# Phenolic Analysis for Classification of Mulberry (Morus spp.) Leaves according to Cultivar and Leaf Age

Authors: Pitchaya Pothinuch; Sasitorn Tongchitpakdee
Journal: Journal of Food Quality
 (2019)

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## Abstract

Phenolic compounds in mulberry leaves harvested from three cultivars (Buriram 60, BR 60; Sakonnakhon, SK; and Khunphai, KH) at different leaf ages (tips, young, and old leaves) were identified and quantified using HPLC-DAD and HPLC-ESI/MS. A total of 13 phenolic compounds, which were mainly as caffeoylquinic acids and flavonol glycosides, were detectable. Predominant phenolic compounds were 5-O-caffeoylquinic acid (3.5–13.1 mg/g dry weight), 4-O-caffeoylquinic acid (1.3–2.4 mg/g dry weight), and quercetin-3-O-rutinoside (1.0–4.4 mg/g dry weight). Qualitative and quantitative differences in phenolic compounds in mulberry leaves were investigated among cultivars and leaf ages. Principal component analysis and hierarchical cluster analysis were used for classification of the mulberry leaves. Based on the similarity of phenolic compounds, mulberry leaves were clustered into three groups: (1) tips of leaves from all cultivars; (2) young and old leaves of mulberry cv. BR 60; (3) young and old leaves of mulberry cv. SK and KH. Therefore, according to phenolic compounds in mulberry leaves, tips of leaves from all cultivars should be intended for production of functional healthy foods.


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## Body

## 1. Introduction

Mulberry is a fast-growing plant belonging to genusMorus of family Moraceae. Mulberry has been widely cultivated in Asian countries such as China, Japan, Korea, and Thailand in order to utilize its leaves as food for silkworms in sericulture. The leaves are also used as supplements for feeding livestock to improve milk yield and quality [1]. Mulberry leaves have been traditionally applied as folk medicine to treat fever, protect the liver, strengthen the joints, facilitate the discharge of urine, and lower blood pressure. Moreover, the leaves have been processed as human food such as mulberry leaf tea and seasoning power. Several research studies have revealed pharmacological activities of mulberry leaves including hypoglycemic effect [2] and anti-inflammatory [3] and antihypertensive properties [4] which might be due to their bioactive compounds. Significant amounts of phenolic compounds, 1-deoxynojirimycin (DNJ) and melatonin [5–7], were quantified in mulberry leaves. Therefore, mulberry leaves could be a potent functional ingredient for production of healthy foods. To our knowledge, there is limited information of mulberry leaf selection for their utilization. Selection of mulberry leaves is an important approach that could affect the quality of mulberry leaf-based products. Most of previous studies focused on only the effect of cultivars on phenolic compounds in mulberry leaves. However, there has been reported that melatonin content in mulberry leaves were influenced by the combination effects of cultivars and leaf ages [7]. Moreover, both cultivars and leaf ages were also affected on phenolics in spinach and berries [8, 9]. Considering these two factors, this study aims to investigate phenolic profiles and contents of mulberry leaves harvested from three commercial cultivars in Thailand including Buriram 60 (BR 60), cv. Sakonnakhon (SK), and cv. Khunphai (KH) at different three stages of leaf ages (tips, young, and old leaves) using HPLC-DAD and HPLC-ESI/MS. Furthermore, chemometric analysis (principle component analysis (PCA) and hierarchical cluster analysis (HCA)) was also used for classification of mulberry leaves. The information obtained from this study could benefit both growers and food processers who would like to use the mulberry leaves as an alternative healthy ingredient for development of functional foods and supplements.


## 2. Materials and Methods

### 2.1. Plant Materials

Mulberry leaves from three commercial Thai cultivars (BR 60, SK, and KH) were manually harvested at Queen Sirikit Sericulture Center, Saraburi, Thailand (latitude 14°41′18.6″N × longitude 100°53′39.4″E) in March 2012. For each cultivar, three stages of leaf ages (tips, young, and old leaves) were collected. The tips of leaves were selected from the leaves at positions 1 to 3 from the top of each branch; young leaves were from positions 4 to 6; and old leaves were from positions 7 to 10. The averages of horizontal and vertical lengths of leaves were 8.4 ± 1.6 and 11.5 ± 2.5 cm for tips, 12.2 ± 2.0 and 16.1 ± 2.8 cm for young leaves, and 15.1 ± 2.4 and 18.8 ± 3.0 cm for old leaves. For all cultivars, a thousand leaves of each leaf age were collected. The sampling was carried out in duplicate for each sample. For sample preparation, the leaves were washed, cut into small pieces, immediately frozen in liquid nitrogen, and freeze-dried (Model: Gamma 2–16 LSC, Christ, Germany; freezing condition: temperature −50°C, pressure 0.1 mbar). The dried sample was ground, sieved with a 40 mesh sieve, and stored at −20°C.


### 2.2. Chemicals

The chemicals, 5-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic acid), quercetin-3-O-rutinoside (rutin), and kaempferol-3-O-rutinoside, were purchased from Sigma Aldrich Corp. (St. Louis, MO, USA). Analytical ethanol was purchased from Lab-scan (Bangkok, Thailand). HPLC grade acetonitrile was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). The water used in all experiments was purified using a Milli-Q system from Millipore Corp. (Bedford, MA, USA).


### 2.3. Extraction

The extraction method was modified from Pothinuch et al. [10]. Freeze-dried powder of sample (1 g) was extracted twice with 80% ethanol (25 mL) using ultrasonication for 30 min while cooled with ice. After centrifugation at 10,000 g at 4°C for 30 min, the supernatant was filtered and evaporated under vacuum. The aqueous extract was stored at −20°C until use. Each sample was extracted in triplicate.


### 2.4. Identification and Quantification of Phenolic Compounds

Phenolic compounds were identified and quantified using HPLC-DAD and HPLC-ESI/MS systems, following the methods of Pothinuch et al. [10]. The HPLC-PDA system consisted of a reverse phase 5 μm Symmetry® (4.6 × 250 mm) column (Waters, Milford, MA, USA) and an HPLC (Waters, Milford, MA, USA) equipped with a Waters 2707 autosampler, a Waters 600 pump, and a Waters 2998 PDA detector. The mobile phase used was acetonitrile (A) and 1% formic acid in water (B). The solvent gradient was performed as follows: 0–5 min with 5–10% A; 5–10 min with 10–15% A; 10–30 min with 15–25% A; 30–40 min with 25–50% A; 40–45 min with 50–5% A; and 45–50 min with 5% A. The flow rate was 1.0 mL/min, and injection volume of extract was 20 μL. The detection wavelength of the PDA detector was set in range of 200–500 nm. Mass spectral (MS) analysis was carried out with HPLC-ESI/MS under API-ES positive and negative modes. The ionization condition was set at 350°C and 3,000 V for capillary temperature and voltage, respectively. The nebulizer pressure was 60 psig, and flow rate of nitrogen gas was 13 L/min. The full scan mass was range from m/z 50 to m/z 1000. Identification of phenolic compounds was based on retention times, UV spectra, and MS information. Quantification of phenolic compounds was performed using reference standards of 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside. The contents were expressed as mg/g DW and determined in triplicate.


### 2.5. Statistical Analysis

Data were expressed as mean ± SEM. Statistical analysis was performed by using a two-way analysis of variance (ANOVA) followed by a Tukey simultaneous test. Significant difference was statistically considered atp<0.05. PCA and HCA were performed to standardize data in order to classify different mulberry leaf samples. PCA was used to observe interrelationships of sample and analyzed parameters. HCA was applied to cluster different samples by considering hierarchical associations using Euclidean distance and Ward’s method as dissimilarity measure and agglomeration method, respectively.


## 2.1. Plant Materials

Mulberry leaves from three commercial Thai cultivars (BR 60, SK, and KH) were manually harvested at Queen Sirikit Sericulture Center, Saraburi, Thailand (latitude 14°41′18.6″N × longitude 100°53′39.4″E) in March 2012. For each cultivar, three stages of leaf ages (tips, young, and old leaves) were collected. The tips of leaves were selected from the leaves at positions 1 to 3 from the top of each branch; young leaves were from positions 4 to 6; and old leaves were from positions 7 to 10. The averages of horizontal and vertical lengths of leaves were 8.4 ± 1.6 and 11.5 ± 2.5 cm for tips, 12.2 ± 2.0 and 16.1 ± 2.8 cm for young leaves, and 15.1 ± 2.4 and 18.8 ± 3.0 cm for old leaves. For all cultivars, a thousand leaves of each leaf age were collected. The sampling was carried out in duplicate for each sample. For sample preparation, the leaves were washed, cut into small pieces, immediately frozen in liquid nitrogen, and freeze-dried (Model: Gamma 2–16 LSC, Christ, Germany; freezing condition: temperature −50°C, pressure 0.1 mbar). The dried sample was ground, sieved with a 40 mesh sieve, and stored at −20°C.


## 2.2. Chemicals

The chemicals, 5-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic acid), quercetin-3-O-rutinoside (rutin), and kaempferol-3-O-rutinoside, were purchased from Sigma Aldrich Corp. (St. Louis, MO, USA). Analytical ethanol was purchased from Lab-scan (Bangkok, Thailand). HPLC grade acetonitrile was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). The water used in all experiments was purified using a Milli-Q system from Millipore Corp. (Bedford, MA, USA).


## 2.3. Extraction

The extraction method was modified from Pothinuch et al. [10]. Freeze-dried powder of sample (1 g) was extracted twice with 80% ethanol (25 mL) using ultrasonication for 30 min while cooled with ice. After centrifugation at 10,000 g at 4°C for 30 min, the supernatant was filtered and evaporated under vacuum. The aqueous extract was stored at −20°C until use. Each sample was extracted in triplicate.


## 2.4. Identification and Quantification of Phenolic Compounds

Phenolic compounds were identified and quantified using HPLC-DAD and HPLC-ESI/MS systems, following the methods of Pothinuch et al. [10]. The HPLC-PDA system consisted of a reverse phase 5 μm Symmetry® (4.6 × 250 mm) column (Waters, Milford, MA, USA) and an HPLC (Waters, Milford, MA, USA) equipped with a Waters 2707 autosampler, a Waters 600 pump, and a Waters 2998 PDA detector. The mobile phase used was acetonitrile (A) and 1% formic acid in water (B). The solvent gradient was performed as follows: 0–5 min with 5–10% A; 5–10 min with 10–15% A; 10–30 min with 15–25% A; 30–40 min with 25–50% A; 40–45 min with 50–5% A; and 45–50 min with 5% A. The flow rate was 1.0 mL/min, and injection volume of extract was 20 μL. The detection wavelength of the PDA detector was set in range of 200–500 nm. Mass spectral (MS) analysis was carried out with HPLC-ESI/MS under API-ES positive and negative modes. The ionization condition was set at 350°C and 3,000 V for capillary temperature and voltage, respectively. The nebulizer pressure was 60 psig, and flow rate of nitrogen gas was 13 L/min. The full scan mass was range from m/z 50 to m/z 1000. Identification of phenolic compounds was based on retention times, UV spectra, and MS information. Quantification of phenolic compounds was performed using reference standards of 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside. The contents were expressed as mg/g DW and determined in triplicate.


## 2.5. Statistical Analysis

Data were expressed as mean ± SEM. Statistical analysis was performed by using a two-way analysis of variance (ANOVA) followed by a Tukey simultaneous test. Significant difference was statistically considered atp<0.05. PCA and HCA were performed to standardize data in order to classify different mulberry leaf samples. PCA was used to observe interrelationships of sample and analyzed parameters. HCA was applied to cluster different samples by considering hierarchical associations using Euclidean distance and Ward’s method as dissimilarity measure and agglomeration method, respectively.


## 3. Results and Discussion

Mulberry cv. BR 60, cv. SK, and cv. KH are commercially promoted by The Queen Sirikit Department of Sericulture, Ministry of Agriculture and Cooperatives, Thailand. BR 60 is a hybrid cultivar between mulberry cv. Liu Jio 44 (male) and cv. Noi (native Thai cultivar, female), while SK is a hybrid cultivar between mulberry cv. Luin Jio No. 40 (male) and cv. KH (native Thai cultivar, female). KH is a native Thai cultivar. The leaf production of mulberry cv. BR 60, cv. SK, and cv. KH were 6.9, 4.0, and 3.2 kg/km2/year, respectively. Mulberry cv. BR 60 can grow only in the area that has sufficient amount of water, while mulberry cv. SK and cv. KH can be cultivated in any area in Thailand. All cultivars also have highly resistant against leaf mosaic and root rot diseases. In this study, HPLC profile and contents of phenolic compounds were investigated in order to characterize mulberry leaves according to cultivars and leaf ages.


### 3.1. Identification of Phenolic Compounds in Mulberry Leaves

Phenolic compounds in mulberry leaves were identified using HPLC-DAD and HPLC-ESI/MS. HPLC chromatograms of mulberry leaves from three cultivars at different three stages of leaf ages are illustrated in Figures1(a)–1(i). A total of 13 peaks were detected at a wavelength of 350 nm in young and old leaves of mulberry cv. BR 60, whereas other mulberry leaf samples showed only 9 peaks in their chromatograms. The combination of distinct UV spectra and MS characteristics in both positive and negative ionization modes was used to identify individual phenolic compounds in mulberry leaves. External reference standards including 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside were used for spiking and confirmation of the authenticity of peaks 2, 3, 8, and 10, respectively. For other peaks, comparison of λmax and MS data with data obtained from previous studies was used for identification. The results showed that two groups of phenolic compounds were identified in the mulberry leaves: (1) caffeoylquinic acids and (2) flavonol glycosides. Peak identification of individual compounds is shown in Table 1 and mentioned below:(1)
Caffeoylquinic Acids. Peaks 1, 2, 3, and 5 were identified as caffeoylquinic acid isomers. These peaks were detected in the first 20 min of running time and had similar UV spectra with λmax at 237–241 and 317–325 nm. Observation of the base ions at m/z 355 in the positive mode ([M + H]+) and 353 in the negative mode ([M − H]−) in the four peaks indicates that their molecular weight equals 354, corresponding to the molecular weight of monocaffeoylquinic acid. Peaks 2 and 3 were identified as 5-O-caffeoylquinic acid (chlorogenic acid) and 4-O-caffeoylquinic acid (cryptochlorogenic acid), respectively, by comparing retention time of reference standards and confirming spiking of the reference standard. To differentiate individual isomers of caffeoylquinic acid, MS data were used by considering patterns of negative fragment ions identified by LC-MS4, as explained in the previous study [11]. Peak 1 was likely to be 3-caffeoylquinic acid (neochlorogenic acid) due to the existence of the fragment ion at m/z 179, and this peak was first eluted from the C-18 column, similarly to the results of Nakatani et al. [12] who used a similar system of reverse phase HPLC. Peak 5 was also assigned as an isomer of caffeoylquinic acid. This peak produced same fragment ions as found in peak 2. These two peaks might be different in three-dimensional orientation of their atoms (cis- and trans-forms), which could not be differentiated based on our analysis. According to the results reported by Xie et al. [13], peak 5 might be assigned as the cis-form of 5-O-caffeoylquinic acid since cis-form of 5-O-caffeoylquinic acid retained longer than its trans-form. The cis-5-O-caffeoylquinic acid has not been reported in mulberry leaves, but this compound was found in mulberry fruits [14]. Naturally, the trans-form of 5-O-caffeoylquinic acid was predominantly produced in plant tissue over its cis-form; however, under UV light exposure, the trans-form could be converted to the cis-form as observed in tobacco leaves [15, 16].(2)
Flavonol Glycosides. Peaks 4 and 6–13 were identified as glycosides of flavonols (quercetin and kaempferol). These peaks were eluted after 20 min of running time, except for peak 4. Peaks 6, 8, 9, and 11 were considered to be quercetin derivatives since these peaks had similar UV spectra with λmax at 256 and 353-354 and also produced a positive ion at m/z 303, suggesting the existence of quercetin residue. Peak 8 was identified as quercetin-3-O-rutinoside by spiking and comparing with the reference standard. Also, production of [M + H]+ at m/z 611 and [M − H]− at m/z 609 in peak 8 indicated its molecular weight of 610 which matched quercetin-3-O-rutinoside. Peak 6 generated similar positive fragment ions as detected in peak 8. Moreover, peak 6 possibly produced the fragment ion at m/z 611 by cleavage rhamnose (molecular mass 146 amu) from [M + H]+. Thus, peak 6 might consist of quercetin-rutinoside and rhamnosyl residues. Regarding MS data and its possible molecular weight of 756, peak 6 might match quercetin-3-(rhamnosyl-glucoside)-7-O-rhamnoside or quercetin-3-O-(2-rhamnosyl)rutinoside [17], which have never reported in mulberry leaves. Peak 9 produced [M + H]+ and [M − H]− at m/z 465 and 463, representing a molecular weight equal to 464, respectively. The fragment ion at m/z 303 was also found in this peak, which possibly was generated by loss of hexoside residue (a molecular mass of 162 amu) from [M + H]+. Therefore, this peak was supposed to contain quercetin and hexosyl group in its structure, which could be either quercetin-glucoside or quercetin-galactoside. However, only quercetin-3-O-glucoside has been reported in mulberry leaves [18–20]. Peak 11 was tentatively identified as quercetin-(malonyl)hexoside by comparing with the results of Ruiz et al. [21]. This peak generated [M + H]+ and [M − H]− at m/z 551 and 549, respectively, indicating its molecular weight of 550. Its molecular weight might belong to quercetin-(malonyl)glucoside or quercetin-(malonyl)galactoside. However, only quercetin-(malonyl)glucoside was determined in mulberry leaves [5, 19, 20, 22]. Peaks 4, 7, 10, 12 and 13 were identified as kaempferol derivatives because these peaks had similar UV spectra with λmax at 265 and 345–347 nm and also produced the fragment ion at m/z 287 (kaempferol residue). Peak 10 was identified as kaempferol-3-O-rutinoside by comparing with the reference standard and confirming by spiking with its reference standard. Based on MS data, peak 7 was likely to be derivative of kaempferol-rutinoside because this peak produced same fragment ions as presented in peak 10. Also, the fragment ion at m/z 611 was produced by loss of rhamnose (molecular mass 146 amu). Thus, peak 7 possibly consisted of kaempferol-rutinoside and rhamnosyl residues [18]. The fragment ion at m/z 287 was also found in peak 12, which possibly was generated by loss of the hexosyl group (molecular mass 162 amu) from its [M + H]+. It indicated that peak 12 was supposed to contain the hexosyl group, which might be kaempferol-glucoside or kaempferol-galactoside. Only kaempferol-3-O-glucoside has been identified in mulberry leaves [23], whereas there have been no previous studies reporting kaempferol-galactoside in mulberry leaves. Peak 4 produced similar fragment ions as found in peak 12. It is possible that peak 4 might be the kaempferol-hexoside derivative. Moreover, fragment ion at m/z 611 was also found in this peak, which is possibly generated by cleavage of rhamnose (molecular mass 146 amu) from [M + H]+. The results suggested that peak 4 was possibly comprised of kaempferol-hexoside and rhamnosyl residue. Comparing with previous results reported by El-Desoky et al. [24] and Stobiecki et al. [25], this peak might be kaempferol-3-O-glucosyl-(1 ⟶ 2)-rhamnoside-7-O-glucoside or kaempferol-3-O-glucosyl-glucoside-7-O-rutinoside. Based on survey of literatures, there was no study that identified this compound in mulberry leaves. Peak 13 generated [M + H]+ at m/z 535 and [M − H]− at m/z 533, indicating molecular weight equal to 534. The fragment ion at m/z 489 was also yielded in the negative mode. When compared with the results from a previous study reported by Llorach et al. [26], this peak might be kaempferol-(malonyl)hexoside. However, only kaempferol-(malonyl)glucoside was found in mulberry leaves [20]. Thus, peak 10 was tentatively identified as kaempferol-(malonyl)glucoside.Figure 1
Typical HPLC chromatograms of phenolic compounds of different mulberry leaves: tip of leaves cv. Buriram 60 (a), young leaves cv. Buriram 60 (b), old leaves cv. Buriram 60 (c), tip of leaves cv. Sakonnakhon (d), young leaves cv. Sakonnakhon (e), old leaves cv. Sakonnakhon (f), tip of leaves cv. Khunphai (g), young leaves cv. Khunphai (h), and old leaves cv. Khunphai (i), detected at 350 nm and zoom-in spectrum range of 10–40 min. The peak numbers are corresponding to UV spectrum and MS data in Table1.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Table 1
Identification of phenolic compounds of mulberry leaves and the data taken from HPLC-DAD and HPLC-MS.
Peak
RT (min)
λ
max (nm)
Mass spectra
Tentative compound∗
Positive ion (m/z)
Negative ion (m/z)
[M + H]+
Fragment ions
[M − H]−
Fragment ions
Caffeoylquinic acid isomers
1
13.1
239, 325
355
163, 135
353
191, 179
Caffeoylquinic acid isomer I2
2
16.5
241, 325
355
163, 135
353
191
5-O-Caffeoylquinic acid1
3
17.4
240, 325
355
163, 135
353
191, 179, 173
4-O-Caffeoylquinic acid1
5
19.5
237, 317
355
163, 135
353
191
Caffeoylquinic acid isomer II2
Flavonol derivatives
4
18.2
265, 347
757
611, 287, 449
755
609
Kaempferol-hexoside derivative2
6
21.4
256, 354
757
303, 611, 627, 465
755
625
Quercetin-rutinoside derivative2
7
23.7
265, 354
741
595, 287, 449
739
—
Kaempferol-rutinoside derivative2
8
25.6
256, 354
611
465, 303
609
—
Quercetin-3-O-rutinoside1
9
27.1
256, 353
465
303
463
—
Quercetin-hexoside2
10
29.1
265, 347
595
449, 287
593
—
Kaempferol-3-O-rutinoside1
11
29.6
256, 354
551
303
549
505
Quercetin-(malonyl)-hexoside2
12
31.0
265, 347
449
287
447
—
Kaempferol-hexoside2
13
34.3
265, 347
535
287
533
489
Kaempferol-(malonyl)-hexoside2
∗Identification of phenolic compound. 1According to UV spectrum and MS data in combination with retention time of the reference standard. 2According to UV spectral and MS data comparison with the data obtained from previous studies.


### 3.2. Quantification of Phenolic Compounds in Mulberry Leaves

Quantitative analysis of phenolic compounds in mulberry leaves is shown in Table2. The 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside were quantified using external reference standards. Caffeoylquinic acid isomers I and II were quantified as chlorogenic acid equivalents. Quercetin derivatives were quantified as quercetin-3-O-rutinoside equivalents, whereas kaempferol derivatives were reported as kaempferol-3-O-rutinoside equivalents. The results showed that total caffeoylquinic acid and total flavonol derivative contents of mulberry leaves ranged from 5.7 to 16.3 and 2.9 to 9.1 mg/g DW, respectively (Table 2). Among all samples, tip of leaves from mulberry cv. SK had the highest content of total caffeoylquinic acids and total flavonol glycosides. For young leaves, total caffeoylquinic acids and total flavonol derivatives were higher in mulberry leaves cv. BR 60 than other cultivars. No significant difference of total caffeoylquinic acids was found in old leaves; however, total flavonol glycosides of old leaves were significantly higher in mulberry leaves cv. BR 60 when compared to cv. SK and KH. Total caffeoylquinic acids and total flavonol derivatives decreased as mulberry leaves aged. The results indicated that phenolic compounds in mulberry leaves were distinctly influenced by cultivars and leaf ages, which is in agreement with the results determined in pear leaves [27]. Variation of phenolic compounds in different cultivars could be related to genetic diversity which might be involved in synthesis and metabolism of phenolic compounds [28]. Concerning the effect of leaf ages, decreasing phenolic contents as leaves age might lessen the activity of phenylalanine ammonia-lyase, a key enzyme in phenolic synthesis and metabolites, as reported in apple leaves [29]. The result also showed that 5-O-caffeoylquinic acid is the most abundant phenolic compound of mulberry leaves, contributing up to 57%. Similarly, this compound was predominantly found in the leaves of mulberry cultivated in Korea [18], Spain [19], and Tunisia [20]. The mulberry leaves in this study contained greater 5-O-caffeoylquinic acid content than that contained in the Korean and Tunisian mulberry leaves but lower than that of the Spanish mulberry leaves. Quercetin-3-O-rutinoside and 4-O-caffeoylquinic acid were also major phenolic compounds, which contributed up to 19.2% and 16.1%, respectively. Quercetin-(malonyl)hexoside (12.2–13.8%) and kaempferol-(malonyl)hexoside (10.2–12.4%) were contributed only in young and old leaves of mulberry cv. BR 60. Quercetin 3-(malonyl)glucoside was reported as a major phenolic compound in Japanese and Korean mulberry reported by Katsube et al. [5] and Lee and Choi [18], respectively. Furthermore, quercetin-rutinoside derivative and kaempferol-rutinoside derivative were observed only in young and old leaves of mulberry cv. BR 60 with very low amount (<1%). Considering individual phenolic compounds, their contents significantly decreased when the leaves aged, except for caffeoylquinic acid isomer I (3-O-caffeoylquinic acid).Table 2
Quantification of phenolic compounds detected in mulberry leaves from 3 commercial Thai cultivars at different leaf ages.
Parameter
Buriram 60
Sakonnakhon
Khunphai
Tips
Young leaves
Old leaves
Tips
Young leaves
Old leaves
Tips
Young leaves
Old leaves
Caffeoylquinic acid isomer I1
0.32 ± 0.01B,c
0.60 ± 0.01NS,a
0.55 ± 0.01B,b
0.39 ± 0.01A,b
0.68 ± 0.02NS,a
0.68 ± 0.02A,a
0.33 ± 0.01B,b
0.64 ± 0.06NS,a
0.73 ± 0.01A,a
5-O-Caffeoylquinic acid
11.03 ± 0.53B,a
6.76 ± 0.14A,b
3.88 ± 0.12NS,c
13.12 ± 0.24A,a
5.04 ± 0.80B,b
3.50 ± 0.36NS,c
10.72 ± 0.13B,a
5.73 ± 0.85B,b
5.03 ± 0.91NS,b
4-O-Caffeoylquinic acid
1.91 ± 0.11B,a
2.13 ± 0.05NS,a
1.26 ± 0.03B,b
2.45 ± 0.05A,a
1.80 ± 0.25NS,b
1.38 ± 0.11B,c
1.84 ± 0.01B,ns
1.99 ± 0.09NS,ns
1.88 ± 0.17A,ns
Caffeoylquinic acid isomer II1
0.23 ± 0.02B,a
0.18 ± 0.00A,b
0.10 ± 0.00NS,c
0.33 ± 0.02A,a
0.13 ± 0.02B,b
0.10 ± 0.01NS,b
0.23 ± 0.02B,a
0.15 ± 0.01B,b
0.14 ± 0.02NS,b
Total caffeoylquinic acids
13.49 ± 0.66B,a
9.68 ± 0.20A,b
5.79 ± 0.17NS,c
16.29 ± 0.16A,a
7.59 ± 0.45B,b
5.65 ± 0.47NS,c
13.13 ± 0.08B,a
8.51 ± 0.69A,b
7.78 ± 0.97NS,b
Kaempferol-hexoside derivative3
0.30 ± 0.02A,a
0.09 ± 0.01B,b
0.07 ± 0.00C,b
0.32 ± 0.00A,a
0.13 ± 0.01
0.11 ± 0.01
0.29 ± 0.00
0.16 ± 0.00
0.01 ± 0.00
Quercetin-rutinoside derivative2
ND
0.14 ± 0.00ns
0.15 ± 0.02ns
ND
ND
ND
ND
ND
ND
Kaempferol-rutinoside derivative3
ND
0.10 ± 0.00b
0.13 ± 0.00a
ND
ND
ND
ND
ND
ND
Quercetin-3-O-rutinoside
3.65 ± 0.26B,a
1.55 ± 0.05B,b
1.02 ± 0.04B,b
4.39 ± 0.05A,a
1.97 ± 0.18B,b
1.56 ± 0.12B,c
3.45 ± 0.02B,a
2.42 ± 0.20A,b
2.22 ± 0.29A,b
Quercetin-hexoside2
1.00 ± 0.08B,a
0.28 ± 0.02B,b
0.05 ± 0.00B,c
2.03 ± 0.05A,a
0.46 ± 0.05A,b
0.38 ± 0.05A,b
0.94 ± 0.03B,a
0.36 ± 0.05B,b
0.36 ± 0.05A,b
Kaempferol-3-O-rutinoside
1.11 ± 0.07B,a
0.61 ± 0.02B,b
0.41 ± 0.01C,c
1.59 ± 0.01A,a
1.07 ± 0.09A,b
0.84 ± 0.07B,c
1.05 ± 0.00B,ns
1.14 ± 0.08A,ns
1.11 ± 0.09A,ns
Quercetin-(malonyl)-hexoside2
ND
2.00 ± 0.08a
1.46 ± 0.06b
ND
ND
ND
ND
ND
ND
Kaempferol-hexoside3
0.25 ± 0.02B,a
0.22 ± 0.01NS,a
0.14 ± 0.01NS,b
0.73 ± 0.02A,a
0.17 ± 0.01NS,b
0.14 ± 0.00NS,b
0.23 ± 0.01B,ns
0.20 ± 0.04NS,ns
0.19 ± 0.04NS,ns
Kaempferol-(malonyl)-hexoside3
ND
1.67 ± 0.20a
1.31 ± 0.17b
ND
ND
ND
ND
ND
ND
Total flavonols
6.31 ± 0.42B,a
6.66 ± 0.22A,a
4.74 ± 0.17A,b
9.06 ± 0.14A,a
3.67 ± 0.28B,b
2.92 ± 0.22B,b
5.67 ± 0.05B,a
4.12 ± 0.33B,b
3.88 ± 0.47B,b
Values are expressed as mean ± SEM. Different capital letters indicate significant differences between cultivars within the same leaf age atp<0.05. Different small letters indicate significant differences between leaf ages within the same cultivar at p<0.05. ND: none detectable; NS: no significant difference among cultivars at p<0.05; ns: no significant difference among leaf age at p<0.05. 1Content expressed as mg chlorogenic acid equivalent/g dry weight. 2Content expressed as mg quercetin-3-rutinoside equivalent/g dry weight. 3Content expressed as mg kaempferol-3-rutinoside equivalent/g dry weight.


### 3.3. Classification of Mulberry Leaves

PCA was applied to the data set of 9 different samples of mulberry leaves harvested from three cultivars (BR 60, SK, and KH) at different 3 stages of leaf ages (tips, young, and old leaves) in order to achieve understanding of characteristics of these leaf samples. The results showed that the first two principal components (PCs) explained 84.79% of the total variation of data set. PC1 explained 61.34% of total variance and had high contribution of 5-O-caffeoylquinic acid, quercetin-3-O-rutinoside, caffeoylquinic acid isomer II, and total caffeoylquinic acids (Figure 2(a)). The nine samples of mulberry leaves from different cultivars and leaf ages were clearly divided into three groups (Figure 2(b)). The first group had very high positive scores for PC1, which consisted of tips of leaves from all cultivars. This group can be best described by high content of phenolic compounds, especially 5-O-caffeoylquinic acid (>10 mg/g DW) and quercetin-3-O-rutinoside (>3.4 mg/g DW). The second group had negative scores for PC1 and positive scores for PC2 including young and old leaves of mulberry cv. BR 60. This group was best characterized by the presence of quercetin-(malonyl)hexoside and kaempferol-(malonyl)hexoside. The third group contained young and old leaves of mulberry cv. SK and KH, which had negative scores for PC1 and PC2. This group corresponds to low phenolic contents. HCA also applied for grouping the mulberry leaves based on the similarities of phenolic compounds. Three groups of mulberry leaves were clustered by HCA (Figure 3). It showed clearly that the results from HCA were very similar to the results from PCA. Summarizing the PCA and HCA, these chemometric analyses revealed that the predominant phenolic compounds, 5-O-caffeoylquinic acid and quercetin-3-O-rutinoside, can be used to classify tips of leaves from young and old leaves. However, the difference of tips of leaves among cultivars could not be classified. Besides, the results from this study also suggested that mulberry leaf samples in the same group might be used interchangeably because the leaves provided similar phenolic compounds. Previous study reported that mulberry leaves were clustered by genetic characters such clones and species [19, 30]. This study exhibited that cultivars and leaf ages were firstly considered for classification of mulberry leaves which is the new evidence exhibiting the important of the two factors in mulberry leaf utilization.Figure 2
Principal component analysis based on phenolic compounds of different 9 samples of mulberry leaves: (a) scattering plot of loadings on principle components PC 1 and PC 2; (b) sample map of scores on PC1 and PC 2 as function of cultivars and leaf age. 4-CQA: 4-O-caffeoylquinic acid; 5-CQA: 5-O-caffeoylquinic acid; CQA isomers I, II: caffeoylquinic acid isomers I, II; total CQA: total caffeoylquinic acids; K-H: kaempferol-hexoside; Q-H: quercetin-hexoside; Q-3-R: quercetin-3-O-rutinoside; K-3-R: kaempferol-3-O-rutinoside; K-M-H: kaempferol-(malonyl)hexoside; Q-M-H: quercetin-(malonyl)hexoside; K-R: kaempferol-rutinoside; Q-R: quercetin-rutinoside. Cultivar’s codes are BR 60: Buriram 60, SK: Sakonnakhon, and KH: Khunphai. Leaf age’s codes are T: tips, Y: young leaves, and O: old leaves.
(a)
(b)Figure 3
Dendrogram of hierarchical cluster analysis among different samples of mulberry leaves from three cultivars at different mulberry leaves. Cultivar’s codes are BR 60: Buriram 60, SK: Sakonnakhon, and KH: Khunphai. Leaf age’s codes are T: tips, Y: young leaves, and O: old leaves.


## 3.1. Identification of Phenolic Compounds in Mulberry Leaves

Phenolic compounds in mulberry leaves were identified using HPLC-DAD and HPLC-ESI/MS. HPLC chromatograms of mulberry leaves from three cultivars at different three stages of leaf ages are illustrated in Figures1(a)–1(i). A total of 13 peaks were detected at a wavelength of 350 nm in young and old leaves of mulberry cv. BR 60, whereas other mulberry leaf samples showed only 9 peaks in their chromatograms. The combination of distinct UV spectra and MS characteristics in both positive and negative ionization modes was used to identify individual phenolic compounds in mulberry leaves. External reference standards including 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside were used for spiking and confirmation of the authenticity of peaks 2, 3, 8, and 10, respectively. For other peaks, comparison of λmax and MS data with data obtained from previous studies was used for identification. The results showed that two groups of phenolic compounds were identified in the mulberry leaves: (1) caffeoylquinic acids and (2) flavonol glycosides. Peak identification of individual compounds is shown in Table 1 and mentioned below:(1)
Caffeoylquinic Acids. Peaks 1, 2, 3, and 5 were identified as caffeoylquinic acid isomers. These peaks were detected in the first 20 min of running time and had similar UV spectra with λmax at 237–241 and 317–325 nm. Observation of the base ions at m/z 355 in the positive mode ([M + H]+) and 353 in the negative mode ([M − H]−) in the four peaks indicates that their molecular weight equals 354, corresponding to the molecular weight of monocaffeoylquinic acid. Peaks 2 and 3 were identified as 5-O-caffeoylquinic acid (chlorogenic acid) and 4-O-caffeoylquinic acid (cryptochlorogenic acid), respectively, by comparing retention time of reference standards and confirming spiking of the reference standard. To differentiate individual isomers of caffeoylquinic acid, MS data were used by considering patterns of negative fragment ions identified by LC-MS4, as explained in the previous study [11]. Peak 1 was likely to be 3-caffeoylquinic acid (neochlorogenic acid) due to the existence of the fragment ion at m/z 179, and this peak was first eluted from the C-18 column, similarly to the results of Nakatani et al. [12] who used a similar system of reverse phase HPLC. Peak 5 was also assigned as an isomer of caffeoylquinic acid. This peak produced same fragment ions as found in peak 2. These two peaks might be different in three-dimensional orientation of their atoms (cis- and trans-forms), which could not be differentiated based on our analysis. According to the results reported by Xie et al. [13], peak 5 might be assigned as the cis-form of 5-O-caffeoylquinic acid since cis-form of 5-O-caffeoylquinic acid retained longer than its trans-form. The cis-5-O-caffeoylquinic acid has not been reported in mulberry leaves, but this compound was found in mulberry fruits [14]. Naturally, the trans-form of 5-O-caffeoylquinic acid was predominantly produced in plant tissue over its cis-form; however, under UV light exposure, the trans-form could be converted to the cis-form as observed in tobacco leaves [15, 16].(2)
Flavonol Glycosides. Peaks 4 and 6–13 were identified as glycosides of flavonols (quercetin and kaempferol). These peaks were eluted after 20 min of running time, except for peak 4. Peaks 6, 8, 9, and 11 were considered to be quercetin derivatives since these peaks had similar UV spectra with λmax at 256 and 353-354 and also produced a positive ion at m/z 303, suggesting the existence of quercetin residue. Peak 8 was identified as quercetin-3-O-rutinoside by spiking and comparing with the reference standard. Also, production of [M + H]+ at m/z 611 and [M − H]− at m/z 609 in peak 8 indicated its molecular weight of 610 which matched quercetin-3-O-rutinoside. Peak 6 generated similar positive fragment ions as detected in peak 8. Moreover, peak 6 possibly produced the fragment ion at m/z 611 by cleavage rhamnose (molecular mass 146 amu) from [M + H]+. Thus, peak 6 might consist of quercetin-rutinoside and rhamnosyl residues. Regarding MS data and its possible molecular weight of 756, peak 6 might match quercetin-3-(rhamnosyl-glucoside)-7-O-rhamnoside or quercetin-3-O-(2-rhamnosyl)rutinoside [17], which have never reported in mulberry leaves. Peak 9 produced [M + H]+ and [M − H]− at m/z 465 and 463, representing a molecular weight equal to 464, respectively. The fragment ion at m/z 303 was also found in this peak, which possibly was generated by loss of hexoside residue (a molecular mass of 162 amu) from [M + H]+. Therefore, this peak was supposed to contain quercetin and hexosyl group in its structure, which could be either quercetin-glucoside or quercetin-galactoside. However, only quercetin-3-O-glucoside has been reported in mulberry leaves [18–20]. Peak 11 was tentatively identified as quercetin-(malonyl)hexoside by comparing with the results of Ruiz et al. [21]. This peak generated [M + H]+ and [M − H]− at m/z 551 and 549, respectively, indicating its molecular weight of 550. Its molecular weight might belong to quercetin-(malonyl)glucoside or quercetin-(malonyl)galactoside. However, only quercetin-(malonyl)glucoside was determined in mulberry leaves [5, 19, 20, 22]. Peaks 4, 7, 10, 12 and 13 were identified as kaempferol derivatives because these peaks had similar UV spectra with λmax at 265 and 345–347 nm and also produced the fragment ion at m/z 287 (kaempferol residue). Peak 10 was identified as kaempferol-3-O-rutinoside by comparing with the reference standard and confirming by spiking with its reference standard. Based on MS data, peak 7 was likely to be derivative of kaempferol-rutinoside because this peak produced same fragment ions as presented in peak 10. Also, the fragment ion at m/z 611 was produced by loss of rhamnose (molecular mass 146 amu). Thus, peak 7 possibly consisted of kaempferol-rutinoside and rhamnosyl residues [18]. The fragment ion at m/z 287 was also found in peak 12, which possibly was generated by loss of the hexosyl group (molecular mass 162 amu) from its [M + H]+. It indicated that peak 12 was supposed to contain the hexosyl group, which might be kaempferol-glucoside or kaempferol-galactoside. Only kaempferol-3-O-glucoside has been identified in mulberry leaves [23], whereas there have been no previous studies reporting kaempferol-galactoside in mulberry leaves. Peak 4 produced similar fragment ions as found in peak 12. It is possible that peak 4 might be the kaempferol-hexoside derivative. Moreover, fragment ion at m/z 611 was also found in this peak, which is possibly generated by cleavage of rhamnose (molecular mass 146 amu) from [M + H]+. The results suggested that peak 4 was possibly comprised of kaempferol-hexoside and rhamnosyl residue. Comparing with previous results reported by El-Desoky et al. [24] and Stobiecki et al. [25], this peak might be kaempferol-3-O-glucosyl-(1 ⟶ 2)-rhamnoside-7-O-glucoside or kaempferol-3-O-glucosyl-glucoside-7-O-rutinoside. Based on survey of literatures, there was no study that identified this compound in mulberry leaves. Peak 13 generated [M + H]+ at m/z 535 and [M − H]− at m/z 533, indicating molecular weight equal to 534. The fragment ion at m/z 489 was also yielded in the negative mode. When compared with the results from a previous study reported by Llorach et al. [26], this peak might be kaempferol-(malonyl)hexoside. However, only kaempferol-(malonyl)glucoside was found in mulberry leaves [20]. Thus, peak 10 was tentatively identified as kaempferol-(malonyl)glucoside.Figure 1
Typical HPLC chromatograms of phenolic compounds of different mulberry leaves: tip of leaves cv. Buriram 60 (a), young leaves cv. Buriram 60 (b), old leaves cv. Buriram 60 (c), tip of leaves cv. Sakonnakhon (d), young leaves cv. Sakonnakhon (e), old leaves cv. Sakonnakhon (f), tip of leaves cv. Khunphai (g), young leaves cv. Khunphai (h), and old leaves cv. Khunphai (i), detected at 350 nm and zoom-in spectrum range of 10–40 min. The peak numbers are corresponding to UV spectrum and MS data in Table1.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Table 1
Identification of phenolic compounds of mulberry leaves and the data taken from HPLC-DAD and HPLC-MS.
Peak
RT (min)
λ
max (nm)
Mass spectra
Tentative compound∗
Positive ion (m/z)
Negative ion (m/z)
[M + H]+
Fragment ions
[M − H]−
Fragment ions
Caffeoylquinic acid isomers
1
13.1
239, 325
355
163, 135
353
191, 179
Caffeoylquinic acid isomer I2
2
16.5
241, 325
355
163, 135
353
191
5-O-Caffeoylquinic acid1
3
17.4
240, 325
355
163, 135
353
191, 179, 173
4-O-Caffeoylquinic acid1
5
19.5
237, 317
355
163, 135
353
191
Caffeoylquinic acid isomer II2
Flavonol derivatives
4
18.2
265, 347
757
611, 287, 449
755
609
Kaempferol-hexoside derivative2
6
21.4
256, 354
757
303, 611, 627, 465
755
625
Quercetin-rutinoside derivative2
7
23.7
265, 354
741
595, 287, 449
739
—
Kaempferol-rutinoside derivative2
8
25.6
256, 354
611
465, 303
609
—
Quercetin-3-O-rutinoside1
9
27.1
256, 353
465
303
463
—
Quercetin-hexoside2
10
29.1
265, 347
595
449, 287
593
—
Kaempferol-3-O-rutinoside1
11
29.6
256, 354
551
303
549
505
Quercetin-(malonyl)-hexoside2
12
31.0
265, 347
449
287
447
—
Kaempferol-hexoside2
13
34.3
265, 347
535
287
533
489
Kaempferol-(malonyl)-hexoside2
∗Identification of phenolic compound. 1According to UV spectrum and MS data in combination with retention time of the reference standard. 2According to UV spectral and MS data comparison with the data obtained from previous studies.


## 3.2. Quantification of Phenolic Compounds in Mulberry Leaves

Quantitative analysis of phenolic compounds in mulberry leaves is shown in Table2. The 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside were quantified using external reference standards. Caffeoylquinic acid isomers I and II were quantified as chlorogenic acid equivalents. Quercetin derivatives were quantified as quercetin-3-O-rutinoside equivalents, whereas kaempferol derivatives were reported as kaempferol-3-O-rutinoside equivalents. The results showed that total caffeoylquinic acid and total flavonol derivative contents of mulberry leaves ranged from 5.7 to 16.3 and 2.9 to 9.1 mg/g DW, respectively (Table 2). Among all samples, tip of leaves from mulberry cv. SK had the highest content of total caffeoylquinic acids and total flavonol glycosides. For young leaves, total caffeoylquinic acids and total flavonol derivatives were higher in mulberry leaves cv. BR 60 than other cultivars. No significant difference of total caffeoylquinic acids was found in old leaves; however, total flavonol glycosides of old leaves were significantly higher in mulberry leaves cv. BR 60 when compared to cv. SK and KH. Total caffeoylquinic acids and total flavonol derivatives decreased as mulberry leaves aged. The results indicated that phenolic compounds in mulberry leaves were distinctly influenced by cultivars and leaf ages, which is in agreement with the results determined in pear leaves [27]. Variation of phenolic compounds in different cultivars could be related to genetic diversity which might be involved in synthesis and metabolism of phenolic compounds [28]. Concerning the effect of leaf ages, decreasing phenolic contents as leaves age might lessen the activity of phenylalanine ammonia-lyase, a key enzyme in phenolic synthesis and metabolites, as reported in apple leaves [29]. The result also showed that 5-O-caffeoylquinic acid is the most abundant phenolic compound of mulberry leaves, contributing up to 57%. Similarly, this compound was predominantly found in the leaves of mulberry cultivated in Korea [18], Spain [19], and Tunisia [20]. The mulberry leaves in this study contained greater 5-O-caffeoylquinic acid content than that contained in the Korean and Tunisian mulberry leaves but lower than that of the Spanish mulberry leaves. Quercetin-3-O-rutinoside and 4-O-caffeoylquinic acid were also major phenolic compounds, which contributed up to 19.2% and 16.1%, respectively. Quercetin-(malonyl)hexoside (12.2–13.8%) and kaempferol-(malonyl)hexoside (10.2–12.4%) were contributed only in young and old leaves of mulberry cv. BR 60. Quercetin 3-(malonyl)glucoside was reported as a major phenolic compound in Japanese and Korean mulberry reported by Katsube et al. [5] and Lee and Choi [18], respectively. Furthermore, quercetin-rutinoside derivative and kaempferol-rutinoside derivative were observed only in young and old leaves of mulberry cv. BR 60 with very low amount (<1%). Considering individual phenolic compounds, their contents significantly decreased when the leaves aged, except for caffeoylquinic acid isomer I (3-O-caffeoylquinic acid).Table 2
Quantification of phenolic compounds detected in mulberry leaves from 3 commercial Thai cultivars at different leaf ages.
Parameter
Buriram 60
Sakonnakhon
Khunphai
Tips
Young leaves
Old leaves
Tips
Young leaves
Old leaves
Tips
Young leaves
Old leaves
Caffeoylquinic acid isomer I1
0.32 ± 0.01B,c
0.60 ± 0.01NS,a
0.55 ± 0.01B,b
0.39 ± 0.01A,b
0.68 ± 0.02NS,a
0.68 ± 0.02A,a
0.33 ± 0.01B,b
0.64 ± 0.06NS,a
0.73 ± 0.01A,a
5-O-Caffeoylquinic acid
11.03 ± 0.53B,a
6.76 ± 0.14A,b
3.88 ± 0.12NS,c
13.12 ± 0.24A,a
5.04 ± 0.80B,b
3.50 ± 0.36NS,c
10.72 ± 0.13B,a
5.73 ± 0.85B,b
5.03 ± 0.91NS,b
4-O-Caffeoylquinic acid
1.91 ± 0.11B,a
2.13 ± 0.05NS,a
1.26 ± 0.03B,b
2.45 ± 0.05A,a
1.80 ± 0.25NS,b
1.38 ± 0.11B,c
1.84 ± 0.01B,ns
1.99 ± 0.09NS,ns
1.88 ± 0.17A,ns
Caffeoylquinic acid isomer II1
0.23 ± 0.02B,a
0.18 ± 0.00A,b
0.10 ± 0.00NS,c
0.33 ± 0.02A,a
0.13 ± 0.02B,b
0.10 ± 0.01NS,b
0.23 ± 0.02B,a
0.15 ± 0.01B,b
0.14 ± 0.02NS,b
Total caffeoylquinic acids
13.49 ± 0.66B,a
9.68 ± 0.20A,b
5.79 ± 0.17NS,c
16.29 ± 0.16A,a
7.59 ± 0.45B,b
5.65 ± 0.47NS,c
13.13 ± 0.08B,a
8.51 ± 0.69A,b
7.78 ± 0.97NS,b
Kaempferol-hexoside derivative3
0.30 ± 0.02A,a
0.09 ± 0.01B,b
0.07 ± 0.00C,b
0.32 ± 0.00A,a
0.13 ± 0.01
0.11 ± 0.01
0.29 ± 0.00
0.16 ± 0.00
0.01 ± 0.00
Quercetin-rutinoside derivative2
ND
0.14 ± 0.00ns
0.15 ± 0.02ns
ND
ND
ND
ND
ND
ND
Kaempferol-rutinoside derivative3
ND
0.10 ± 0.00b
0.13 ± 0.00a
ND
ND
ND
ND
ND
ND
Quercetin-3-O-rutinoside
3.65 ± 0.26B,a
1.55 ± 0.05B,b
1.02 ± 0.04B,b
4.39 ± 0.05A,a
1.97 ± 0.18B,b
1.56 ± 0.12B,c
3.45 ± 0.02B,a
2.42 ± 0.20A,b
2.22 ± 0.29A,b
Quercetin-hexoside2
1.00 ± 0.08B,a
0.28 ± 0.02B,b
0.05 ± 0.00B,c
2.03 ± 0.05A,a
0.46 ± 0.05A,b
0.38 ± 0.05A,b
0.94 ± 0.03B,a
0.36 ± 0.05B,b
0.36 ± 0.05A,b
Kaempferol-3-O-rutinoside
1.11 ± 0.07B,a
0.61 ± 0.02B,b
0.41 ± 0.01C,c
1.59 ± 0.01A,a
1.07 ± 0.09A,b
0.84 ± 0.07B,c
1.05 ± 0.00B,ns
1.14 ± 0.08A,ns
1.11 ± 0.09A,ns
Quercetin-(malonyl)-hexoside2
ND
2.00 ± 0.08a
1.46 ± 0.06b
ND
ND
ND
ND
ND
ND
Kaempferol-hexoside3
0.25 ± 0.02B,a
0.22 ± 0.01NS,a
0.14 ± 0.01NS,b
0.73 ± 0.02A,a
0.17 ± 0.01NS,b
0.14 ± 0.00NS,b
0.23 ± 0.01B,ns
0.20 ± 0.04NS,ns
0.19 ± 0.04NS,ns
Kaempferol-(malonyl)-hexoside3
ND
1.67 ± 0.20a
1.31 ± 0.17b
ND
ND
ND
ND
ND
ND
Total flavonols
6.31 ± 0.42B,a
6.66 ± 0.22A,a
4.74 ± 0.17A,b
9.06 ± 0.14A,a
3.67 ± 0.28B,b
2.92 ± 0.22B,b
5.67 ± 0.05B,a
4.12 ± 0.33B,b
3.88 ± 0.47B,b
Values are expressed as mean ± SEM. Different capital letters indicate significant differences between cultivars within the same leaf age atp<0.05. Different small letters indicate significant differences between leaf ages within the same cultivar at p<0.05. ND: none detectable; NS: no significant difference among cultivars at p<0.05; ns: no significant difference among leaf age at p<0.05. 1Content expressed as mg chlorogenic acid equivalent/g dry weight. 2Content expressed as mg quercetin-3-rutinoside equivalent/g dry weight. 3Content expressed as mg kaempferol-3-rutinoside equivalent/g dry weight.


## 3.3. Classification of Mulberry Leaves

PCA was applied to the data set of 9 different samples of mulberry leaves harvested from three cultivars (BR 60, SK, and KH) at different 3 stages of leaf ages (tips, young, and old leaves) in order to achieve understanding of characteristics of these leaf samples. The results showed that the first two principal components (PCs) explained 84.79% of the total variation of data set. PC1 explained 61.34% of total variance and had high contribution of 5-O-caffeoylquinic acid, quercetin-3-O-rutinoside, caffeoylquinic acid isomer II, and total caffeoylquinic acids (Figure 2(a)). The nine samples of mulberry leaves from different cultivars and leaf ages were clearly divided into three groups (Figure 2(b)). The first group had very high positive scores for PC1, which consisted of tips of leaves from all cultivars. This group can be best described by high content of phenolic compounds, especially 5-O-caffeoylquinic acid (>10 mg/g DW) and quercetin-3-O-rutinoside (>3.4 mg/g DW). The second group had negative scores for PC1 and positive scores for PC2 including young and old leaves of mulberry cv. BR 60. This group was best characterized by the presence of quercetin-(malonyl)hexoside and kaempferol-(malonyl)hexoside. The third group contained young and old leaves of mulberry cv. SK and KH, which had negative scores for PC1 and PC2. This group corresponds to low phenolic contents. HCA also applied for grouping the mulberry leaves based on the similarities of phenolic compounds. Three groups of mulberry leaves were clustered by HCA (Figure 3). It showed clearly that the results from HCA were very similar to the results from PCA. Summarizing the PCA and HCA, these chemometric analyses revealed that the predominant phenolic compounds, 5-O-caffeoylquinic acid and quercetin-3-O-rutinoside, can be used to classify tips of leaves from young and old leaves. However, the difference of tips of leaves among cultivars could not be classified. Besides, the results from this study also suggested that mulberry leaf samples in the same group might be used interchangeably because the leaves provided similar phenolic compounds. Previous study reported that mulberry leaves were clustered by genetic characters such clones and species [19, 30]. This study exhibited that cultivars and leaf ages were firstly considered for classification of mulberry leaves which is the new evidence exhibiting the important of the two factors in mulberry leaf utilization.Figure 2
Principal component analysis based on phenolic compounds of different 9 samples of mulberry leaves: (a) scattering plot of loadings on principle components PC 1 and PC 2; (b) sample map of scores on PC1 and PC 2 as function of cultivars and leaf age. 4-CQA: 4-O-caffeoylquinic acid; 5-CQA: 5-O-caffeoylquinic acid; CQA isomers I, II: caffeoylquinic acid isomers I, II; total CQA: total caffeoylquinic acids; K-H: kaempferol-hexoside; Q-H: quercetin-hexoside; Q-3-R: quercetin-3-O-rutinoside; K-3-R: kaempferol-3-O-rutinoside; K-M-H: kaempferol-(malonyl)hexoside; Q-M-H: quercetin-(malonyl)hexoside; K-R: kaempferol-rutinoside; Q-R: quercetin-rutinoside. Cultivar’s codes are BR 60: Buriram 60, SK: Sakonnakhon, and KH: Khunphai. Leaf age’s codes are T: tips, Y: young leaves, and O: old leaves.
(a)
(b)Figure 3
Dendrogram of hierarchical cluster analysis among different samples of mulberry leaves from three cultivars at different mulberry leaves. Cultivar’s codes are BR 60: Buriram 60, SK: Sakonnakhon, and KH: Khunphai. Leaf age’s codes are T: tips, Y: young leaves, and O: old leaves.


## 4. Conclusions

Caffeoylquinic acids and flavonol glycosides were main phenolic compounds in mulberry leaves. The predominant compounds were 5-O-caffeoylquinic acid and quercetin-3-O-rutinoside. Cultivars and leaf ages significantly influenced phenolic compounds in mulberry leaves; therefore, the two factors should be used as criteria for selection of mulberry leaves.According to the similarity of phenolic compounds, three groups of mulberry leaves were classified using PCA and HCA. Tips of mulberry leaves from all cultivars should be intended for production of functional healthy because of their higher phenolic contents. Therefore, cultivars and leaf ages can be used as the quality index for selection of mulberry leaves in their utilization.



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*Source: 2807690-2019-10-20.xml*