a quaternary gradient low pressure pump, the CTO-20AC column oven, a SPD-M20A UV-detector, and a SIL-20AC autosampler. The data were acquired and processed using LC-solution chromatography data processing system.Two chromatographic separation steps were all performed on a Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) at a column temperature of 25°C. The flow rate of mobile phase was 0.8 mL·min−1. The UV detection wavelength was set at 270 nm. All the injection volumes were 10 μL. The mobile phase consisted of acetonitrile-1% phosphoric acid (20 : 80, v/v). The HPLC chromatograms of the standard solution and the extract of sample were shown in Figure 1.Figure 1
HPLC chromatograms of sample solution (A) and standard solution (B); quercitrin (1); 3-hydroxyphloridzin (2); and phloridzin (3).


## 2.5. Analysis of Nutritional Components

### 2.5.1. Determination of Crude Protein Content

0.5010 g (40 mesh) ofM. halliana tea powder was accurately weighed. The extraction method of crude protein was determined according to the method as described in Chinese National Standard (GB 5009.5-2016) and ultraviolet spectrophotometric method was used to measure the protein content. At the same time, blank experiment was carried out with the same method to calculate the total nitrogen content. The protein content formula is as follows: protein content = total nitrogen × 6.25.


### 2.5.2. Determination of Crude Fat Content

5.001 g ofM. hallianatea powder was accurately weighed. Crude fat was measured by the soxhlet extraction method as described in Chinese National Standard (GB 5009.6-2016) previously. The fat content formula is as follows: fat content = (m1-m0)/m2×100, where m1 refers to quality of absorption bottle and crude fat (g); m0 represents the quality of absorption bottle (g); m2 is the quality of sample (g).


### 2.5.3. The Content Determination of Reducing Sugar

M. hallianatea powder was accurately weighed as 4.0036 g. The reducing sugar extraction method was determined according to the method described in Chinese National Standard (GB 5009.7-2016) and we used direct titration method for calibration. The reducing sugar formula is as follows: the reducing sugar content %=[m1/(m×V/250×1000)]×100.m
1 is quality of the reducing sugar (mg); V is consumption volume of sample solution in calibration (mL); m is quality of sample (g).


### 2.5.4. The Content Determination of Total Soluble Sugar

0.1 g ofM. halliana tea powder was accurately weighed and extracted. The total soluble sugar content was determined according to the method described previously [20]. Meanwhile, the method for determining the total soluble sugar was the same as that used for determining the reducing sugar content. The total soluble sugar formula is as follows: the mass percentage of total sugar = (c×f×VSampletotal/106×m)×10  (c is sugar concentration of standard curve (μg/mL); f is dilution ratio; VSampletotal is total volume of sample (mL); m is quality of sample (g)).


## 2.5.1. Determination of Crude Protein Content

0.5010 g (40 mesh) ofM. halliana tea powder was accurately weighed. The extraction method of crude protein was determined according to the method as described in Chinese National Standard (GB 5009.5-2016) and ultraviolet spectrophotometric method was used to measure the protein content. At the same time, blank experiment was carried out with the same method to calculate the total nitrogen content. The protein content formula is as follows: protein content = total nitrogen × 6.25.


## 2.5.2. Determination of Crude Fat Content

5.001 g ofM. hallianatea powder was accurately weighed. Crude fat was measured by the soxhlet extraction method as described in Chinese National Standard (GB 5009.6-2016) previously. The fat content formula is as follows: fat content = (m1-m0)/m2×100, where m1 refers to quality of absorption bottle and crude fat (g); m0 represents the quality of absorption bottle (g); m2 is the quality of sample (g).


## 2.5.3. The Content Determination of Reducing Sugar

M. hallianatea powder was accurately weighed as 4.0036 g. The reducing sugar extraction method was determined according to the method described in Chinese National Standard (GB 5009.7-2016) and we used direct titration method for calibration. The reducing sugar formula is as follows: the reducing sugar content %=[m1/(m×V/250×1000)]×100.m
1 is quality of the reducing sugar (mg); V is consumption volume of sample solution in calibration (mL); m is quality of sample (g).


## 2.5.4. The Content Determination of Total Soluble Sugar

0.1 g ofM. halliana tea powder was accurately weighed and extracted. The total soluble sugar content was determined according to the method described previously [20]. Meanwhile, the method for determining the total soluble sugar was the same as that used for determining the reducing sugar content. The total soluble sugar formula is as follows: the mass percentage of total sugar = (c×f×VSampletotal/106×m)×10  (c is sugar concentration of standard curve (μg/mL); f is dilution ratio; VSampletotal is total volume of sample (mL); m is quality of sample (g)).


## 3. Results and Discussion

### 3.1. Analysis of Chemical Components

#### 3.1.1. Linearity

The stock standard solutions (0.2, 1, 5, 10, 15, 20, and 25μL) were accurately injected to chromatographic instrument for the construction of calibration curves, respectively, and the corresponding sample sizes of the injected quercitrin were 0.0972, 0.486, 2.43, 4.86, 7.29, 9.72, and 12.15 μg, respectively; the corresponding sample sizes of the injected 3-hydroxyphloridzin were 0.0932, 0.466, 2.33, 4.66, 6.99, 9.32, and 11.65 μg, respectively; the corresponding sample sizes of the injected phloridzin were 0.9, 4.5, 22.5, 45.00, 67.5, 90.00, and 112.5 μg, respectively. The calibration curves were constructed by plotting the peak areas versus the injected sample quality (μg) of each compound. The results were presented in Table 1. The r values were in the range from 0.9991 to 0.9998, which indicated that the methods displayed good linearity.Table 1
Regression equations and linear ranges for three compounds.
Compound
Regressive equation
r
Linear range/μg
Phloridzin
Y= 1165813.0631X + 976247.0924
0.9996
0.9~112.5
Quercitrin
Y= 1306866.7475X + 58713.4808
0.9998
0.0972~12.15
3-Hydroxyphloridzin
Y
=654528.6618X+41738.1201
0.9991
0.0932~11.65
HereY is the peak area; X refers to injection amount (μg).


#### 3.1.2. Precision

10μL of each the stock standard solution was accurately injected to chromatographic instrument. The stock solutions were analyzed in six replicates within one day for determining the precision of the developed assay. The relative standard deviations (RSDs) of peak areas for the three compounds were 0.35%, 0.35%, and 0.79%, respectively. The results indicated that the methods were precise for quantitative analysis of phloridzin, 3-hydroxyphloridzin, and quercitrin.


#### 3.1.3. Stability

The sample solutions were prepared under the optimum extraction conditions and placed at room temperature, and then 10μL of sample solution was injected into chromatographic instrument at 0, 3, 6, 9, 12, and 24 h, respectively. The RSDs of peak areas for the three compounds were 0.25%, 0.45%, and 0.41%, respectively. The results indicated that the sample solution was basically stable at room temperature within 24 h.


#### 3.1.4. Repeatability

Six test sample solutions were processed under the optimum extraction conditions, and 10μL of each solution was then injected to chromatographic instrument for analyzing. The RSDs of peak areas for the three compounds were 0.33%, 2.38%, and 1.24%, respectively, indicating that the analytical methods have the acceptable level of repeatability.


#### 3.1.5. Recovery

Nine batches of leaves and flowers samples ofM. hallianawere prepared and divided into three groups, respectively. Then three standard substances at three different amounts were added to the leaves and flowers samples. The spiked samples were prepared according to the optimum extraction conditions. All the calculated recovery values of the analytes ranged from 98.19 to 99.35% and the RSDs were 1.6%, 1.4%, and 1.9%, respectively. The results demonstrated that the methods were reasonable and feasible.


#### 3.1.6. Sample Analysis

The mass fractions of phloridzin, quercitrin, and 3-hydroxyphloridzin in original leaves and tea were presented in Table2  (n=3), respectively.Table 2
The contents of three flavonoids inM. halliana tea and original leaves for different periods (mg/g).
Compound
Period
I
II
III
Tea
Original leaves
Tea
Original leaves
Tea
Original leaves
Phloridzin
448.118
382.687
299.486
301.121
504.680
251.456
Quercitrin
9.919
4.237
14.877
7.077
7.412
7.182
3-Hydroxyphloridzin
143.630
112.306
110.055
91.911
137.408
84.023


### 3.2. Analysis of Nutritional Components

#### 3.2.1. The Content Determination of Crude Protein

According to the above experimental method, the standard curve of crude protein was drawn. We took extract ofM. hallianatea and measured its crude protein content. The field experiments were carried out with six replications and then the data were expressed as mean.The absorbance ofM. hallianatea was 0.0136, when it was substituted into the standard curve and the crude protein content ofM. hallianatea was 4.362 g/100 g. Protein played an important role in the body, which provided power and energy for the protein metabolism and maintained the normal operation of all kinds of tissues. It also can strengthen the body resistance, hypoxia, and fatigue resistance and have high nutritional value, which is the important nutritional protein for old body [21]. The crude protein content ofM. hallianatea was 4.362 g/100 g which explained that it had more amino acids, which could promote the formation of aroma in tea. And the stated amino acids colloid in tea soup, which played an important role in keeping clear of the beverage and the stability of the colloidal solution. It also had offset function of the nervous system excitement caused by caffeine. SoM. hallianatea can enhance the body resistance, hypoxia, and fatigue resistance and have high nutritional value.


#### 3.2.2. The Content Determination of Crude Fat

According to the method and formula, the crude fat ofM. hallianatea was 2.399 g/100 g. The lipid composition in tea played a positive role in the formation of the tea aroma, which could accelerate the absorption of fat-soluble vitamins and prevent vitamin deficiency disease. The fat content in tea was less confirmed by medical research, but it had function of improving human immunity, preventing cancer, and so on. Therefore, the special population who had liver disease, hypertension, hyperlipidemia, and pancreatic function not congruent also easily drinks this in addition to the ordinary people.


#### 3.2.3. Determination of Reducing Sugar Content

The cupric tartrate solution consumption ofM. hallianatea was 9.98. According to the formula, the reducing sugar ofM. halliana tea was 8.5 mg/100 g.


#### 3.2.4. Determination of Total Soluble Sugar Content

According to the above method, the standard glucose was diluted into six concentrations with gradient dilutions; then the series of glucose standard solution concentrations were taken as the abscissa and absorbance was taken as the ordinate to make a standard curve and calculate the regression equation; the equation wasy=0.003×X+0.005  (R2=0.999).The absorbance of total soluble sugar was taken into the equation to draw standard curve for the total sugar contents of sample, and all the field experiment results were carried out with six replications and then the data were expressed as mean. According to the method and formula, the total soluble sugar ofM. hallianatea was 6.8 g/100 g.The contents of total sugar and reducing sugar, which are taken as the criteria for evaluating the plant nutrition, are the important components in food flavor and nutrition. Tea polysaccharide had antidiabetic, hypolipidemic, antioxidant, and immunoregulation function [22]. From the results of total soluble sugar and reducing sugar,M. hallianatea can be developed as a health care food in the prevention and treatment of those diseases.


## 3.1. Analysis of Chemical Components

### 3.1.1. Linearity

The stock standard solutions (0.2, 1, 5, 10, 15, 20, and 25μL) were accurately injected to chromatographic instrument for the construction of calibration curves, respectively, and the corresponding sample sizes of the injected quercitrin were 0.0972, 0.486, 2.43, 4.86, 7.29, 9.72, and 12.15 μg, respectively; the corresponding sample sizes of the injected 3-hydroxyphloridzin were 0.0932, 0.466, 2.33, 4.66, 6.99, 9.32, and 11.65 μg, respectively; the corresponding sample sizes of the injected phloridzin were 0.9, 4.5, 22.5, 45.00, 67.5, 90.00, and 112.5 μg, respectively. The calibration curves were constructed by plotting the peak areas versus the injected sample quality (μg) of each compound. The results were presented in Table 1. The r values were in the range from 0.9991 to 0.9998, which indicated that the methods displayed good linearity.Table 1
Regression equations and linear ranges for three compounds.
Compound
Regressive equation
r
Linear range/μg
Phloridzin
Y= 1165813.0631X + 976247.0924
0.9996
0.9~112.5
Quercitrin
Y= 1306866.7475X + 58713.4808
0.9998
0.0972~12.15
3-Hydroxyphloridzin
Y
=654528.6618X+41738.1201
0.9991
0.0932~11.65
HereY is the peak area; X refers to injection amount (μg).


### 3.1.2. Precision

10μL of each the stock standard solution was accurately injected to chromatographic instrument. The stock solutions were analyzed in six replicates within one day for determining the precision of the developed assay. The relative standard deviations (RSDs) of peak areas for the three compounds were 0.35%, 0.35%, and 0.79%, respectively. The results indicated that the methods were precise for quantitative analysis of phloridzin, 3-hydroxyphloridzin, and quercitrin.


### 3.1.3. Stability

The sample solutions were prepared under the optimum extraction conditions and placed at room temperature, and then 10μL of sample solution was injected into chromatographic instrument at 0, 3, 6, 9, 12, and 24 h, respectively. The RSDs of peak areas for the three compounds were 0.25%, 0.45%, and 0.41%, respectively. The results indicated that the sample solution was basically stable at room temperature within 24 h.


### 3.1.4. Repeatability

Six test sample solutions were processed under the optimum extraction conditions, and 10μL of each solution was then injected to chromatographic instrument for analyzing. The RSDs of peak areas for the three compounds were 0.33%, 2.38%, and 1.24%, respectively, indicating that the analytical methods have the acceptable level of repeatability.


### 3.1.5. Recovery

Nine batches of leaves and flowers samples ofM. hallianawere prepared and divided into three groups, respectively. Then three standard substances at three different amounts were added to the leaves and flowers samples. The spiked samples were prepared according to the optimum extraction conditions. All the calculated recovery values of the analytes ranged from 98.19 to 99.35% and the RSDs were 1.6%, 1.4%, and 1.9%, respectively. The results demonstrated that the methods were reasonable and feasible.


### 3.1.6. Sample Analysis

The mass fractions of phloridzin, quercitrin, and 3-hydroxyphloridzin in original leaves and tea were presented in Table2  (n=3), respectively.Table 2
The contents of three flavonoids inM. halliana tea and original leaves for different periods (mg/g).
Compound
Period
I
II
III
Tea
Original leaves
Tea
Original leaves
Tea
Original leaves
Phloridzin
448.118
382.687
299.486
301.121
504.680
251.456
Quercitrin
9.919
4.237
14.877
7.077
7.412
7.182
3-Hydroxyphloridzin
143.630
112.306
110.055
91.911
137.408
84.023


## 3.1.1. Linearity

The stock standard solutions (0.2, 1, 5, 10, 15, 20, and 25μL) were accurately injected to chromatographic instrument for the construction of calibration curves, respectively, and the corresponding sample sizes of the injected quercitrin were 0.0972, 0.486, 2.43, 4.86, 7.29, 9.72, and 12.15 μg, respectively; the corresponding sample sizes of the injected 3-hydroxyphloridzin were 0.0932, 0.466, 2.33, 4.66, 6.99, 9.32, and 11.65 μg, respectively; the corresponding sample sizes of the injected phloridzin were 0.9, 4.5, 22.5, 45.00, 67.5, 90.00, and 112.5 μg, respectively. The calibration curves were constructed by plotting the peak areas versus the injected sample quality (μg) of each compound. The results were presented in Table 1. The r values were in the range from 0.9991 to 0.9998, which indicated that the methods displayed good linearity.Table 1
Regression equations and linear ranges for three compounds.
Compound
Regressive equation
r
Linear range/μg
Phloridzin
Y= 1165813.0631X + 976247.0924
0.9996
0.9~112.5
Quercitrin
Y= 1306866.7475X + 58713.4808
0.9998
0.0972~12.15
3-Hydroxyphloridzin
Y
=654528.6618X+41738.1201
0.9991
0.0932~11.65
HereY is the peak area; X refers to injection amount (μg).


## 3.1.2. Precision

10μL of each the stock standard solution was accurately injected to chromatographic instrument. The stock solutions were analyzed in six replicates within one day for determining the precision of the developed assay. The relative standard deviations (RSDs) of peak areas for the three compounds were 0.35%, 0.35%, and 0.79%, respectively. The results indicated that the methods were precise for quantitative analysis of phloridzin, 3-hydroxyphloridzin, and quercitrin.


## 3.1.3. Stability

The sample solutions were prepared under the optimum extraction conditions and placed at room temperature, and then 10μL of sample solution was injected into chromatographic instrument at 0, 3, 6, 9, 12, and 24 h, respectively. The RSDs of peak areas for the three compounds were 0.25%, 0.45%, and 0.41%, respectively. The results indicated that the sample solution was basically stable at room temperature within 24 h.


## 3.1.4. Repeatability

Six test sample solutions were processed under the optimum extraction conditions, and 10μL of each solution was then injected to chromatographic instrument for analyzing. The RSDs of peak areas for the three compounds were 0.33%, 2.38%, and 1.24%, respectively, indicating that the analytical methods have the acceptable level of repeatability.


## 3.1.5. Recovery

Nine batches of leaves and flowers samples ofM. hallianawere prepared and divided into three groups, respectively. Then three standard substances at three different amounts were added to the leaves and flowers samples. The spiked samples were prepared according to the optimum extraction conditions. All the calculated recovery values of the analytes ranged from 98.19 to 99.35% and the RSDs were 1.6%, 1.4%, and 1.9%, respectively. The results demonstrated that the methods were reasonable and feasible.


## 3.1.6. Sample Analysis

The mass fractions of phloridzin, quercitrin, and 3-hydroxyphloridzin in original leaves and tea were presented in Table2  (n=3), respectively.Table 2
The contents of three flavonoids inM. halliana tea and original leaves for different periods (mg/g).
Compound
Period
I
II
III
Tea
Original leaves
Tea
Original leaves
Tea
Original leaves
Phloridzin
448.118
382.687
299.486
301.121
504.680
251.456
Quercitrin
9.919
4.237
14.877
7.077
7.412
7.182
3-Hydroxyphloridzin
143.630
112.306
110.055
91.911
137.408
84.023


## 3.2. Analysis of Nutritional Components

### 3.2.1. The Content Determination of Crude Protein

According to the above experimental method, the standard curve of crude protein was drawn. We took extract ofM. hallianatea and measured its crude protein content. The field experiments were carried out with six replications and then the data were expressed as mean.The absorbance ofM. hallianatea was 0.0136, when it was substituted into the standard curve and the crude protein content ofM. hallianatea was 4.362 g/100 g. Protein played an important role in the body, which provided power and energy for the protein metabolism and maintained the normal operation of all kinds of tissues. It also can strengthen the body resistance, hypoxia, and fatigue resistance and have high nutritional value, which is the important nutritional protein for old body [21]. The crude protein content ofM. hallianatea was 4.362 g/100 g which explained that it had more amino acids, which could promote the formation of aroma in tea. And the stated amino acids colloid in tea soup, which played an important role in keeping clear of the beverage and the stability of the colloidal solution. It also had offset function of the nervous system excitement caused by caffeine. SoM. hallianatea can enhance the body resistance, hypoxia, and fatigue resistance and have high nutritional value.


### 3.2.2. The Content Determination of Crude Fat

According to the method and formula, the crude fat ofM. hallianatea was 2.399 g/100 g. The lipid composition in tea played a positive role in the formation of the tea aroma, which could accelerate the absorption of fat-soluble vitamins and prevent vitamin deficiency disease. The fat content in tea was less confirmed by medical research, but it had function of improving human immunity, preventing cancer, and so on. Therefore, the special population who had liver disease, hypertension, hyperlipidemia, and pancreatic function not congruent also easily drinks this in addition to the ordinary people.


### 3.2.3. Determination of Reducing Sugar Content

The cupric tartrate solution consumption ofM. hallianatea was 9.98. According to the formula, the reducing sugar ofM. halliana tea was 8.5 mg/100 g.


### 3.2.4. Determination of Total Soluble Sugar Content

According to the above method, the standard glucose was diluted into six concentrations with gradient dilutions; then the series of glucose standard solution concentrations were taken as the abscissa and absorbance was taken as the ordinate to make a standard curve and calculate the regression equation; the equation wasy=0.003×X+0.005  (R2=0.999).The absorbance of total soluble sugar was taken into the equation to draw standard curve for the total sugar contents of sample, and all the field experiment results were carried out with six replications and then the data were expressed as mean. According to the method and formula, the total soluble sugar ofM. hallianatea was 6.8 g/100 g.The contents of total sugar and reducing sugar, which are taken as the criteria for evaluating the plant nutrition, are the important components in food flavor and nutrition. Tea polysaccharide had antidiabetic, hypolipidemic, antioxidant, and immunoregulation function [22]. From the results of total soluble sugar and reducing sugar,M. hallianatea can be developed as a health care food in the prevention and treatment of those diseases.


## 3.2.1. The Content Determination of Crude Protein

According to the above experimental method, the standard curve of crude protein was drawn. We took extract ofM. hallianatea and measured its crude protein content. The field experiments were carried out with six replications and then the data were expressed as mean.The absorbance ofM. hallianatea was 0.0136, when it was substituted into the standard curve and the crude protein content ofM. hallianatea was 4.362 g/100 g. Protein played an important role in the body, which provided power and energy for the protein metabolism and maintained the normal operation of all kinds of tissues. It also can strengthen the body resistance, hypoxia, and fatigue resistance and have high nutritional value, which is the important nutritional protein for old body [21]. The crude protein content ofM. hallianatea was 4.362 g/100 g which explained that it had more amino acids, which could promote the formation of aroma in tea. And the stated amino acids colloid in tea soup, which played an important role in keeping clear of the beverage and the stability of the colloidal solution. It also had offset function of the nervous system excitement caused by caffeine. SoM. hallianatea can enhance the body resistance, hypoxia, and fatigue resistance and have high nutritional value.


## 3.2.2. The Content Determination of Crude Fat

According to the method and formula, the crude fat ofM. hallianatea was 2.399 g/100 g. The lipid composition in tea played a positive role in the formation of the tea aroma, which could accelerate the absorption of fat-soluble vitamins and prevent vitamin deficiency disease. The fat content in tea was less confirmed by medical research, but it had function of improving human immunity, preventing cancer, and so on. Therefore, the special population who had liver disease, hypertension, hyperlipidemia, and pancreatic function not congruent also easily drinks this in addition to the ordinary people.


## 3.2.3. Determination of Reducing Sugar Content

The cupric tartrate solution consumption ofM. hallianatea was 9.98. According to the formula, the reducing sugar ofM. halliana tea was 8.5 mg/100 g.


## 3.2.4. Determination of Total Soluble Sugar Content

According to the above method, the standard glucose was diluted into six concentrations with gradient dilutions; then the series of glucose standard solution concentrations were taken as the abscissa and absorbance was taken as the ordinate to make a standard curve and calculate the regression equation; the equation wasy=0.003×X+0.005  (R2=0.999).The absorbance of total soluble sugar was taken into the equation to draw standard curve for the total sugar contents of sample, and all the field experiment results were carried out with six replications and then the data were expressed as mean. According to the method and formula, the total soluble sugar ofM. hallianatea was 6.8 g/100 g.The contents of total sugar and reducing sugar, which are taken as the criteria for evaluating the plant nutrition, are the important components in food flavor and nutrition. Tea polysaccharide had antidiabetic, hypolipidemic, antioxidant, and immunoregulation function [22]. From the results of total soluble sugar and reducing sugar,M. hallianatea can be developed as a health care food in the prevention and treatment of those diseases.


## 4. Conclusions

The contents of glycosides, being made up of sugar and nonsaccharide components, are higher than those in other kinds of medicinal materials such as root, peel, flower, and fruit. Glycosides as the active ingredients coexisted with a variety of enzymes in many traditional Chinese medicines. Thus, they can be hydrolyzed by those enzymes. Therefore, Chinese traditional medicine containing glycosides can inhibit enzyme activity and protect glycosides by frying, cooking, and other methods, which can effectively control the glycosidase solution [23].Figure2 showed that the three flavonoids contents of all the wholeM. hallianatea were higher than that of the original leaves, the phloridzin contents of which were the highest. The possible reason was that glycosides digestion ability is reduced and glycosides component content ofM. hallianatea after stir-frying increased, or there might be some components by dissolution and decomposition and they might turn into new compositions. DifferentM. hallianateas came from different original leaf source, and the contents of 3-hydroxyphloridzin and phloridzin presented a decreasing and then increasing trend, and the content of quercitrin displayed an increasing and then reducing trend. Compared with our group previously related research [15], we found that the significant difference in the contents of three flavonoids existed in different harvest periods.Figure 2
The contents of three flavonoids inM. halliana tea and original leaves for different periods.
(a)
(b)Phloridzin as the main ingredient inM. halliana [11], which had antidiabetics, improving memory, antioxidant, anticancer, estrogenic, and antiestrogenic activities [24–26], was a competitive inhibitor of glucose transport [27]. Phloridzin has extensive application and helps in development in food, new drugs, and natural health food because of the low toxicity characteristic.M. hallianais one of China’s unique plants and cultivated in various regions, but mainly as ornamental plant. Because of its rich resources, wide distribution, and easiness of breeding, cutting, and grafting, it brings favorable conditions for mass production. The processing tea ofM. hallianawas similar to green tea production process and it could be harvested in summer and autumn. Our study developedM. hallianatea as a health care drinking and established a HPLC method for simultaneous determination of quercitrin, 3-hydroxyphloridzin, and phloridzin; meanwhile, the analysis of the same compositions’ change ofM. hallianatea before and after processing was contrasted.Through the analysis of the main nutrients ofM. hallianatea, we found that diversity of nutrients, containing the total sugar, reducing sugar, crude protein, crude fat, and other nutrients, is the tea nutrition characteristic. Therefore, the nutrient contents ofM. halliana tea are quite high, and some nutrients are quite rich, which is helpful to comprehensive understanding of the benefits of drinking tea. This study showed thatM. hallianatea had certain development in food and nutrition health care value. We believed that it will become very popular after comprehensive analysis of the main nutrition compositions and efficacy components ofM. hallianatea.



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*Source: 7950137-2017-10-11.xml*