Therapeutic Potential of Peucedanum japonicum Thunb. and Its Active Components in a Delayed Corneal Wound Healing Model Following Blue Light Irradiation-Induced Oxidative Stress.

Blue light is reported to be harmful to eyes by inducing reactive oxygen species (ROS). Herein, the roles of Peucedanum japonicum Thunb. leaf extract (PJE) in corneal wound healing under blue light irradiation are investigated. Blue-light-irradiated human corneal epithelial cells (HCECs) show increased intracellular ROS levels and delayed wound healing without a change in survival, and these effects are reversed by PJE treatment. In acute toxicity tests, a single oral administration of PJE (5000 mg/kg) does not induce any signs of clinical toxicity or body weight changes for 15 days post-administration. Rats with OD (oculus dexter, right eye) corneal wounds are divided into seven treatment groups: NL (nonwounded OS (oculus sinister, left eye)), NR (wounded OD), BL (wounded OD + blue light (BL)), and PJE (BL + 25, 50, 100, 200 mg/kg). Blue-light-induced delayed wound healing is dose-dependently recovered by orally administering PJE once daily starting 5 days before wound generation. The reduced tear volume in both eyes in the BL group is also restored by PJE. Forty-eight hours after wound generation, the numbers of inflammatory and apoptotic cells and the expression levels of interleukin-6 (IL-6) largely increase in the BL group, but these values return to almost normal after PJE treatment. The key components of PJE, identified by high-performance liquid chromatography (HPLC) fractionation, are CA, neochlorogenic acid (NCA), and cryptochlorogenic acid (CCA). Each CA isomer effectively reverses the delayed wound healing and excessive ROS production, and their mixture synergistically enhances these effects. The expression of messenger RNAs (mRNAs) related to ROS, such as SOD1, CAT, GPX1, GSTM1, GSTP1, HO-1, and TRXR1, is significantly upregulated by PJE, its components, and the component mixture. Therefore, PJE protects against blue-light-induced delayed corneal wound healing via its antioxidative, anti-inflammatory, and antiapoptotic effects mechanistically related to ROS production.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species.

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S.indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S.indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Peucedanum japonicum Thunberg and Its Active Components Mitigate Oxidative Stress, Inflammation and Apoptosis after Urban Particulate Matter-Induced Ocular Surface Damage.

We previously demonstrated that urban particulate matter (UPM) exposure decreases the migration activity and survival of human corneal epithelial cells (HCECs). Herein, we investigated the potential to improve the corneal wound-healing ability of Peucedanum japonicum Thunb. leaf extract (PJE) and its active components on UPM-induced ocular surface damage in vitro and in vivo. PJE effectively assisted wound healing without altering HCEC survival and enhanced catalase (CAT), heme oxygenase 1 (HO1) and glutathione peroxidase 1 (GPX1) antioxidant gene expression. A corneal wound was uniformly induced on the right eye in all experimental animals and divided into eight groups such as two control groups (wounded right eye group-NR and non-wounded left eye group-NL), UPM treated group and PJEs (25, 50, 100, 200, 400 mg/kg) treated groups. Corneal abrasion model rats exposed to UPM showed delayed wound healing compared to unexposed rats, but wound healing was dose-dependently enhanced by PJE oral administration. Seventy-two hours after wound generation, inflammatory cells, apoptotic cells and interleukin-6 (IL-6) expression were increased substantially after UPM exposure, but PJE treatment significantly reduced the wound to an almost normal level while enhancing re-epithelialization without changing corneal thickness. Next, we tried to identify the key molecules for enhancing wound healing through fractionation. The major compounds in the fraction, confirmed by high-performance liquid chromatography (HPLC), were chlorogenic acid (CA), neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA). Each type of CA isomers showed slightly different half maximal effective (EC50) and maximal effective (ECmax) concentrations, and their mixtures synergistically enhanced HCEC migration. Thus, corneal abrasion wound recovery after UPM exposure improved after PJE treatment, and the active PJE components were identified, providing an important basis to develop therapeutics for ocular surface damage using PJE.

SR-5, the specific ratio of Korean multi-herbal formula: An evaluation of antiulcerogenic effects on experimentally induced gastric ulcers in mice.

PURPOSE: Previously, we demonstrated that the specific ratio of Korean multi-herbal formula (SR-5) exhibits hepatoprotective properties against ethanol-induced hepatic damage in rats. Chronic and excessive alcohol consumption is a major etiological factor involved in gastric disease and ulcer development induced by the inflammatory response and oxidative stress. METHODS: The present study evaluated the gastroprotective effects of SR-5 (100, 150, and 200 mg/kg) against hydrochloride acid/ethanol (HCl/EtOH)-induced and indomethacin/hydrochloride acid (INDO/HCl)-induced gastritis in a mouse model and the mechanisms involved. RESULTS: All the tested doses of SR-5 significantly inhibited gastric lesions in the HCl/EtOH-induced ulcer model mice. Similarly, all the tested doses of SR-5 significantly inhibited gastric lesions in the INDO/HCl-induced ulcer model mice. Furthermore, mice pretreated with SR-5 had significantly increased gastric levels of enzymatic and nonenzymatic antioxidants, namely, catalase (CAT) and glutathione (GSH), with concomitant reductions in malondialdehyde (MDA) and reactive oxygen species (ROS) levels compared with those in the HCl/EtOH or INDO/HCl group. SR-5 suppressed the expression of nuclear factor-kappa B (NF-κB)/p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) to their normal values. CONCLUSION: These findings are the first to demonstrate the powerful protective effect of SR-5 against gastric injury development and provide hope for clinical application.

Combined antihypertensive effect of unripe Rubus coreanus Miq. and Dendropanax morbiferus H. Lév. Extracts in 1 kidney-1 clip hypertensive rats and spontaneously hypertensive rats.

BACKGROUND: We previously showed that enzymatically hydrolyzed Dendropanax morbiferus H. Lév. leaf (Hy-DP) and unripe Rubus coreanus Miq. (5-uRCK) extracts exhibit potent vasodilator effects on isolated aortic rings from rats partly through endothelium-dependent and endothelium-independent mechanisms. These two extracts have different mechanisms of action; however, their combined effect on antihypertensive activity has not been explored. METHODS: The present study aims to investigate the effect of a chronic optimized mixture (HDR-2, composed of Hy-DP and 5-uRCK in a 2:1 mass ratio) on vascular tension and blood pressure in two different hypertensive rat models. RESULTS: The results showed that HDR-2 concentration-dependently relaxed endothelium-intact and endothelium-denuded aortic rings precontracted with phenylephrine. Antihypertensive effects were assessed in vivo on a 1 kidney-1 clip (1 K-1C) rat model of hypertension and spontaneously hypertensive rats (SHRs). Acute HDR-2 treatment significantly decreased systolic blood pressure (SBP) 3 h posttreatment in both models. Chronic HDR-2 administration also significantly decreased SBP in the hypertensive rat models. Moreover, HDR-2 increased eNOS protein expression and phosphorylation levels in the aorta. CONCLUSION: Chronic HDR-2 administration may effectively improve vascular function by decreasing plasma angiotensin-converting enzyme (ACE) activity and AngII levels. HDR-2 significantly improved acetylcholine (ACh)-induced aortic endothelium-dependent relaxation and affected sodium nitroprusside (SNP)-induced endothelium-independent relaxation in SHRs.

Long-Term Exposure to Urban Particulate Matter on the Ocular Surface and the Incidence of Deleterious Changes in the Cornea, Conjunctiva and Retina in Rats.

We investigated the time-dependent deleterious ocular changes induced by urban particulate matter (UPM) in vitro and in vivo. UPM treatment decreased human corneal epithelial cell migration and survival. Fluorescein scores were consistently increased by UPM application for 16 weeks. One week of rest at 2 or 4 weeks led to a recovery trend, whereas two weeks of rest at 8 weeks induced no change. UPM treatment decreased the tear film break-up time at 2 weeks, which was thereafter maintained until 16 weeks. No changes were found after periods of rest. UPM-treated eyes exhibited greater corneal epithelium thickness than normal eyes at 2 weeks, which recovered to normal at 4 and 8 weeks and was significantly decreased at 16 weeks. Apoptotic cell number in the epithelium was increased at 2 weeks, which remained constant except at 8 weeks. IL-6 expression in the cornea of the right eye continually increased for 16 weeks, and significant recovery was only observed at 8 weeks after 2 weeks of rest. Ocular pressure was significantly increased in the right eye at 12 and 16 weeks. Topical UPM application to the eye induced deleterious changes to various closely related parts of the eye.

Unripe Rubus coreanus Miquel Extract Containing Ellagic Acid Regulates AMPK, SREBP-2, HMGCR, and INSIG-1 Signaling and Cholesterol Metabolism In Vitro and In Vivo.

Our previous study demonstrated that a 5% ethanol extract of unripe Rubus coreanus (5-uRCK) has hypo-cholesterolemic and anti-obesity activity. However, the molecular mechanisms of its effects are poorly characterized. We hypothesized that 5-uRCK and one of its major bioactive compounds, ellagic acid, decrease cellular and plasma cholesterol levels. Thus, we investigated the hypocholesterolemic activity and mechanism of 5-uRCK in both hepatocytes and a high-cholesterol diet (HCD)-induced rat model. Cholesterol in the liver and serum was significantly reduced by 5-uRCK and ellagic acid. The hepatic activities of HMG-CoA and CETP were reduced, and the hepatic activity of LCAT was increased by both 5-uRCK extract and ellagic acid, which also caused histological improvements. The MDA content in the aorta and serum was significantly decreased after oral administration of 5-uRCK or ellagic acid. Further immunoblotting analysis showed that AMPK phosphorylation in the liver was induced by 5-uRCK and ellagic acid, which activated AMPK, inhibiting the activity of HMGCR by inhibitory phosphorylation. In contrast, 5-uRCK and ellagic acid suppressed the nuclear translocation and activation of SREBP-2, which is a key transcription factor in cholesterol biosynthesis. In conclusion, our results suggest that 5-uRCK and its bioactive compound, ellagic acid, are useful alternative therapeutic agents to regulate blood cholesterol.

Aqueous Extract of Perilla frutescens var. acuta Relaxes the Ciliary Smooth Muscle by Increasing NO/cGMP Content In Vitro and In Vivo.

The leaves of Perilla frutescens var. acuta (PFA) are commonly used as a traditional medicine in Korea, Japan, and China. We previously showed that PFA attenuates eye fatigue by improving visual accommodation through a clinical study. However, detailed mechanisms and chemical compounds have not been studied. In this study, we analyzed the active compounds in an aqueous extract of PFA involved in ciliary muscle relaxation in vitro and in vivo. NMR and MS analyses showed that the PFA extract contained mainly luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide. The composition after freeze-drying and spray-drying was similar. Freeze-dried PFA (50 µg/mL, 100 µg/mL, and 200 µg/mL) increased nitric oxide and cGMP levels in ciliary muscle cells isolated from the eyes of rats. [Ca2+]i decreased in a dose-dependent manner. Furthermore, Sprague-Dawley rats treated with freeze-dried PFA (200 mg/kg, orally) showed significantly increased cGMP levels compared with the control group and irradiated with white light. Our results suggest that PFA extract has the potential to reduce eye fatigue by relaxing ciliary muscles.

Molecular Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by InDel Markers of Chloroplast DNA.

The tuber of Cynanchum wilfordii (Baekshuoh Radix in Korean) is an important medicinal herb in Korea and China; however, it is difficult to differentiate C. wilfordii from a related medicinal herb, C. auriculatum (Baishouwu Radix in Chinese). We sought to develop a molecular method that could be used to distinguish between the tubers of C. wilfordii and C. auriculatum. We aligned the chloroplast genome sequences (available in the NCBI database) of the two species and identified three species-specific insertion and deletion (InDel) sites in the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 intergenic spacer (IGS) regions. To confirm the presence of these three InDels and validate their use as markers, we designed three primer pairs to amplify the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 IGS regions. Polymerase chain reaction (PCR) amplification of the trnQ-psbK IGS region yielded a 249 bp fragment for C. wilfordii, and 419 bp fragment for C. auriculatum, whereas the rps2-rpoC2 IGS primers produced a 629 bp fragment from C. wilfordii and a 282 bp fragment from C. auriculatum. In the psaJ-rpl33 IGS region, allele fragments of 342 and 360 bp in length were amplified from C. wilfordii, whereas 249 and 250 bp fragment were amplified from C. auriculatum. We propose these three InDel markers as a valuable, simple, and efficient tool for identifying these medicinal herbs and will thus reduce adulteration of these herbal materials in commercial markets.

Anti-Inflammatory Effects of a Stauntonia hexaphylla Fruit Extract in Lipopolysaccharide-Activated RAW-264.7 Macrophages and Rats by Carrageenan-Induced Hind Paw Swelling.

The fruit of Stauntoniahexaphylla is commonly used as a traditional anthelmintic in Korea, Japan, and China. However, its anti-inflammatory activity and the underlying mechanisms have not been studied systematically. In the present study, we examined the anti-inflammatory activities of an aqueous extract of S. hexaphylla fruit (SHF) in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The SHF extract contained anti-inflammatory compounds, such as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid. The extract inhibited protein levels of inducible nitric oxide synthase and the activity of cyclooxygenase enzyme, with concomitant reductions in the production of nitric oxide and prostaglandin E2 in LPS-activated RAW 264.7 cells. Additionally, the SHF extract reduced the production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. The SHF extract attenuated LPS-induced nuclear factor-κB (NF-κB) activation by decreasing the phosphorylation of its inhibitor, IκBα. Furthermore, the SHF extract showed a significant anti-inflammatory effect in vivo by reducing the volume of carrageenan-induced paw edema in rats. Our results suggest that the SHF extract exerts potential anti-inflammatory properties against LPS-activated RAW 254.7 cells, and in an animal model of inflammation.

Cynanchum wilfordii Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia by Regulating 5α-Reductase and Androgen Receptor Activities in a Rat Model.

Benign prostatic hyperplasia (BPH) is characterized by uncontrolled proliferation of the prostate gland. Cynanchum wilfordii has been reported to improve sexual behavior in male rats. In this study, we investigated the protective effect of an aqueous extract of C. wilfordii (CWW) against BPH development in a testosterone-induced BPH rat model. The rats were divided into the following six groups: sham/vehicle; BPH/vehicle; BPH/finasteride; and three CWW doses (50, 100, and 200 mg/kg). After a 4-week treatment with CWW, the rats were euthanized at scheduled times, and their prostates were weighed, followed by a histopathological examination. Prostate growth inhibition rates in rats administered CWW 50, 100, and 200 mg/kg were 54.5%, 51.8%, and 50.1%, respectively. The BPH/CWW group showed decreased serum testosterone and dihydrotestosterone (DHT) levels compared to the BPH/vehicle group. Furthermore, the BPH/CWW group showed reduced prostate testosterone and DHT levels compared to the BPH/vehicle group. Mechanistically, the reverse transcription-polymerase chain reaction revealed downregulated mRNA expression levels of the androgen receptor, 5α-reductase, and B-cell lymphoma-2 (Bcl-2) in the BPH/CWW200 group compared with those in the testosterone-induced groups. In conclusion, these findings show the effectiveness of CWW in slowing the progression of testosterone-induced BPH in rats.

Development of User-Friendly Method to Distinguish Subspecies of the Korean Medicinal Herb Perilla frutescens Using Multiplex-PCR.

Perilla (Perilla frutescens) is an economically and culturally important plant in East Asia. Plant breeding between cultivars has enhanced the genetic diversity of perilla overall, but means that functionally diverse subspecies are more difficult to identify and distinguish. In this study, we developed gene-based DNA markers to distinguish between the Korean herbal medicinal perilla varieties. We identified informative simple sequence repeat (SSR) regions on the promoter regions of the Myb-P1 and dihydroflavonol 4-reductase (DFR) genes, as well as a large insertion-deletion (indel) region in the limonene synthase (LS) gene, and developed markers to characterize the distinct subspecies differences (PfMyb-P1pro, PfDFRpro, and PfLS, respectively). Using the PfLS primers, a 430-bp region could be amplified from P. frutescens var. acuta, crispa, and f. viridis (known as Jasoyeop, Jureum-soyeop, and Chungsoyeop, respectively), but not from P. frutescens var. japonica (Dlggae). The PfMybpro primers resulted in PCR products of 314 or 316, 330, 322, and 315 bp from Dlggae, Jasoyeop, Jureum-soyeop, and Chungsoyeop, respectively, and the PfDFRpro primers resulted in products of 189 or 202, 187 or 189, 185 or 189, and 193bp, respectively, for the four perilla subspecies. Combining these three reactions into a single multiplex PCR approach resulted in subspecies-specific PCR band patterns for six common types of commercial perilla, distinguishing between three varieties of Dlggae (Cham-Dlggae, Ip-Dlggae, and Bora-Dlggae), as well as identifying Jasoyeop, Jureum-soyeop, and Chungsoyeop. These user-friendly markers will be valuable as a simple and efficient method for identifying the Korean medicinal herb Jasoyeop, as well as distinguishing between other functionally distinct subspecies, which may have broad applications in the Korean herbal industry.

Antithrombosis activity of protocatechuic and shikimic acids from functional plant Pinus densiflora Sieb. et Zucc needles.

Pine needle extract (PE) and fermented pine needle extract (FPE) have been reported to show various biological and pharmacological activities such as antioxidant, anti-inflammatory, anti-bacterial, anti-cholesterol, gastrointestinal motility control, and fibrinolytic effect. The aims of our research were to isolate fibrinolytic compounds from PE and FPE and evaluate their antithrombotic activity in vitro and in vivo. Protocatechuic (1) and shikimic (2) acids were isolated and identified from FPE. 1 and 2 not only have fibrinolysis activity but also inhibit fibrin formation similar to aspirin. Lysis of fibrin clots by 1 and 2 occurred completely at pH 2-4. Results of SDS-PAGE showed that fibrin polypeptide chains (Aα, Bß, γ) lysed by 1 and 2 were intact. The antithrombotic effects of 1 and 2 were confirmed by models of carrageenan-induced tail thrombosis, collagen and epinephrine-induced pulmonary thromboembolism in mice, and FeCl3-induced carotid arterial thrombus. Moreover, 1 and 2 did not induce hemorrhage in the tail veins of mice, unlike common antithrombotic compounds. We also measured changes in the quantities of 1 and 2 obtained from FPE. As fermentation progressed, we demonstrated that the quantity of 1 steadily increased, while the quantity of 2 did not significantly change. We therefore demonstrated that FPE is an excellent resource for 1 and 2 and can be produced inexpensively in sufficient quantities for industrial-scale extraction.