The Contents of Polyphenols in Perilla frutescens (L.) Britton var. frutescens (Egoma) Leaves are Determined by Vegetative Stage, Spatial Leaf Position, and Timing of Harvesting during the Day

: The leaf of Perilla frutescens (L.) Britton var. frutescens (egoma) is a rich source of polyphenolic compounds, including rosmarinic acid. However, there is still a lack of detailed information concerning the content of phenolic compounds in these leaves. Since some flavonoids were found as a conjugated form, leaves were used untreated or hydrolyzed using β-glucuronidase for analysis. Enzymatic hydrolysis method successfully identified some polyphenols, which have not been reported before. Scutellarin, a flavone glucuronide with a molecular mass similar to that of luteolin 7- O -glucuronide, was present in egoma leaves. Scutellarin was the second most abundant polyphenolic compound, after rosmarinic acid. Egoma leaves at the top of the plant contained a higher amount of rosmarinic acid and scutellarin compared to that in the leaves below. The difference in plant growth stage also influenced the rosmarinic acid and scutellarin contents, while the time of harvesting during the day did rosmarinic acid contents only. This is the first time that scutellarin, a traditional Chinese medicine, widely used for the treatment of cerebrovascular disease, was quantitatively determined in egoma leaves. The present study may help adding value to egoma leaves, developing dietary supplements, functional foods, and cosmetics.

applications. Its global demand as a dietary supplement has been increasing recently 5 .
However, there is still a lack of detailed quantitative information of other phenolic compounds except rosmarinic acid in the mint family Lamiaceae leaves, which are presumably influenced by the timing of harvesting during the day and plant growth.
The aim of this study was to determine the most abundant phenolic compounds other than rosmarinic acid in egoma leaves, using enzymatic hydrolysis as a pretreatment step. Applying HPLC, the phenolic content was quantified at different plant growth stages, at specific harvesting times of the day, and using leaves of different spatial positions.

Materials
Leaves of egoma were obtained from Ecobito Farm & Company Kanzaki, Japan . Rosmarinic acid and bovineliver-derived β-glucuronidase were purchased from Sigma-Aldrich GK. Tokyo, Japan . Scutellarin and scutellarein were obtained from Toronto Research Chemicals Toronto, Canada and Tokyo Chemical Industry Co., Ltd Tokyo, Japan , respectively.

Preparation of egoma leaf powder
Egoma leaves were rinsed in water, and subsequently blanched, simmering in boiling water ratio of 1:5 for 10 s. The blanched leaves were drained of water and cooled to room temperature. All the blanched samples were vacuumdried EYELA vacuum oven, VOS-451SD, Tokyo Rikakikai Co., Ltd . The dried leaves were crushed and ground to make a leaf powder using a CMT Vibrating Sample Mill T1-100 CMT Co., Ltd., Fukushima, Japan .

Egoma leaf extracts for quantification of phenolic
compounds An aliquot of egoma leaf powder 0.1 g was extracted twice with 10 mL of solvent acetone/water/acetic acid 60/39.8/0.2 v/v , heated to 80 for 1 h using a heat block Dry Thermo unit DTU-2CN, Taitec, Saitama, Japan , and then centrifuged with a Kubota 5100 centrifuge Kubota Corporation, Tokyo, Japan at 1600 g for 15 min. The combined extract was transferred to a measuring flask and filled up to 20 mL. The leaf extracts were filtered through a 0.45 µm filter for HPLC analysis.

Egoma leaf extracts for hydrolysis
An aliquot of leaf powder 0.1 g was extracted twice with 10 mL of 80 ethanol, shaken at room temperature for 1 h using a tube mixer CM-1000 Cute Mixer, EYELA, Tokyo, Japan , and then centrifuged with a Kubota 5100 centrifuge Kubota Corporation, Tokyo, Japan at 1600 g for 15 min. The combined extract was poured into a measuring flask and filled up to 20 mL. The extract was then transferred to a tube and dried by centrifugal evaporation MiVac Quattro Concentrator, Genevac Ltd., Ipswitch, Suffolk, UK . The dried perilla extract was finally dissolved in 80 ethanol 50 mg/mL .

Hydrolysis of egoma leaf extracts
Fifty µL 50 mg/mL of leaf extract and 50 µL of 100 U/ mL of β-glucuronidase were mixed with 400 µL of an acetate buffer of pH 5.0 in a 2 mL screw-capped glass vial. After tightening the caps firmly, the vials were incubated at 37 for 24 h. Then, the hydrolysis process was terminated, heating the vials up to 100 for 5 min. The hydrolyzed samples were precipitated by adding 500 µL of 100 acetonitrile and then filtered through a 0.45 µm filter for HPLC analysis.

Determination of phenolic compounds
HPLC analysis was carried out with a Dionex TM UltiMate TM 3000 HPLC system Thermo Fisher Scientific Inc., Sunnyvale, CA, USA . Prior to the HPLC analysis, the egoma extracts were filtered through a 0.45 µm filter. Then, 3 µL of hydrolyzed and unhydrolyzed perilla leaf extracts were injected onto an analytical Unison UK-C18 Imtakt Crop 2Ø 250 mm column at 40 . The mobile phases were composed of 0.1 v/v of formic acid in water eluent A and 0.1 v/v of formic acid in acetonitrile eluent B . The gradient program was as follows: 10 B 0 min , 50 B 20 min , 100 B 21 to 27 min , and 10 B 28 min to 40 min . The total run-time was 40 min. Absorbance at 330 nm was measured to detect phenolic compounds. A standard with known retention time was used to calculate the phenolic content by comparing the peak area with that of the standard. The concentration of the standards ranged from 6.25 to 100 µg/mL.

Statistics
All values were expressed as means SE. Statistical analysis was performed by one-way ANOVA with Tukey-Kramer s post hoc test, and a p-value 0.05 was consid-ered significant Graph Pad Prism 6 version 6.07 .

Results and Discussion
Leaves of egoma were collected at different spatial positions and growth stages of the plant. The growth cycle of perilla is defined as the period from leaf formation to leaf senescence. Classified growth stages were: early vegetative, vegetative, before flowering, during flowering, seed maturation time, and senescence. Phenolic compounds, extracted from egoma leaves with or without enzymatic hydrolysis, were analyzed with HPLC. Their chromatograms are shown in Fig. 1. Six peaks were detected before hydrolysis. One major peak was rosmarinic acid. We first attempted to identify unknown phenolic compounds using available standards, comparing retention times, UV and LC-MS spectra of the leaf extracts. Four peaks in the unhydrolyzed samples disappeared and three new peaks of aglycon apigenin, luteolin, and scutellarein developed after hydrolysis. This suggests that polyphenolic compounds in egoma leaves consist mainly glucuronides of apigenin, luteolin, and scutellarein. Scutellarein was noticeable as the most abundant aglycon obtained after hydrolyzation.
Scutellarin is a flavone glucuronide predominantly detected in Erigeron breviscapus vant. Hand. Mazz., utilized in pharmacological applications such as antioxidants, anti-inflammatories, or antitumor agents 11,12 . Scutellarin has also been used for clinical treatments of cerebrovascular and cardiovascular diseases, such as strokes, and exerts protective effects on brain ischemia or ischemia/reperfusion 13,14 . This suggests that egoma leaves extracts could also be used as supplements to prevent cardiovascular diseases.
Quantitative analysis of rosmarinic acid and scutellarin in leaves of different plant growth stages was performed with HPLC, as presented in Fig. 2. The highest rosmarinic acid content 81.9 2.4 mg/g dry weight dw was determined at the time of flowering, while a significantly higher scutellarin content was analyzed at the early vegetative stage. We observed further that the total polyphenol content was highest during flowering time data not shown , when polyphenols act as antioxidants to protect plants from insects and oxidative stress 15 . It is further suggested that polyphenol synthesis is induced in leaves during the reproductive stage. Rosmarinic acid and scutellarin remained in senescent leaves at the time of harvesting the seeds. The amounts detected were 53.3 3.3 mg/g dw and 14.2 0.45 mg/g dw,  respectively, indicating that senescent egoma leaves could also be an alternative source of health-promoting supplements. Specific spatial leaf positions seem to play a vital role in accumulating polyphenolic compounds during the vegetative stage. As shown in Fig. 3, the levels of rosmarinic acid in the first pair of leaves at the top of the plant 66.7 1.04 mg/g dw were higher than those of the third and fourth pairs of leaves. The first pair of leaves had higher scutellarin levels 17.7 0.24 mg/g dw than those of the second, third, and fourth pairs. Thus, regular harvesting of the upper fully-grown leaves during the vegetative stage resulted in egoma leaves rich in rosmarinic acid and scutellarin.
To determine the best harvesting time of the day, the first pair of leaves was picked four times daily, continuously for three days. The effect of harvesting time on accumulated phenolics is presented in Fig. 4. The rosmarinic acid levels were higher in the samples collected at 06:00 am and 10:00 am compared to those collected at 02:00 pm and 06:00 pm. In contrast, no harvesting time effect was found for scutellarin. The contents of rosmarinic acid and scutellarin in the mornings were 51.4 3.79 mg/g dw and 16.9 0.58 mg/g dw, respectively, and decreased to 42.1 3.97 mg/g dw and 13.0 1.43 mg/g dw, respectively, in the afternoon. This indicates that perilla leaves should be picked in the morning to obtain higher amounts of phenolic compounds.

Conclusion
Scutellarin is the second most abundant polyphenolic compound in egoma leaves. Glucuronides of apigenin and luteolin were also present. Higher amounts of rosmarinic acid and scutellarin, both of which are known to have physiological properties such as anti-inflammatory action, were detected in the upper leaves during the vegetative stage, with a higher scutellarin content observed at the  early vegetable stage. The content of rosmarinic acid was highest at the time of flowering. Scutellarin and rosmarinic acid extracts may find potential value adding applications as dietary supplements, functional foods, or as cosmetic ingredient.