Functional Evaluation of Sasa Makino et Shibata Leaf Extract as Group III OTC Drug

Kampo Medicines, classified as Group II, are usually available as hot water extracts of more than two different plant species. Recently, the presentation of the detailed compositional analysis by HPLC has become mandatory for the publication of the biological activity of Kampo Medicines. However, we often experience the loss of biological activity of Kampo medicines during the purification steps, thus making it difficult to assign the active princi‐ ples. Herb extracts are classified into Group II and Group III. Three major products of bam‐


Introduction
Over the counter (OTC) drugs in Japan are classified into three groups (I, II and III), based on the safety [1]. Group I drugs have the highest risk of exerting the adverse effects on our health. The intensity of such side effects declines in the order of Group I, II and III. Only Group III drugs with the least side effects can be purchased through the internet. Kampo Medicines, classified as Group II, are usually available as hot water extracts of more than two different plant species. Recently, the presentation of the detailed compositional analysis by HPLC has become mandatory for the publication of the biological activity of Kampo Medicines. However, we often experience the loss of biological activity of Kampo medicines during the purification steps, thus making it difficult to assign the active principles. Herb extracts are classified into Group II and Group III. Three major products of bam-Products B (Sunchlon ® , referred to as "BLE") is an alkaline extract of Sasa Makino et Shibata (dry weight 77.6 mg/ml [4]) that contains Cu (II)-chlorophyllin, but approximately 80% of lignin-carbohydrate complex (LCC) has been removed as precipitate [5].

Safety
Product C (Shojusen®, referred to as "KS) is a hot water extract of the leaves of Sasa krilensis Makino et Sibata (27.0 mg/ml), supplemented with ethanol extract of the leaves of Pinus densiflora Sieb et Zucc. (1.2 mg/ml), ethanol extract of the roots of Panax ginseng C.A. Meyer (0.92 mg/ml) and paraben as a preservative [6] ( Table 2).
Lignins are major class of natural products present in the natural kingdom, and are formed through phenolic oxidative coupling processes in the plant [29]. Lignins are formed by the dehydrogenative polymerization of three monolignols: p-coumaryl, p-coniferyl and sinapyl alcohols [29]. These monolignols were produced from L -phenylalanine by general phenyl-propanoid pathway [30]. Some polysaccharides in the cell walls of lignified plants are linked to lignin to form lignin-carbohydrate complexes (LCCs). Considering that both of SE and LCC are prepared by extraction with alkaline solution, it is not surprising that they display common biological activities with each other. Furthermore, we have recently identified the anti-UV substances of SE as p-coumaric acid derivative(s), one of lignin precursors [31]. Alkaline extraction step that is necessary for the preparation of SE provides higher amounts of LCC as compared with hot-water extracted Kampo medicines. One or two-order higher anti-HIV activity of both SE and LCC over tannins and flavonoids suggest their possible applicability towards virally-induced diseases.
However, there is a possibility that the components from SE and other plants are associated with each other, thus modify their biological activities. Also, SE components may inhibit the activity of CYP3A4, the most abundant drug-metabolizing enzyme, so as to increase the bioavailability of co-administered drugs (especially, CYP3A4 substrates). Lastly, the clinical evidences that demonstrate how the treatment of SE products improves the patient's conditions are limited.
Based on these circumstances, we review the functional analysis of SE products as alternative medicines, citing the literatures of other groups and ours, focusing on the following points: (i) component analysis, (ii) spectrum of reported biological activities in comparison with those of Kampo medicines, (iii) possibility of complex formation between the compo-

Component analysis
Components of SE are listed in Table 3 According to this information, we have fractionated the LCC into the following three fractions Fr I, II and III by repeated acid precipitation and solubization with NaHCO 3 or NaOH solution, and polysaccharide fraction was recovered as Fr. IV by addition of equal volume of ethanol in Figure 2.

Anti-bacterial activity
Product B (BLE) significantly reduced the bacterial growth and lactate production in vitro in the total saliva [9].
Product A (SE) showed a bacteriostatic, but not a bactericidal effect on Fusobacterium nucleatum and Prevotella intermedia ( Figure 7A, 7B). The MIC 50 for the Fusobacterium nucleatum and Prevotella intermedia was calculated to be 0.63 and 1.25%, respectively, and at the highest concentration (2.5%), 12.0 and 17.2% of the bacteria remained viable, respectively.
Gas chromatography demonstrated that these bacteria produced H 2 S and CH 3 SH, but not (CH 3 ) 2 H. SE more efficiently reduced the production of H 2 S in Fusobacterium nucleatum, with a 50% inhibitory concentration (IC 50 ) of 0.04% ( Figure 7C). On the other hand, SE more efficiently reduced the production of CH 3

Antitumor activity
Oral administration of SE (ad lib.) significantly delayed the development and growth of mammary tumors in a mammary tumor strain of virgin SHN mice [25]. Oral administration of SE (ad lib.) significantly inhibited spontaneous mammary tumorigenesis, reduced tumor multiplicity, inhibited the mammary duct branching, side bud development and angiogenesis in another mouse model of human breast cancer, transgenic FVB-Her2/NeuN mouse model [26].  (Table 5). Although SE did not show high tumor-specific cytotoxicity, it was highly cytotoxic to three human myelogenous leukemic cell lines (HL-60, ML-1, KG-1) and one T-cell leukemic cell line (MT-4). The type of cell death induced by SE remains to be investigated [2].
SE, Fr. 3 and Fr. 5 showed the surfactant action by reducing the surface tension. These substances did not significantly affect the phase-transtion temperature of dipalmitoyl phosphatidylcholine (DPPC)-liposome bilayer nor the membrane-fluidity. These data suggest that the membrane-stabilizing activity of SE may be generated by polysaccharide, lignin, or chlorophyll present in Fr. 3, 4 and 5.  We next compared the hepatocyte protective effect of SE, other Herbal extracts and tinctures (Aloe, Gambir, Swertiae, Plantaginis, Geranii, Houttuyniae extracts). Aloe extract rather enhanced the leakage of liver enzymes, whereas SE and Gambir extract were inhibitory. SE more significantly inhibited the enzyme leakage, as compared with other herbal extracts and tinctures, suggesting that the hepatocyte protective activity of SE may be more potent that other herbal extracts [13].
Single oral administration of SE (10~20 ml =0.6~1.2 mg/kg) slightly reduced the vasopermeability in ddY male mice (assessed by Whittle method). Single oral administration of SE (5 ml=0.3 mg/kg) slightly inhibited the formation of carrageenin-induced edema in SD male rats at 1 h, but rather enhanced the formation of edema at 3 h and thereafter ( Figure 9A). Single oral administration of SE (5 ml=0.3 mg/kg) inhibited the formation of formalin-induced edema at 3 h ( Figure 9B). Repeated oral administration of SE (1, 5, 10 ml/kg/day×7 or 9days) stimulated the growth of fibroblasts and neovascularization, in contrast to the enhanced formation of collagen fiber. This suggests that SE may stimulate the regeneration of normal tissue during the restoration process of inflammatory tissues [12].
Oral administration of SE slightly increased the phagocytic index (assessed by carbon clearance method) after 3~5 h, but did not affect the phagocytic index at 7 days, suggesting that SE does not reduce the function of reticuloendothelial system. SE also inhibited the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) from the LPS-activated mouse macrophage-like cells RAW264.7 via inhibition of the expression of iNOS and COX-2 at protein and mRNA levels [14].
Products A, B and C dose-dependently reduced the intensity of superoxide anion (O 2 -) (detected as DMPO-OOH) generated by hypoxanthine and xanthine oxidase reaction. The potency of O 2 -scavenging activity of the three products was comparable: product A (IC 50 =0.46 mg/ml), product B (IC 50 =0.52 mg/ml) and product C (IC 50 =0.54 mg/ml) ( Table 6) [4].
Products A, B and C dose-dependently reduced the intensity of hydroxyl radical ( OH) (detected as DMPO-OH) generated by the Fenton reaction. The potency of products A and C was comparable with each other (IC 50 =2.1 and 1.9 mg/ml, respectively), but 4-fold higher than that of product B (IC 50 =8.0 mg/ml) ( Table 6)

Anti-UV activity
UV irradiation (6 J/m 2 /min) for 1 min followed by 48 h culture resulted in extensive cell death (closed circles in Figure 11). Popular antioxidants, N-acetyl-L-cysteine (NAC) and catalase (enzyme that degrades hydrogen peroxide), could not prevent the UV-induced cellular damage, suggesting that hydrogen peroxide may not be involved in the UV-induced cytotoxicity, but the type of radical species produced by UV irradiation remains to be identified (Exp. 1, Table 7). SE dose-dependently inhibited the UV-induced cytotoxicity in a bell-shaped fashion ( Figure 11). The viability of the cells was recovered to 50% by the addition of 0.53 mg/ml SE (=EC 50   SE (product A) showed higher anti-UV activity (SI=20) than other Sasa senanensis Rehder leaf products B (SI=4) (that has lower amounts of LCC) and C (SI=13) (that contains ginseng extract and pine (Pinus densiflora) leaf extract) [4], suggesting the importance of LCC for the anti-UV activity. A granulated powder of Sasa senanensis Rehder leaf extract (SE-10) (SI=129) showed approximately three-fold higher anti-UV activity than SE (Exp. 2, Table 7) [19], suggesting some synergistic effect of SE and other components present in SE-10.

Synergistic action with vitamin C
Vitamin C exhibited either antioxidant or prooxidant activity, depending on the concentration [46]. We have reported that ascorbate derivatives that produced the doublet signal of ascorbate radical (sodium-L -ascorbate, L -ascorbic acid, D -isoascorbic acid, 6-β-D -galactosyl-Lascorbate, sodium 5,6-benzylidene-L -ascorbate) induced apoptosis (characterized by internucleosomal DNA fragmentation and an increase in the intracellular Ca 2+ concentration) in HL-60 cells. On the other hand, ascorbate derivatives that did not produce radicals ( L -ascorbic acid-2-phosphate magnesium salt, L -ascorbic acid 2-sulfate and dehydroascorbic acid) did not induce apoptosis [47,48]. This suggests the possible involvement of the ascorbate radical in apoptosis-induction by ascorbic acid-related compounds.
We accidentally found that LCCs from the pine cone of Pinus parviflola Sieb et Zucc, pine cone of Pinus elliottii var. Elliotti, leaf of Ceriops decandra (Griff.) Ding Hou and, thorn apple of Crataegu Cuneata Sieb. et Zucc modulated the radical intensity of ascorbate bi-phasically, depending on the concentrations. At higher concentration, LCCs strongly enhanced the radical intensity of sodium ascorbate, which rapidly decayed, possibly due to the breakdown of ascorbic acid or to the consumption of ascorbyl radical. LCCs, not only from pine cones (Fr. VI), but also from Catuaba bark, pine seed shell, A. nikoense Maxim. and C. Cuneata Sieb. et Zucc. enhanced the radical intensity and cytotoxic activity of sodium ascorbate [27]. On the other hand, tannins such as gallic acid, EGCG, and tannic acid counteracted the radical intensity and cytotoxic activity of sodium ascorbate [49].
Sodium ascorbate rapidly reduced the oxygen concentration in the culture medium, possibly due to oxygen consumption via its pro-oxidation action. Simultaneous addition of LCCs further enhanced the ascorbate-stimulated consumption of oxygen [50]. These data suggest that the synergistic enhancement of the cytotoxic activity of LCCs and ascorbate might be due at least in part to the stimulated induction of hypoxia.
Similarly, SE and vitamin C synergistically enhanced the activity that scavenging superoxide anion radical (determined by the intensity of DMPO-OOH) and hydroxyl radical (determined by the intensity of DMPO-OH radical) (

Inhibition of CYP3A4 activity
CYP3A4 activity was measured by β-hydroxylation of testosterone in human recombinant CYP3A4. Products A, B and C dose-dependently inhibited the β-hydroxylation of testosterone, generally used for the assay of CYP3A4 activity. Product C exhibited the highest CYP3A4-inhibitory activity (IC 50 =58 μg/ml), followed by product B (IC 50 =124 μg/ml) and then product A (IC 50 =403 μg/ml). Product B inhibited the CYP3A4 to an extent similar to that attained by Cu (II)-chlorophyllin; product A inhibited CYP3A4 to lower extent than that achieved by grapefruit juice (Figure 12) [4]. SE-10 and SE dose-dependently inhibited the β-hydroxylation of testosterone, generally used for the assay of CYP3A4 activity. SE-10 (IC 50 = 0.516 μg SE equivalent/ml) had an approximately 16% lower CYP3A4-inhibitory activity (IC 50 = 0.445 μg/ml) [19].Combined with our recent report [4], CYP3A4 inhibitory activity increases in the following order (from lower to higher): SE-10 < SE < products B and C. SE-10 and SE seem likely to be safer drugs as compared with products B and C, since the latter are expected to enhance the side-effects of CYP3A4-metabolizable drugs more potently.

Possibility of complex formation between the components
Solvent fractionation of SE demonstrated that the majority of chlorophyllin and the activity that inhibited the NO production by macrophages were recovered from the water layer that contains majority of compounds (more than 81%) [18]. This suggests the possibility that chlorophyllin in SE may be associated with hydrophilic substances, especially LCC in the native state or after extraction with alkaline solution, since the preparative method of SE is the same with that of LCC. This was supported by the observation that LCC isolated from SE has greenish color (absorption peak = 655 nm), characteristic to chlorophyllin (absorption peak = 629 nm), expected to contain 1.7-2.6% chlorophyllin in the molecule, and that 68.5% of SE eluted as a single peak at the retention time of 22.175 min in HPLC [18]. Upon binding to chlorophyllin, LCC may obtain the activity of inhibiting the NO production by activated macrophages.

Clinical application for the treatment of oral diseases
Oral intake of product B (BLE) slightly but significantly reduced the gingival crevicular fluid (determined by Periometer ® ), and tended to reduce gingival index in the experimentally induced gingivitis patients [9].
Lichen planus is a chronic mucocutaneous disease that affects the skin, tongue, and oral mucosa. The most common presentation of oral lichen planus is the reticular form that manifests as white lacy streaks on the mucosa (known as Wickham's striae) or as smaller papules (small raised areas). The cause of lichen planus is not known. Some lichen planus-type rashes occur as allergic reactions to medications and a complication of chronic hepatitis C virus infection [51]. Hepatitis C virus has been reported to occasionally replicate in oral lichen tissue and contribute to mucosal damage [52,53]. It has been reported that the Epstein-Barr virus is more frequently detected in oral lesions such as oral lichen planus and oral squamous cell carcinoma in comparison with healthy oral epithelium [54].
Potent antiviral, antibacterial, and anti-inflammatory activity of SE prompted us to investigate whether SE is effective on oral lichenoid dysplasia and osteoclastogenesis. A male patient with white lacy streaks in the oral mucosa was orally administered SE three times a day for ten months. Long-term treatment cycle of SE progressively reduced both the area of white steaks ( Figure 13) and the base-line levels of salivary interleukin-6 and 8 ( Figure 14) [55]. IL-8 concentration after SE treatment was below the initial level throughout the experimental period. This was accompanied by the improvement of patient's symptoms. Before the SE treatment, the patient felt that the mucosa is uneven, rough and cut by touching with his tongue. Three weeks after the treatment, such feeling reduced and the mucosa became much smooth. At four weeks, the rough mucosa was narrowed into smaller area, and the patient could eat without pungent feeling on the oral mucosa. Oral intake of SE also improved the patient's symptom of pollen allergy, and loose teeth, giving an impression that the oral mucosa became much tighter. SE significantly inhibited the RANKL-induced differentiation of mouse macrophage-like RAW264.7 cells towards osteoclasts (evaluated by TRAP-positive multinuclear cell formation). These pilot clinical study suggests the therapeutic potentiality of SE against oral diseases [55].

Conclusion
SE (Sasa Health ® ), alkaline extract of Sasa senanensis Rehder extract has shown diverse biological activities including membrane stabilizing, anti-leukemia, anti-inflammatory, radical scavenging, anti-UV, bacteriostatic, antiviral, anti-stomatitis, and anti-lichen planus activity Alternative Medicine 190 ( Figure 15). Among these biological activities, antiviral, anti-UV and synergism with vitamin C are unique properties to SE as well as LCC ( Figure 15). Figure 13. Time-dependent effect of SE on the oral lichenoid dysplasia. One 51 years old male patient with lichenoid dysplasia was treated for 0, 1 or 10 months with 13.3 ml of 50% diluted SE (containing 33 mg dried material/ml) at each meal, 3 times a day. Intraoral photographs in the right side (upper panel) and left side (lower panel) of buccal mucosa were taken. It should be noted that the SE treatment progressively reduced the area of the white streaks (a → d → g, b → e → h in the right side of buccal mucosa and c → f → i in the left side of buccal mucosa). Cited from [55] with permission.

Alternative Medicine
SE as well as LCCs, which are efficiently extracted with alkaline solution, showed higher anti-HIV and anti-UV activity, as compared with hot water extract of many plant species including Kampo medicines ( Figure 16). Antitumor activity of polysaccharide fractions of pine cone extracts against ascites tumor cells transplanted in mice also increased with acidity (binding strength to DEAE-cellulose column) [56], suggesting the potency of alkaline extract against certain types of diseases. We have reported broad antiviral spectrum of LCC ranging from HIV [57][58][59], influenza virus [60][61][62], herpes simplex virus [63][64][65]. Oral administration of LCC from pine cone extract significantly improved the symptom of HSV-infected patients [66,67], and lichenoid dysplasia patient [55]. These data suggest the possible application of SE to virally-induced diseases. Considering to low absorption through the intestinal tract [68], the application through the mucosa membrane is recommended. We are now studying the interaction between SE, antibacterial agent and charcoal to optimize the therapeutic potential of SE for the main component of toothpaste.