Skin-Related Properties and Constituents from the Aerial Parts Extract of Persicaria senticosa

In the course of screening for cosmetic ingredients by measuring antioxidant and antiwrinkle and whitening and anti-inflammatory activities, skin-related activity was tested using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, elastase inhibition, tyrosinase inhibition, and nitric oxide assay. Several Polygonaseae extracts were found to show potent activity. The results showed that the Persicaria senticosa methanolic extract has the 1,1diphenyl-2picrylhydrazyl (DPPH) and ABTS radical scavenging activities (IC50 61.0 and 17.5 μg/mL). In the elastase inhibition assay and nitric oxide assay, the IC50 of methanolic extract of Persicaria senticosa was 739.7 μg/mL and 71.8 μg/mL. The Persicaria senticosa 70% ethanolic extract partitioned with n-hexane, CH2Cl2, EtOAc, n-BuOH, and aqueous fractions. The purification of EtOAc soluble layer was by column chromatography separation and MPLC analysis of Compounds 1-7. It was identified as loliolide (1), quercetin-3-O-glucoside (2), quercetin-3-O-glucuronide (3), 4-methoxy caftraric acid (4), kaempferol-3-(6-methylglucuronide) (5), quercetin-3-(6-methylglucuronide) (6), and quercetin (7). Structure was elucidated by a combination of 1D and 2D NMR and MS spectrometry as well as comparison with reported literatures. Radical scavenging effect on DPPH, tyrosinase inhibition, and nitric oxide assay on several compounds from Persicaria senticosa was found to show potent activity. The results showed that Compound 7 has the NO assay (IC5029.7 μM). For DPPH, the IC50 of Compounds 2, 3, 5, and 7 was 39.6, 31.2, 37.0, and 22.7 μM. In tyrosinase inhibitory activity, the IC50 of Compound 7 was 14.3 μM.


Introduction
Persicaria senticosa is an annual plant in the family Polygonaseae, which is distributed in the whole of Korea. Since early times, this plant has been used as folk medicine with beneficial effects for the treatment of various diseases such as removing the swelling parts of the wound or carbuncles and cellulitis and circulating blood and removing blood stagnation. Recent studies have shown that Persicaria senticosa had anti-inflammatory effects [1]; however, this plant has not been reported as bioactive cosmetic ingredients. And previous phytochemical studies on the flowers, stem, and roots of the genus Polygonum have revealed that various flavonoids and phenolic compounds such as hydroxybenzoic acid, rutin, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, and luteolin-7-O-rutinoside were considered the major active components [2,3]. These active compounds exhibit diverse pharmacological effects, such as anti-inflammatory, antiulcer, antihypertensive, and anticancer effects [4]. During the screening for cosmetic ingredients by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH), ABTS radical scavenging, elastase inhibition, tyrosinase inhibition and nitric oxide assay, several Polygonum extracts were found to show potent activity.
2.2. NO Assay. RAW 264.7 cells were seeded in 96-well plates (5 × 10 4 cells/well) and were treated with sample for 1 h prior to LPS (1 μg/mL) stimulation for 24 h. The negative control was treated with serum-free media. The amount of nitrite, a stable metabolite of NO, was measured by using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid). Absorbance was subsequently measured at 540 nm using an ELISA reader. The quantity of nitrite was determined from a standard curve for sodium nitrite [5][6][7].
2.3. Cell Cytotoxicity Assay. RAW 264.7 cells were plated at a density of 5 × 10 4 cells/well in 96-well plates. Cells were treated with samples for 1 h prior to LPS (1 μg/mL) stimulation for 24 h. MTT (5 mg/mL in PBS) was added to each well and incubated for 2 hr. The medium was removed from the wells by aspiration, DMSO was added to each well, and the plate was shaken. The absorbance of each well was measured at a wavelength of 540 nm using an ELISA reader. Data are presented as the mean ± standard deviation of three replicates.

DPPH Radical
Scavenging Activity Assay. DPPH radical scavenging activity was measured by using the method described by Blois [8] and Ozgen et al. [9]. DPPH solution dissolved in methanol was added to the sample, which was diluted to the required concentration, and the reaction was carried out at room temperature for 30 min. Absorbance was measured at 517 nm using an ELISA reader. The antioxidant butylated hydroxyanisole (BHA) was used as a positive control, and the IC 50 value of the sample was determined.
2.5. ABTS Radical Scavenging Activity. ABTS radical scavenging activity was measured by using a previously described method [10,11]. ABTS+ was formed by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate (K 2 S 2 O 8 ) solution with ABTS: K 2 S 2 O 8 (2 : 1 ratio) for 12-16 h to form a cation (ABTS + ). The absorbance was measured at 734 nm using an ELISA reader. BHA, an antioxidant, was used as the positive control.
2.6. Tyrosinase Inhibition Assay. Tyrosinase inhibitory activity was measured by using the method described by Yagi et al. [12]. The reaction was carried out in 0.1 M potassium phosphate buffer (pH 6.5) containing 1.5 mM L-tyrosine and 1250 units/mL mushroom tyrosinase. The reaction mixture was incubated at 37°C for 20 min. The test samples were assayed for tyrosinase inhibition by measuring its effect on tyrosinase activity using SpectraMax 190PC microplate ELISA reader at 490 nm. Arbutin and kojic acid were used as a positive control, and the IC 50 value of the sample was determined.
2.7. Elastase Inhibition Assay. The reaction was carried out in 0.5 mM Tris buffer (pH 8.5) containing 1 mg/mL N-succinyl-(Ala) 3 -p-nitroanilide and 0.6 unit/mL elastase. The reaction mixture was incubated at 25°C for 10 min. The test samples were assayed for elastase inhibition by measuring its effect on elastase activity using an ELISA reader at 405 nm. Ursolic acid was used as a positive control, and the IC 50 value of the sample was determined [13].

Statistical
Analysis. Statistical analysis of the data was performed by PRIZM5 software (GraphPad, CA, USA), and data are presented as mean ± SD. One-tailed Student's t-test was used for analyzing the significance of difference between groups, and P < 0:05 was considered statistically significant.

Skin-Related Properties of Persicaria senticosa Fractions.
Radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl(DPPH), ABTS radical scavenging, elastase inhibition, and nitric oxide assay on several Persicaria senticosa fractions was found to show potent activity. The results showed that 2 Oxidative Medicine and Cellular Longevity  /mL, respectively). The DPPH and ABTS radical scavenging activities of EtOAc fractions were 13.7 and 5.0 μg/mL. In the elastase inhibition assay, the IC 50 of nhexane and CH 2 Cl 2 fractions was under 100 μg/mL (Table 2).

Conclusions
Persicaria senticosa is an annual plant in the family Polygonaseae which is distributed in whole Korea. Since early times, this plant has been used as folk medicine with beneficial effects for the treatment of various diseases. This study evaluated skin-related properties and constituents from the aerial part extract of Persicaria senticosa and thirty-four Polygonaseae plants. In the course of screening for cosmetic ingredients by measuring the radical scavenging effect on DPPH, ABTS radical scavenging, antiwrinkle was evaluated using elastase inhibition, whitening was studied by tyrosinase inhibition, and anti-inflammatory was tested on nitric oxide assay. Several Polygonum extracts were found to show potent activity. The results showed that the Persicaria senticosa methanolic extract has the DPPH and ABTS radical scavenging activities (IC 50 61.0 and 17.5 μg/mL). In the elastase inhibition assay and nitric oxide assay, the IC 50 of methanolic extract of Persicaria senticosa was 241.5 μg/mL and 71.8 μg/mL, respectively. The Persicaria senticosa 70% ethanolic extract was partitioned with n-hexane, CH 2 Cl 2 , EtOAc, n-BuOH, and aqueous fractions.
EtOAc soluble fraction showed a potent skin-related activity ( Figure 2 and Table 3). These results suggested that    (Table 2). Thus, bioassay-guided purification of three active fractions, i.e., the EtOAc soluble fraction of Persicaria senticosa was conducted to purify the active principles responsible for the skin-related activity followed by the process described in Scheme 1, respectively. 100 g/mL 50 g/mL 25 g/mL 12.5 g/mL 6.25 g/mL 3.12 g/mL
This present study demonstrated that Persicaria senticosa and Polygonaseae contain chemical compounds with good skin-related activities and could be interesting as a novel source of bioactive agents for cosmetic industries.

Data Availability
The data used to support the findings of this study are included in the Supplemental Information File.

Conflicts of Interest
The authors have no conflicts of interest to declare.