Preparation of test samples

The ninety-one herbal plants used for screening in this study are shown in Table 1. Ethanol extracts of the herbal plants were obtained from Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan) and Koei Kogyo Co., Ltd. (Tokyo, Japan). These ethanol extracts were evaporated under reduced pressure and lyophilized as test samples. Lyophilized samples were reconstituted at 100 mg/mL with DMSO, and each test sample was diluted with basal medium at a concentration in the range of 0.02–500 μg/mL and used in each experiment.

Table 1

A list of herbal plants used in screening of Sema3A inducers.

No	Scientific name	Part	No.	Scientific name	Part
1	Uncaria gambir	Leaf, burgeon	47	Ziziphus jujuba	Fruit
2	Arnica montana	Flower	48	Thymus vulgaris	Aerial part
3	Aloe ferox, Aloe africana	Leaf	49	Syzygium aromaticum	Bud
4	Aloe arborescens	Leaf	50	Citrus unshiu	Pericarp
5	Ginkgo biloba	Leaf	51	Capsicum annuum	Fruit
6	Urtica thunbergiana	Leaf	52	Angelica acutiloba	Root
7	Artemisia capillaris	Flower head	53	Calendula officinalis	Flower
8	Foeniculum vulgare	Fruit	54	Panax ginseng	Root
9	Thea sinensis	Leaf	55	Lonicera japonica	Leaf, stem
10	Malva sylvestris L.	Flower	56	Eriobotrya japonica	Leaf
11	Rosa multiflora	Fruit	57	Tussilago farfata	Flower, leaf
12	Rabdosia japonica	Aerial part	58	Poria cocos	Sclerotium
13	Scutellaria baicalensis	Root	59	Ruscus aculeatus	Rootstock
14	Phellodendron amurense	Bark	60	Carthamus tinctorius	Flower
15	Fucus vesiculosus	Whole alga	61	Mentha piperita	Leaf
16	Glycyrrhiza glabra, Glycyrrhiza uralensis	Root, rootstock	62	Paeonia suffruticosa	Root bark
17	Cinchona succirubra	Bark	63	Humulus lupulus	Female Flower
18	Prunus armeniaca	Seed	64	Aesculus hippocastanum	Seed
19	Mallotus philippinensis	Bark	65	Melissa officinalis	Leaf
20	Sasa veitchii	Leaf	66	Prunus persica	Leaf
21	Matricaria chamomilla	Flower	67	Eucalyptus globulus	Leaf
22	Sophora flavescens	Root	68	Saxifraga stolonifera	Whole plant
23	Citrus paradisi	Fruit	69	Citrus junos	Fruit
24	Cinnamomum cassia	Bark	70	Lavandula angustifolia	Flower
25	Gentiana lutea	Root, rootstock	71	Thea sinensis	Leaf
26	Geranium thunbergii	Aerial part	72	Ganoderma lucidum	Fruit body
27	Saccharomyces cerevisiae	Whole	73	Rosa canina	Fruit
28	Arctium lappa	Root	74	Rosmarinus officinalis	Leaf
29	Oryza sativa	Seed coat	75	Thymus serpyllum	Aerial part
30	Asiasarum sieboldii	Root, rootstock	76	Hydrangea serrata var. thunbergii	Leaf
31	Salvia officinalis	Leaf	77	Coptis japonica	Rootstock
32	Crataegus cuneata	Fruit	78	Hypericum perforatum	Aerial part
33	Gardenia jasminoides	Fruit	79	Artemisia princeps	Leaf
34	Zanthoxylum piperitum	Pericarp	80	Lonicera japonica	Flower
35	Rehmannia glutinosa	Root	81	Thea sinensis var. assamica	Leaf
36	Perilla frutescens var. Crispa	Leaf	82	Acorus calamus	Rootstock
37	Tilia cordata	Flower	83	Achillea millefolium	Whole plant
38	Paeonia lactiflora	Root	84	Morus alba	Root bark
39	Houttuynia cordata	Aerial part	85	Luffa cylindrica	Aerial part
40	Sanguisorba officinalis	Root, rootstock	86	Lilium candidum	Bulb
41	Zingiber officinale	Rootstock	87	Coix lachryma-jobi var. ma-yuen stapf	Seed
42	Betula pendula	Bark	88	Cordyceps sinensis	Whole
43	Pinctada fucate	Nacre	89	Machilus odoratissima	Bark
44	Equisetum arvense	Whole	90	Punica granatum	Pericarp
45	Cnidium officinale	Rootstock	91	Vitis vinifera	Leaf
46	Swertia japonica	Whole plant

Cell culture

NHEKs derived from adult epidermis (Lonza, Basel, Switzerland) were cultured in keratinocyte basal medium (KBM-Gold; Lonza, Basel, Switzerland) supplemented with KGM-Gold SingleQuot (Lonza), containing 0.15 mM calcium, at 37°C with 5% CO₂. Cells were passaged at 60–70% confluence, and the experiments were performed using sub-confluent cells within four passages.

Transfection and measurement of promoter activity

NHEKs were seeded in 24-well flat bottom plates for 24 hours. The culture medium was replaced with antibiotic-free medium and the cells were co-transfected with 500 ng/well pGL3-Sema3A vector containing the firefly luciferase gene and 50 ng/well pRL3-TK vector containing the Renilla luciferase gene (Promega, Madison, WI, USA) using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s instructions. pGL3-Sema3A vector was produced in accordance with the methods reported by Kamata et al. [14]. At 24 hours after the start of transfection, the medium was replaced with fresh medium containing the test samples shown in Table 1, apigenin, baicalein, baicalin, chrysin, or wogonin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at various concentrations, and NHEKs were further incubated for 24 or 48 hours at 37°C. The cells were washed and lysed with passive lysis buffer, and luciferase activities were analyzed using a dual-luciferase reporter assay system with a 2030 ARVO-X4 Multi label Plate Reader (Perkin Elmer, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Relative luciferase activity was expressed as a relative ratio of experimental data to untreated control data after normalization with firefly luminescence/Renilla luminescence ratio for each sample well. Cultured cells were observed using a microscopic and checked for morphologically damaged cells by test sample treatment. Data obtained from damaged cells were excluded from the results.

Measurement of Sema3A at mRNA and protein levels in cultured NHEKs

NHEKs were incubated with five plant extracts, Arnica montana (sample No. 2), Artemisia capillaris (No. 7), Malva sylvestris L. (No. 10), Scutellaria baicalensis (No. 13), or Hydrangea serrata var. thunbergii (No. 76), at a final concentration of 50 μg/mL for 3 or 6 hours at 37°C. The cells were subsequently harvested and transcription levels of the human Sema3A gene were quantified using quantitative real-time PCR. Quantification was performed using an Applied Biosystems Fast Real-Time PCR system (Applied Biosystems) using PrimeScript^™ One Step RT-PCR Kit (Takara Bio Inc., Shiga, Japan) and the primers 5’-ACCCAACTATCAATGGGTGCCTTA-3’ (forward) and 5’-AACACTGGATTGTACATGGCTGGA-3’ (reverse). As an internal control, ribosome protein S18 (RPS18) mRNA was amplified using the primers 5′-TTTGCGAGTACTCAACACC AACATC-3′ (forward) and 5′-GAGCATA TCTTCGGCCCACAC-3′ (reverse). The amounts of mRNA were normalized relative to those of RPS18 and expressed relative to ratios of the untreated controls.

The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol. Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Cell counting assay

A Cell Counting Kit (CCK)- 8 (Dojindo, Kumamoto, Japan) was used to assess the cell toxicity of each compound on NHEKs. Cells were seeded at 1 × 10⁴ cells per well in 96-well culture plates. After incubation overnight at 37°C, an extract of S. baicalensis or A. montana (50 μg/mL) was added to each well, then incubated for 6 or 24 hours at 37°C. At each time point, CCK-8 solution was added to each well and incubated for 3 hours at 37°C. Cell viability was estimated by measuring the absorbance at 450 nm using a 2030 ARVO-X4 Multi label Plate Reader (Perkin Elmer).

Reconstructed human epidermis (RHE) models

RHE model (EPISKIN, SA, Lyon, France) is an in vitro reconstructed human epidermis from normal human keratinocytes cultured on an inert polycarbonate filter at the air-liquid interface, in a chemically defined medium, featuring normal ultrastructure and functionality similar to human tissue in vivo [15]. RHE models were cultured in accordance with the manufacturer’s protocol. Briefly, inserts containing the RHE models were shipped at room temperature in a multi-well plate filled with an agarose-nutrient solution in which they were embedded. The inserts were carefully taken out of the multi-well plate, any remaining agarose adhering to the outer sides of the insert was removed, and the inserts were placed in a plate in which each well has been filled with Maintenance Medium (EPISKIN, Lyon, France) overnight at 37°C, 5% CO₂. Next, 100 μL of the extract of A. montana or S. baicalensis (100 or 500 μg/mL) was applied from the stratum corneum side of the reconstructed epidermis samples and cultured for 24 hours. The concentration of Sema3A protein in the culture supernatants was measured by ELISA as described above.

Statistical analyses

Unless otherwise indicated, values were presented as the standard deviation (S.D.) of the mean. Statistical analyses were performed using one-way or two-way ANOVA followed by Dunnett’s multiple comparison tests with GraphPad Prism 8 (GraphPad Software, Inc., CA, USA), with p < 0.05 defined as statistically significant.

Screening of Sema3A inducers from herbal plant extracts using a reporter gene assay

Cultured NHEKs were transfected with luciferase reporter gene constructs containing -1444 bp of the human Sema3A promoter as described previously [14]. Following transfection, cells were treated with various types of herbal plant extracts at a final concentration of 50 μg/mL for 24 hours and the cell lysates were subjected to luciferase assays. The results are shown in Table 2. From 91 different types of herbal plant extracts, 48 extracts exhibited significantly increased relative luciferase activity compared with the untreated controls, with no cell damage morphologically by the addition thereof. The dose–response relationship was examined with the lower concentration range for the top 5 samples (sample Nos. 2, 7, 10, 13, and 76) among the 48 herbal plants extracts showing significantly higher activity compared with the control. The samples of No. 7, 10, and 76 increased activity in a dose-dependent manner (Fig 1). No dose-dependent increases in activity were observed with samples No. 2 and 13, although in sample No. 13, it seemed that its activity probably reached a plateau, because it was the most active at a low concentration of 5 μg/mL.

Fig 1

Effects of five positive hits of plant extracts on Sema3A reporter gene expression.

NHEKs were transfected with pGL3-Sema3A. After transfection, the cells were incubated with five plant extracts, namely No. 2, 7, 10, 13, and 76 at a final concentration of 5, 15, and 50 μg/mL for 48 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in Materials and methods. All results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Table 2

A summary of Sema3A reporter gene activity by herbal plant extracts.

No.	Mean	S.D.	Significant difference	P value	No.	Mean	S.D.	Significant difference	P value
1	1.60	0.07	***	0.0007	47	1.05	0.06	n.s.	0.9994
2	2.21	0.20	****	< 0.0001	48	1.24	0.14	n.s.	0.367
3	1.62	0.18	***	0.000	49	1.43	0.12	*	0.0108
4	1.53	0.07	**	0.004	50	1.02	0.01	n.s.	0.9998
5	1.60	0.06	***	0.001	51	1.62	0.10	***	0.0002
6	1.67	0.13	***	0.000	52	1.08	0.05	n.s.	0.999
7	2.81	0.21	****	< 0.0001	53	1.29	0.27	n.s.	0.1762
8	1.73	0.07	****	< 0.0001	54	1.23	0.09	n.s.	0.3833
9	1.55	0.30	**	0.002	55	1.33	0.03	n.s.	0.0841
10	1.97	0.16	****	< 0.0001	56	1.38	0.30	*	0.0348
11	1.44	0.17	*	0.02	57	1.41	0.13	*	0.019
12	1.39	0.17	n.s.	0.05	58	1.29	0.12	n.s.	0.996
13	1.95	0.09	****	< 0.0001	59	1.49	0.14	n.s.	0.9042
14	1.03	0.06	n.s.	1.00	60	1.38	0.08	n.s.	0.9829
15	1.32	0.06	n.s.	0.20	61	1.40	0.21	n.s.	0.9741
16	1.69	0.16	****	< 0.0001	62	1.55	0.14	n.s.	0.8322
17	1.29	0.23	n.s.	0.31	63	0.67	0.05	n.s.	0.992
18	1.19	0.06	n.s.	0.81	64	No data	No data	-	-
19	No data	No data	-	-	65	1.62	0.10	n.s.	0.7277
20	1.40	0.07	*	0.05	66	1.25	0.13	n.s.	0.9991
21	1.25	0.03	****	< 0.0001	67	1.57	0.18	n.s.	0.7978
22	1.20	0.03	****	< 0.0001	68	1.03	0.15	n.s.	0.9999
23	1.03	0.02	n.s.	0.8836	69	1.13	0.04	n.s.	0.9996
24	1.24	0.05	****	< 0.0001	70	1.36	0.26	n.s.	0.9893
25	1.19	0.04	****	< 0.0001	71	1.31	0.11	**	0.0062
26	1.25	0.05	****	< 0.0001	72	1.35	0.01	***	0.0006
27	1.20	0.02	****	< 0.0001	73	1.16	0.04	n.s.	0.2914
28	1.23	0.01	****	< 0.0001	74	1.38	0.07	***	0.0006
29	1.19	0.02	****	< 0.0001	75	1.42	0.13	****	< 0.0001
30	1.23	0.02	****	< 0.0001	76	2.27	0.17	****	< 0.0001
31	1.34	0.02	****	< 0.0001	77	1.29	0.03	**	0.0062
32	1.27	0.00	****	< 0.0001	78	1.38	0.06	***	0.0006
33	1.28	0.05	****	< 0.0001	79	1.54	0.08	****	< 0.0001
34	1.30	0.01	****	< 0.0001	80	1.33	0.04	***	0.0006
35	0.96	0.26	n.s.	0.9993	81	1.32	0.02	**	0.0062
36	1.17	0.03	n.s.	0.2271	82	1.18	0.09	n.s.	0.2914
37	1.10	0.03	n.s.	0.7929	83	1.38	0.13	***	0.0002
38	1.19	0.03	n.s.	0.1783	84	0.02	0.01	****	< 0.0001
39	1.18	0.03	n.s.	0.2098	85	0.99	0.08	n.s.	0.9997
40	1.26	0.03	*	0.0244	86	0.96	0.07	n.s.	0.9534
41	1.26	0.24	*	0.0197	87	0.99	0.08	n.s.	0.9998
42	1.10	0.01	n.s.	0.82	88	0.87	0.04	n.s.	0.8227
43	1.24	0.01	*	0.0368	89	1.01	0.22	n.s.	> 0.9999
44	1.31	0.01	**	0.0037	90	0.85	0.13	n.s.	0.398
45	1.19	0.01	n.s.	0.1383	91	0.83	0.04	n.s.	0.3129
46	1.27	0.07	*	0.0143

NHEKs were transfected with pGL3-Sema3A. After transfection, the cells were incubated with the herbal plant extracts shown in Table 1 at a final concentration of 50 μg/mL for 24 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in the Materials and methods. All results were expressed as mean ± S.D. of triplicate samples.

* p < 0.05,

** p < 0.01,

*** p < 0.001,

**** p < 0.0001, n.s: no significant difference (one-way ANOVA followed by Dunnett’s test). No data: data obtained from cells in which morphological damage was observed by the addition of sample was not used.

Effects of the herbal plant extracts Sema3A induction at mRNA and protein levels in NHEKs

We next examined the induction of Sema3A mRNA and protein levels with the five positive plants extracts (sample No. 2, 7, 10, 13, and 76) by quantitative real-time PCR and ELISA, respectively. Expression of Sema3A mRNA in the NHEKs treated with No. 2, 7, and 13 was markedly increased at both 3 and 6 hours compared with untreated controls (Fig 2). In samples No. 10 and 76, they were slightly increased at 3 and 6 hours, respectively.

Fig 2

Effects of five positive plant extracts on the expression of Sema3A mRNA in cultured NHEKs.

NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3 and 6 hours at 37°C. Expression of Sema3A mRNA was examined using quantitative real-time PCR analysis. The levels of Sema3A mRNA were normalized relative to those of RPS18. All results are expressed as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Sema3A protein levels were measured in the culture supernatants of NHEKs treated with five tested samples using ELISA (Fig 3A). At 3 hours after sample treatment, differences in Sema3A protein levels were hardly noted in the culture supernatants compared with the control group. However, from 6 hours onward, sample treatment led to an overall increase in Sema3A expression, and Sema3A levels were significantly increased with sample No. 13 compared with the controls at 24 hours. In sample No. 2, we noted a tendency to increase, although no statistically significant difference was observed. Increased expression at the protein level was also observed using ELISA with cell lysates of cultured NHEKs with sample No. 13 but not sample No. 2 (Fig 3B). We further tested the effects of samples No. 2 and No. 13 on cell viability of cultured NHEKs using a cell counting assay. In the assay, the cell viabilities of NHEKs treated with these compounds was significantly increased at 6 hours compared with untreated controls, whereas samples No. 2 and No. 13 reduced cell viabilities to 89.8% and 78.2% at 24 hours, respectively (Fig 3C).

Fig 3

Effects of five positive plant extracts on the production of Sema3A proteins in cultured NHEKs.

(A) NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3, 6, and 24 hours at 37°C. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. *p < 0.05, (one-way ANOVA followed by Dunnett’s test). (B) NHEKs were incubated with sample No. 2 (A. montana) or No. 13 (S. baicalensis) at 50 μg/mL for 6 and 24 hours. After that, the cell lysates were prepared with lysis buffer, and then the levels of Sema3A protein in the cell lysates were measured using ELISA. (C) Cell viability of cultured NHEKs treated with the extract of A. montana or S. baicalensis was measured using a cell counting assay. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA followed by Dunnett’s test).

Effect of the plant extracts on Sema3A protein production in RHE models

The Sema3A-inducing activity of the extracts of S. baicalensis and A. montana was evaluated using RHE models. These plant extracts at concentrations of 100 or 500 μg/mL were applied to the stratum corneum side of RHE models. They were cultured for 24 hours, and then Sema3A protein levels in the culture supernatant were measured. In the S. baicalensis-treated group, the concentration of Sema3A increased in a dose-dependent manner, with significantly higher values noted in the 500 μg/mL S. baicalensis-treated group compared with the untreated control (Fig 4). Meanwhile, application of A. montana extract did not affect Sema3A concentration in the culture supernatant.

Fig 4

Effects of S. baicalensis and A. montana extracts on Sema3A production in RHE models.

A 100 μL sample of the extract of S. baicalensis, A. montana (100 or 500 μg/mL) or PBS (vehicle) was applied from the stratum corneum side of cultured RHE samples, and the RHE samples were cultured for a further 24 hours. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. All results are expressed as means ± S.D. of three independent experiments. *p < 0.05, (one-way ANOVA followed by Dunnett’s test).

Bioactive components in S. baicalensis contributing to Sema3A induction

We examined which components in the extract of S. baicalensis contributed to Sema3A induction (Fig 5A). Of the known chemical components in the extract of S. baicalensis, we conducted Sema3A reporter gene assays in NHEKs treated with the five major bioactive flavones, baicalin, baicalein, wogonin, chrysin, and apigenin. The experiment was performed with a maximum concentration of 5 μg/mL and a 3-fold dilution series. The molar concentration of each compound is shown in S1 Table. The extract of S. baicalensis, baicalin, and baicalein all significantly increased the relative luciferase activity at a concentration of 5 μg/mL, and the increase in activity of these samples was dose dependent. Notably, baicalein markedly increased its activity at a lower concentration of 0.56 μg/mL compared with the extract of S. baicalensis and baicalin. Such significant alterations were observed with chrysin and apigenin but were not dose dependent. Meanwhile, wogonin significantly decreased the luciferase activity at a concentration of 5 μg/mL

Fig 5

Effects of S. baicalensis–derived compounds on Sema3A reporter gene expression.

(A) NHEKs were transfected with pGL3-Sema3A vector. After transfection, the cells were incubated with S. baicalensis extract and S. baicalensis-derived compounds: apigenin, baicalein, baicalin, chrysin, and wogonin, at the indicated concentrations, a 3-fold dilution with a maximum concentration of 5 μg/mL, for 24 hours at 37°C. Cell lysates were subjected to the measurement of luciferase activities as described in the Materials and methods. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test). (B) Chemical formulae of major bioactive flavones from S. baicalensis.