Prominent Anti-UV Activity and Possible Cosmetic Potential of Lignin-carbohydrate Complex.

This review article summarizes the recent progress of ultraviolet rays (UV) protective substances, including our original reports. We have established a simple assay method for the determination of anti-UV activity that can be applicable to any kind of adherent cells. This method provides information of both anti-UV activity and cytotoxicity of any kind of samples even though those samples contain unknown amounts of test compounds. We found that lignin-carbohydrate complex (LCC) showed one- or two-order higher anti-UV activity compared to well-known lower molecular weight polyphenols and hot-water extracts of Kampo medicines and tea leaves. Among synthetic compounds, water-soluble azulenes showed the highest anti-UV activity. LCC showed additive or synergistic anti-UV activity with vitamin C. Alkaline extract of Sasa senanensis Rehder leaves (SE), an LCC-rich over-the-counter (OTC) drug, also showed potent antiviral and vitamin C-synergized radical scavenging activity. SE has been utilized to manufacture tooth paste, soap and gel cosmetic to increase the level of quality of life (QOL).

Synergism of Alkaline Extract of the Leaves of Sasa senanensis Rehder and Antiviral Agents.

BACKGROUND: Previous studies have shown a much greater antiviral activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE) against human immunodeficiency virus (HIV), compared to lignin precursors, tannins and flavonoids, suggesting its possible application to oral diseases. Systematic comparative study with herpes simplex virus (HSV) has been limited compared to that with HIV. In the present study, we investigated whether combination of SE with other popular antiviral agents further enhances their individual activity. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected and HSV-infected cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The antiviral activity was evaluated by the selectivity index, defined as the ratio of 50% cytotoxic concentration to 50% effective concentration. Synergy between SE and antiviral agents was evaluated by MacSynerg and CompuSyn software. RESULTS: SE showed potent anti-HIV activity, although its activity was two-orders lower than that of azidothymidine, 2',3'-dideoxycytidine dextran sulfate and curdlan sulfate. Combination of SE with these antiviral agents produced synergistic effects. Using a newly established MTT assay system for anti-HSV activity, SE and acyclovir were found to have synergistic anti-HSV activity. CONCLUSION: The present study suggests the possible efficacy of the clinical application of SE combined with antiviral agents.

Anti-Halitosis Effect of Toothpaste Supplemented with Alkaline Extract of the Leaves of Sasa senanensis Rehder.

BACKGROUND: Previous studies have shown activity against viruses, bacteria, inflammation and oral lichenoid dysplasia of alkaline extract of the leaves of Sasa senanensis Rehder (SE), suggesting its possible application to oral diseases. In the present study, we performed a small-scale clinical test to investigate whether SE is effective against halitosis and in oral bacterial reduction. MATERIALS AND METHODS: A total of 12 volunteers participated in this study. They brushed their teeth immediately after meals three times each day with SE-containing toothpaste (SETP) or placebo toothpaste. Halitosis in the breath and bacterial number on the tongue were measured by commercially available portable apparatuses at a specified time in the morning. RESULTS: Some relationship was observed between halitosis and bacterial number from each individual, especially when those with severe halitosis were included. Repeated experiments demonstrated that SETP significantly reduced halitosis but not the bacterial number on the tongue. CONCLUSION: The present study provides for the first time the basis for anti-halitosis activity of SE.

Biological interaction between Sasa senanensis Rehder leaf extract and toothpaste ingredients.

BACKGROUND: Previous studies have shown antiviral, antibacterial, and anti-inflammatory activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE). In order to manufacture an SE-containing toothpaste for combating oral diseases, we investigated the possible interaction between the candidate ingredients of toothpaste: SE, isopropyl methylphenol (IPMP, antibacterial agent) and charcoal prepared from Sasa senanensis Rehder. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected and UV-irradiated cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Superoxide radical scavenging activity was determined by electron-spin resonance spectroscopy. Antibacterial activity against Porphyromonas gingivalis 381 and Streptococcus mutans ATCC25175 was determined by the turbidity assay. RESULTS: Exposure to less than 50% SE or less than 0.31 mM IPMP for 10 min scarcely damaged human cultured gingival and periodontal ligament fibroblasts. Both SE and IPMP showed bi-modal action, stimulating the bacterial growth at lower concentrations, but synergistically inhibiting it at higher concentrations. Addition of extremely high concentrations of charcoal enhanced both anti-HIV and anti-UV activity of SE. CONCLUSION: Practically, addition of charcoal may not be recommendable, since one or two orders higher concentrations of charcoal as compared with SE, are required to achieve the synergistic effect for anti-HIV and anti-UV activity. Rather, addition of about one tenth of the amount of IPMP may be recommendable for enhancing the antibacterial activity.

Structural characterization of anti-UV components from Sasa senanensis Rehder extract.

BACKGROUND: Previous studies have shown antiviral, antibacterial and anti-inflammatory activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE). However, active components have not been identified. We isolated the substances that exhibit anti-UV activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity from SE and estimated their putative structures. MATERIALS AND METHODS: The anti-UV substances (SEE-1 and SEE-2) were isolated from SE by ethanolic extraction, Wakosil chromatography and recycled high-performance liquid chromatography (HPLC) at a yield of 0.22 and 0.18%, respectively. The structural analysis was carried out with (1)H-nuclear magnetic resonance (NMR), (13)C-NMR and UV absorption. RESULTS: SEE-1 exhibited approximately four-fold higher anti-UV activity and slightly lower DPPH radical-scavenging activity, compared to SE. SEE-1 was identified as p-coumaric acid derivative(s), a lignin precursor. CONCLUSION: The present study demonstrated for the first time the presence of lignin precursors in SE, which may explain why SE exhibits many of the properties of lignin-carbohydrate complexes.

Pilot clinical study of Sasa senanensis Rehder leaf extract treatment on lichenoid dysplasia.

BACKGROUND: Previous studies have shown antiviral, antibacterial, and anti-inflammatory activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE). Here, we investigated whether SE is effective on oral lichenoid dysplasia and osteoclastogenesis. MATERIALS AND METHODS: A male patient with white lacy streaks in the oral mucosa was orally administered SE three times a day for 11 months. The area of white streaks was monitored by intraoral photography. Interleukin-6 and -8 in the saliva were determined by enzyme-linked immunosorbent assay. Osteoclastogenesis of mouse macrophage-like RAW264.7 cells, induced by receptor activator of nuclear factor-κB ligand (RANKL) was monitored by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation. RESULTS: Long-term treatment with SE progressively reduced both the area of white steaks and the levels of salivary interleukin-6 and -8. SE significantly inhibited the macrophage differentiation towards osteoclasts. CONCLUSION: The present study suggests the therapeutic potential of SE towards oral diseases.

Biological activity of SE-10, granulated powder of Sasa senanensis Rehder leaf extract.

BACKGROUND: We have previously reported that alkaline extract of Sasa senanensis leaves (SE) showed potent anti-HIV, anti-UV and radical scavenging activity. In the present study, we investigated the biological activities of SE-10, a granulated powder of SE supplemented with lactose, lactitol, trehalose and tea extract. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected, and UV-irradiated cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Scavenging activity of superoxide anion and hydroxyl radicals was determined by electron-spin resonance spectroscopy. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone in human recombinant CYP3A4. RESULTS: SE-10 had slightly higher anti-HIV and anti-UV activities, but slightly lower radical-scavenging and CYP3A4-inhibitory activities, as compared with SE. CONCLUSION: The present study demonstrates that the biological activities of SE were well preserved during the manufacturing process of SE-10.

Comparative study of biological activity of three commercial products of Sasa senanensis Rehder leaf extract.

BACKGROUND: We have previously reported that alkaline extract of Sasa senanensis leaves (SE) has several biological activities characteristic of lignin-carbohydrate complex (LCC). In the present study, we compared the biological activity of three commercially available products of SE (products A, B and C). MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected, UV-irradiated cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Radical intensity was determined by electron spin resonance spectroscopy. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone in human recombinant CYP3A4. RESULTS: Product A is a pure SE that contains Fe(II)-chlorophyllin, whereas products B and C contain Cu(II)-chlorophyllin and less LCC. Product C is supplemented with ginseng and pine (Pinus densiflora) leaf extracts. Product A exhibited 5-fold higher anti-HIV, 4-fold higher anti-UV, 5-fold higher hydroxyl radical-scavenging, and 3-fold lower CYP3A4 inhibitory activities as compared to those of product B, and 5-fold higher, 1.5-fold higher, comparable, and 7-fold lower activities, respectively, as compared to those of product C. CONCLUSION: The present study demonstrates for the first time the superiority of product A over products B and C, suggesting the beneficial role of LCC and Fe(II)-chlorophyllin.

Anti-UV Activity of Alkaline Extract of the Leaves of Sasa senanensis Rehder.

BACKGROUND: We have previously reported several lignin-like activities of the alkaline extract of the leaves of Sasa senanensis Rehder (SE), such as anti-HIV and radical scavenging activity. As an extension, possible SE protection of cells from ultraviolet (UV)-induced injury (referred to "anti-UV activity") was investigated. MATERIALS AND METHODS: HSC-2, human oral squamous cell carcinoma cells, were exposed to UV irradiation in phosphate-buffered saline containing SE or its fractions, and then incubated for 48 hours in fresh regular culture medium to determine the viable cell number by the MTT method. SE was separated by gel filtration chromatography into the following four fractions: polysaccharide, large and small lignin-carbohydrate complexes and lower molecular weight polyphenol fractions (Fr. I). RESULTS: The anti-UV activity of SE was slightly less than that of sodium ascorbate, but higher than that of gallic acid, (-)-epigallocatechin gallate, chlorophyll a and chlorophyllin. The combination of SE and sodium ascorbate gave synergistic anti-UV activity. On gel filtration fractionation, the majority of the anti-UV activity was recovered from Fr. I, which was eluted as a single peak on high performance liquid chromatography (HPLC). CONCLUSION: The anti-UV activity of SE further suggests its potential as an alternative medicine.

Biological activity of luteolin glycosides and tricin from Sasa senanensis Rehder.

BACKGROUND: In contrast to the several reports of alkaline extracts (Sasa-health, SE), no study of flavonoids from the leaves of S. senanensis has been reported. Four flavonoids were isolated from this plant species and their biological activities were investigated. MATERIALS AND METHODS: Luteolin 6-C-ß-D-glucoside [1], luteolin 7-O-ß-D-glucoside [2], luteolin 6-C-α-L-arabinoside [3] and tricin [4] were extracted from the leaf of S. senanensis with methanol, partitioned with ethyl acetate, separated by Sephadex LH-20 and purified by high-performance liquid chromatography (HPLC). The structure was determined by ultraviolet (UV) spectra, high-resolution mass spectra (HR-MS) and nuclear magnetic resonance (NMR). RESULTS: The luteolin glycosides, 1-3 showed no cytotoxicity against the human normal oral cells and oral squamous cell carcinoma cell lines used up to 0.8 mg/ml, whereas 4 was highly cytotoxic. The luteolin glycosides 1-3 protected the cells from UV induced cytotoxicity, more efficiently than 4. The anti-HIV activity of 4 (Selectivity index, SI=27) was much higher than that of the luteolin glycosides (SI=2-7), but lower than that of SE (SI=40). The scavenging activity of 1-3 against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radicals was comparable with that of quercetin and, much higher than that of 4. CONCLUSION: The luteolin glycosides from S.senanensis show several new biological properties distinct from tricin and the anti-UV activity of the luteolin glycosides may be derived from their radical scavenging activity.

Quest for anti-inflammatory substances using IL-1ß-stimulated gingival fibroblasts.

BACKGROUND: We have previously reported that azulene-related compounds, and alkaline extract of Sasa senanensis Rehder potently inhibited nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophages. We investigated here whether they can inhibit pro-inflammatory cytokine production, by activated human gingival fibroblast (HGF). MATERIALS AND METHODS: HGF was established from the periodontal tissues of extracted tooth. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Production of Prostaglandin E(2) (PGE(2)) and cytokines was determined by enzyme immunoassay, and enzyme-linked immunosorbent assay, respectively. RESULTS: Interleukin (IL)-1ß did not inhibit, but rather slightly stimulated the growth of HGF cells. IL-1ß stimulated the production of PGE(2), IL-6, IL-8 and monocyte chemotactic protein-1 very potently, but not that of nitric oxide and tumor necrosis factor-α. Native LPS and synthetic lipid A from E. coli and P. gingivalis was much less stimulatory. Dexamethasone, not indomethacin, was an efficient inhibitor of IL-8 production. Among five azulene-related compounds, benzo[b]cyclohepta[e][1,4]thiazine most potently inhibited the IL-8 production by HGF cells, as well as NO production by activated RAW264.7 cells. The alkaline extract of Sasa senanensis Rehder significantly inhibited IL-8 production, without affecting the cell viability. CONCLUSION: The present system may be applicable for use in the search for anti-gingivitis substances.

Multiple biological complex of alkaline extract of the leaves of Sasa senanensis Rehder.

Previous studies have shown anti-inflammatory potential of alkaline extract of the leaves of Sasa senanensis Rehder (SE). The aim of the present study was to clarity the molecular entity of SE, using various fractionation methods. SE inhibited the production of nitric oxide (NO), but not tumour necrosis factor-α by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. Lignin carbohydrate complex prepared from SE inhibited the NO production to a comparable extent with SE, whereas chlorophyllin was more active. On successive extraction with organic solvents, nearly 90% of SE components, including chlorophyllin, were recovered from the aqueous layer. Anti-HIV activity of SE was comparable with that of lignin-carbohydrate complex, and much higher than that of chlorophyllin and n-butanol extract fractions. The CYP3A inhibitory activity of SE was significantly lower than that of grapefruit juice and chlorophyllin. Oral administration of SE slightly reduced the number of oral bacteria. When SE was applied to HPLC, nearly 70% of SE components were eluted as a single peak. These data suggest that multiple components of SE may be associated with each other in the native state or after extraction with alkaline solution.

Effect of Sasa senanensis Rehder extract on NO and PGE2 production by activated mouse macrophage-like RAW264.7 cells.

Alkaline extract of Sasa senanensis Rehder (SE) has shown diverse biological activity. As an extension, whether SE affects the function of activated macrophages was investigated. SE inhibited the nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells. Western blot and RT-PCR analyses demonstrated that this was due to the inhibition of inducible NO synthase (iNOS) expression at both protein and mRNA levels. ESR spectroscopy shows that SE dose-dependently scavenged the NO radical produced by NOC-7. In order to confirm the anti-inflammatory potency, possible effects on prostaglandin (PG) E(2) production and expression of enzymes involved in the arachidonic acid pathway were next investigated. It was found that SE effectively inhibited the PGE(2) production by LPS-stimulated RAW264.7 cells, although the extent of inhibition of PGE(2) was slightly less than that of NO production. SE inhibited cyclooxygenase (COX)-2 expression at both protein and mRNA levels, but to much lesser extents as compared with those for iNOS expression. SE contained much lower concentration of arginine, precursor of NO, as compared with the culture medium. These data suggest that SE exerts a weak anti-inflammatory activity.