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

Hiroshi Sakagami, Tomohiko Matsuta, Toshikazu Yasui, Oguchi Katsuji, Madoka Kitajima, Tomoko Sugiura, Hiroshi Oizumi and Takaaki Oizumi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52187

**1. 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.


**Table 1.** Classification of OTC drugs in Japan, based on the safety.

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‐

© 2012 Sakagami et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

boo leaf extract (products A, B, C) are classified into Group III (Table 2), and other drugs are classified into Group I.


**Table 2.** Three major products of Bamboo leaf extract available in the drug store in Japan.

Two bamboos, "Take" and "Sasa" (Japanese names) belong to grasses, but are not strictly distinguished each other botanically. There are 70 genera of bamboos in the world and 14 genera (approximately 600 species) in Japan. Sasa culms are 1-2 m high, 5-8 mm in diameter, robust, ramose at lower portions. Leaf-blades are oblong-lanceolate, 20-25 cm long and 4-5 cm broad (Figure 1A, B). They are distributed into Saghalien, the Kuriles, Hokkaido, Hon‐ shu, Shikoku and Kyushu in Japan. Product A (Sasa Health®, referred to as "SE") (Figure 1C) is a pure alkaline extract of the leaves of *Sasa senanensis* Rehder (dry weight: 58.8 mg/ml [2-4]) that contains Fe (II)-chlorophyllin, in which Mg (II) is replaced by Fe (II) by adding FeCl2. SE-10 (Figure 1D) is a granulated powder of SE supplemented with lactose, lactitol, trehalose and tea extract, and sold as dried and packaged powder in drug stores.

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].

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]. Al‐ kaline 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 an‐ ti-HIV activity of both SE and LCC over tannins and flavonoids suggest their possible ap‐

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

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173

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 condi‐

Based on these circumstances, we review the functional analysis of SE products as alterna‐ tive 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‐

plicability towards virally-induced diseases.

<sup>A</sup> <sup>B</sup><sup>B</sup>

C D

**Figure 1.** The primeval forest of *Sasa senanensis* Rehder (A), its leaves (B), SE (C) and SE-10 (D).

tions are limited.

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 den‐ siflora* 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).

These bamboo leaf products is recognized as being effective in treating various malaises in‐ cluding fatigue, low appetite, halitosis, body odor and stomatitis [7-10]. However, there is no scientific evidence that demonstrates their efficacy due to the lack of appropriate bio‐ markers, although their *in vitro* antiseptic [11], membrane stabilizing [12], anti-inflammatory [13-16], phagocytic [17], radical scavenging [2, 4, 18, 19], anti-oxidant [20-23], antibacterial [2, 9], anti-viral [2, 4, 18, 19, 24] and antitumor activities [2, 25, 26] have been reported. SE showed several common biological properties with LCCs, that is, the prominent anti-HIV, anti-UV and synergistic activity with vitamin C [27, 28].

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‐

**Figure 1.** The primeval forest of *Sasa senanensis* Rehder (A), its leaves (B), SE (C) and SE-10 (D).

boo leaf extract (products A, B, C) are classified into Group III (Table 2), and other drugs are

**Three major products of bamboo leaf extract** Product A (=SE) Fe (II)-chlorophyllin Pure *Sasa senanensis* Rehder extract Product B Cu (II)-chlorophyllin LCC was removed

Product C Cu (II)-chlorophyllin Supplemented with ginseng and pine (*Pinus*

Two bamboos, "Take" and "Sasa" (Japanese names) belong to grasses, but are not strictly distinguished each other botanically. There are 70 genera of bamboos in the world and 14 genera (approximately 600 species) in Japan. Sasa culms are 1-2 m high, 5-8 mm in diameter, robust, ramose at lower portions. Leaf-blades are oblong-lanceolate, 20-25 cm long and 4-5 cm broad (Figure 1A, B). They are distributed into Saghalien, the Kuriles, Hokkaido, Hon‐ shu, Shikoku and Kyushu in Japan. Product A (Sasa Health®, referred to as "SE") (Figure 1C) is a pure alkaline extract of the leaves of *Sasa senanensis* Rehder (dry weight: 58.8 mg/ml [2-4]) that contains Fe (II)-chlorophyllin, in which Mg (II) is replaced by Fe (II) by adding FeCl2. SE-10 (Figure 1D) is a granulated powder of SE supplemented with lactose, lactitol,

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

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 den‐ siflora* Sieb et Zucc. (1.2 mg/ml), ethanol extract of the roots of *Panax ginseng* C.A. Meyer

These bamboo leaf products is recognized as being effective in treating various malaises in‐ cluding fatigue, low appetite, halitosis, body odor and stomatitis [7-10]. However, there is no scientific evidence that demonstrates their efficacy due to the lack of appropriate bio‐ markers, although their *in vitro* antiseptic [11], membrane stabilizing [12], anti-inflammatory [13-16], phagocytic [17], radical scavenging [2, 4, 18, 19], anti-oxidant [20-23], antibacterial [2, 9], anti-viral [2, 4, 18, 19, 24] and antitumor activities [2, 25, 26] have been reported. SE showed several common biological properties with LCCs, that is, the prominent anti-HIV,

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‐

trehalose and tea extract, and sold as dried and packaged powder in drug stores.

lignin-carbohydrate complex (LCC) has been removed as precipitate [5].

(0.92 mg/ml) and paraben as a preservative [6] (Table 2).

anti-UV and synergistic activity with vitamin C [27, 28].

**Table 2.** Three major products of Bamboo leaf extract available in the drug store in Japan.

*densiflora*) leaf extracts.

classified into Group I.

172 Alternative Medicine

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]. Al‐ kaline 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 an‐ ti-HIV activity of both SE and LCC over tannins and flavonoids suggest their possible ap‐ plicability 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 condi‐ tions are limited.

Based on these circumstances, we review the functional analysis of SE products as alterna‐ tive 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‐ nents, (iv) inhibition of CYP3A4 activity and (v) the clinical application for the treatment of oral diseases.

solution, and polysaccharide fraction was recovered as Fr. IV by addition of equal volume of

**Fr Fr. III** 

ppt

), HR-MS *m/z*: 449.0976 (calcd. for C21H21O11,

), HR-MS *m/z*: 419.1027 (calcd. for C20H19O10,

): HR-MS *m/z*: 331.0837 (Calcd. for C17H15O7,

1%NaOH

sup

http://dx.doi.org/10.5772/52187

175

ppt Fr. IV sup

), high-resolution mass spectra

extracted 25 mL of 1.39 % NaHCO3 or 1%NaOH

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

SE 50 mL 2.91 g (100 %) 10000 rpm,10 min,15

10000 rpm,10 min,15

pH 8.47

pH 5 AcOH

sup

ppt

10000 rpm,10 min,15

pH 6.31

**Figure 2.** Fractionation of lignin-carbohydrate complex (LCC) fractions Fr I-III and polysaccharide fraction Fr IV. *Yield of*

Luteolin glycosdes are isolated from the leaves of *Sasa senanensis* Rehder and their structures were identified as decribed below (Figure 3) [32]. Luteolin 6-*C*-β-D-glucoside [compound 1]: yellow amorphous powder, [α]25D +30.7˚ (*c*=0.12, CH3OH), mp 232˚ (dec.), ultraviolet (UV) λmax (MeOH) nm (ε): 348 (22,200), 270 (17,600) and 258 (17,400). Electrospray ionization

Luteolin 7-*O*-β-D-glucoside [compound 2]:yellow amorphous powder, [α]25D -81.1˚ (*c*=0.10, CH3OH), mp 261˚ (dec.), UV λmax (MeOH) nm (ε): 346 (20,500) and 270 (18,400). ESI-TOF-

Luteolin 6-*C*-α-L-arabinoside [compound 3]:yellow amorphous powder, [α]25D +66.0˚ (*c*=0.11, CH3OH), mp > 300˚ (dec.), UV λmax (MeOH) nm (ε): 348 (22,100), 270 (17,600) and 258

Tricin [compound 4]: yellow amorphous powder, UVλmax (MeOH) nm (ε): 349 (41,000) and

10000 rpm,10 min,15

AcOH pH 5

*Frs. I and II represents mean±S.D. from three independent experiments*. Cited from [18], with permission

sup

ppt

**0.59± 0.24 g (20.2%)**

time of flight mass spectra (ESI-TOF-MS) *m/z*: 448 ([M+H]+

), 287 ([aglycon+H]+

(HR-MS) *m/z*: 449.1094 (calcd. for C21H21O11, 449.1084).

(17,400). ESI-TOF-MS *m/z*: 419 ([M+H]+

269 (27,200). ESI-TOF-MS *m/z*: 331 ([M+H]+

ethanol in Figure 2.

**Fr** 

MS *m/z*: 448 ([M+H]+

449.1084).

419.0978).

331.0818).

**0.60± 0.28 g ( 20.7 %)**

## **2. Component analysis**

Components of SE are listed in Table 3. Dietary fibre was the major component of SE. Watersoluble and water-insoluble dietary fibres are present approximately at the 1: 2 ratio.


**Table 3.** Composition of SE. \*corrected, assuming that 1 ml contains 66.1 mg SE. Cited from [19], with permission.

According to this information, we have fractionated the LCC into the following three frac‐ tions Fr I, II and III by repeated acid precipitation and solubization with NaHCO3 or NaOH solution, and polysaccharide fraction was recovered as Fr. IV by addition of equal volume of ethanol in Figure 2.

nents, (iv) inhibition of CYP3A4 activity and (v) the clinical application for the treatment of

Components of SE are listed in Table 3. Dietary fibre was the major component of SE. Water-

Lipid 200 3030 Proline 84 1270 Ash content 900 13600 Glutamic acid 186 2800 Sugar 1200 18200 Serine 21 320 Glucose 90 1360 Threonine 13 200 Arabinose 380 5700 Aspartic acid 159 2400 Xylose 1060 16000 Tryptophan 28 420

Dietary fibre 2100 31800 Folic acid 0.008 0.12 Water-soluble 1400 21200 Lutein 0.3 4.5

Arginine 19 290 Iron 1.02 15 Lysine 59 890 Calcium 1.0 15 Histidine 23 350 Potassium 4.9 74 Phenylalanine 86 1300 Magnesium 0.5 8 Tyrosine 63 950 Zinc 0.08 1.2

Isoleucine 53 800 Vitamin A 0.003 0.05 Methionine 32 480 β-Carotene 0.032 0.5 Valine 95 1440 Vitamin K1 0.006 0.09 Alanine 105 1590 Glycine 99 1500

**Table 3.** Composition of SE. \*corrected, assuming that 1 ml contains 66.1 mg SE. Cited from [19], with permission.

According to this information, we have fractionated the LCC into the following three frac‐ tions Fr I, II and III by repeated acid precipitation and solubization with NaHCO3 or NaOH

**mg/100 ml mg/100 g\* mg/100 ml mg/100 g\***

Sodium 395 5980

soluble and water-insoluble dietary fibres are present approximately at the 1: 2 ratio.

Protein 1500 22700 Glycine

Galactose 180 2700

Water-insoluble 700 10600

Leucine 135 2040

oral diseases.

174 Alternative Medicine

**2. Component analysis**

**Figure 2.** Fractionation of lignin-carbohydrate complex (LCC) fractions Fr I-III and polysaccharide fraction Fr IV. *Yield of Frs. I and II represents mean±S.D. from three independent experiments*. Cited from [18], with permission

Luteolin glycosdes are isolated from the leaves of *Sasa senanensis* Rehder and their structures were identified as decribed below (Figure 3) [32]. Luteolin 6-*C*-β-D-glucoside [compound 1]: yellow amorphous powder, [α]25D +30.7˚ (*c*=0.12, CH3OH), mp 232˚ (dec.), ultraviolet (UV) λmax (MeOH) nm (ε): 348 (22,200), 270 (17,600) and 258 (17,400). Electrospray ionization time of flight mass spectra (ESI-TOF-MS) *m/z*: 448 ([M+H]+ ), high-resolution mass spectra (HR-MS) *m/z*: 449.1094 (calcd. for C21H21O11, 449.1084).

Luteolin 7-*O*-β-D-glucoside [compound 2]:yellow amorphous powder, [α]25D -81.1˚ (*c*=0.10, CH3OH), mp 261˚ (dec.), UV λmax (MeOH) nm (ε): 346 (20,500) and 270 (18,400). ESI-TOF-MS *m/z*: 448 ([M+H]+ ), 287 ([aglycon+H]+ ), HR-MS *m/z*: 449.0976 (calcd. for C21H21O11, 449.1084).

Luteolin 6-*C*-α-L-arabinoside [compound 3]:yellow amorphous powder, [α]25D +66.0˚ (*c*=0.11, CH3OH), mp > 300˚ (dec.), UV λmax (MeOH) nm (ε): 348 (22,100), 270 (17,600) and 258 (17,400). ESI-TOF-MS *m/z*: 419 ([M+H]+ ), HR-MS *m/z*: 419.1027 (calcd. for C20H19O10, 419.0978).

Tricin [compound 4]: yellow amorphous powder, UVλmax (MeOH) nm (ε): 349 (41,000) and 269 (27,200). ESI-TOF-MS *m/z*: 331 ([M+H]+ ): HR-MS *m/z*: 331.0837 (Calcd. for C17H15O7, 331.0818).

19

25

28

Running Title <sup>5</sup>

**3. Biological activities**

tracts [43] (Exp. 6, Table 4).

**3.2. Anti-bacterial activity**

virus activity [24].

the total saliva [9].

Anti-human immunodeficiency virus (HIV) activity was assessed quantitatively by a selec‐ tivity index (SI=CC50/EC50, where CC50 is the 50% cytotoxic concentration against mock-in‐ fected MT-4 cells, and EC50 is the 50% effective concentration against HIV-infected cells). Products A, B and C all effectively and dose-dependently reduced the cytopathic effect of HIV infection (closed symbols in Figure 5), although their anti-HIV activity was much lower than that of positive controls [dextran sulfate (SI=1378), curdlan sulfate (SI=5606), azidothy‐ midine (SI=17746), 2',3'-dideoxycytidine (SI=5123)] (Table 4). The potency of anti-HIV activi‐ ty was in the order of product A (Sasa-Health®, SE) (SI=607) > product C (SI=117) > product B (SI=111) (Exp. I, Table 4) [4]. A granulated powder of *Sasa senanensis* Rehder leaf extract (SE-10) (Figure 1D) (SI=54) showed slightly higher anti-HIV activity than SE (SI=45) (Exp. 2, Table 4) [19]. Among the components of SE, LCC fractions prepared as described in Figure 3 (SI=37~62) showed comparable or slightly higher activity anti-HIV activity than unfractio‐ nated SE (SI=36) (Exp. 3, Table 4) [28]. Luteolin glycosides, luteolin 6-*C*-β-D-glucoside, luteo‐ lin 7-*O*-β-D-glucoside, luteolin 6-*C*-α-L-arabinoside and tricin from *Sasa senanensis* Rehder leaf extract showed somewhat lower anti-HIV activity (SI=2~24) (Exp. 4, Table 4) [32]. The anti-HIV activity of LCCs isolated from SE was comparable with that of LCCs from pine cone, catuaba bark [33], cacao husk [34], cacao mass [35], cultured extract of *Lentinus edodes* mycelia extract [36] and mulberry juice [37, 38], and synthetic lignin (dehydrogenation poly‐ mers of phenylpropanoids) [39], and was generally higher than that of tannins [40], flavo‐ noids [41], gallic acid, (-)-epigallocatechin 3-*O*-gallate (EGCG), curcumin, and chemically modified glucans [42] (Exp. 5, Table 4) and Kampo medicines and its constituent plant ex‐

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177

SE also protected the MDCK cells from the cytopathic effect of influenza virus infection (CC50=0.67%, EC50=0.060%, SI=11) (Figure 6). Tricin showed potent anti-human cytomegalo‐

Product B (BLE) significantly reduced the bacterial growth and lactate production *in vitro* in

Product A (SE) showed a bacteriostatic, but not a bactericidal effect on *Fusobacterium nuclea‐ tum* and *Prevotella intermedia* (Figure 7A, 7B). The MIC50 for the *Fusobacterium nucleatum* and *Prevotella intermedia* was calculated to be 0.63 and 1.25%, respectively, and at the highest

Gas chromatography demonstrated that these bacteria produced H2S and CH3SH, but not (CH3)2H. SE more efficiently reduced the production of H2S in *Fusobacterium nucleatum*, with a 50% inhibitory concentration (IC50) of 0.04% (Figure 7C). On the other hand, SE more effi‐ ciently reduced the production of CH3SH in *Prevotella intermedia*, with an IC50 of 0.16% (Fig‐ ure 7D). A higher concentration of SE (2.5%) completely eliminated both H2S and CH3SH [2].

concentration (2.5%), 12.0 and 17.2% of the bacteria remained viable, respectively.

**3.1. Antiviral activity**

16 Figure 3. Purification of luteolin 6-*C*-β-D-glucoside [compound **1**], luteolin 7-*O*-β-D-glucoside 17 [compound **2**], luteolin 6-*C*-α-L-arabinoside [compound **3**] and tricin [compound **4**] from the leaves 18 of *Sasa senanensis* Rehder. Cited from [32], with permission. **Figure 3.** Purification of luteolin 6-*C*-β-D-glucoside [compound 1], luteolin 7-*O*-β-D-glucoside [compound 2], luteolin 6-*C*-α-L-arabinoside [compound 3] and tricin [compound 4] from the leaves of *Sasa senanensis* Rehder. Cited from [32], with permission.

20 We also isolated substances (SEE-1) that protected the cells from the UV-induced cytotoxicity, by 21 ethanol extraction, Wakosil 40C18 chromatography (H2O elution) and preparative HPLC (Shimadzu 22 LC-10AD pump, Shimadzu SPD-M10AVP photodiode array detector, separation column: Inatsil 23 ODS-3, eluted with H2O : acetonitrile : formic acid (90:10:0.1), and proposed the putative structures 24 as *p*-coumaric acid derivative(s) (Figure 4) [31]. We also isolated substances (SEE-1) that protected the cells from the UV-induced cytotoxici‐ ty, by ethanol extraction, Wakosil 40C18 chromatography (H2O elution) and preparative HPLC (Shimadzu LC-10AD pump, Shimadzu SPD-M10AVP photodiode array detector, sep‐ aration column: Inatsil ODS-3, eluted with H2O : acetonitrile : formic acid (90:10:0.1), and proposed the putative structures as *p*-coumaric acid derivative(s) (Figure 4) [31].

A B

26 Figure 4. Identification of anti-UV substance(s) as *p*-coumaric acid derivative(s).Cited from [31] 27 with permission. **Figure 4.** Identification of anti-UV substance(s) as *p*-coumaric acid derivative(s). Cited from [31] with permission.

## **3. Biological activities**

#### **3.1. Antiviral activity**

Running Title <sup>5</sup>

HO HO

HO HO HOH2C O

OH OH

> OH OH

Luteolin 6-*C*-β-D-glucoside [**1**] Luteolin 7-*O*-β-D-glucoside [**2**]

Luteolin 6-*C*-α-L-arabinoside [**3**] Tricin [**4**]

HO OH

HO

O

OH OH

O

OCH3

OH OCH3

OH

O

O

OH

HO

O OH

O

O

O

OH

OH

HO

O HOH2C

OH

O

O OH

16 Figure 3. Purification of luteolin 6-*C*-β-D-glucoside [compound **1**], luteolin 7-*O*-β-D-glucoside 17 [compound **2**], luteolin 6-*C*-α-L-arabinoside [compound **3**] and tricin [compound **4**] from the leaves

**Figure 3.** Purification of luteolin 6-*C*-β-D-glucoside [compound 1], luteolin 7-*O*-β-D-glucoside [compound 2], luteolin 6-*C*-α-L-arabinoside [compound 3] and tricin [compound 4] from the leaves of *Sasa senanensis* Rehder. Cited from [32],

We also isolated substances (SEE-1) that protected the cells from the UV-induced cytotoxici‐ ty, by ethanol extraction, Wakosil 40C18 chromatography (H2O elution) and preparative HPLC (Shimadzu LC-10AD pump, Shimadzu SPD-M10AVP photodiode array detector, sep‐ aration column: Inatsil ODS-3, eluted with H2O : acetonitrile : formic acid (90:10:0.1), and

Compound **4**

We also isolated substances (SEE-1) that protected the cells from the UV-induced cytotoxicity, by ethanol extraction, Wakosil 40C18 chromatography (H2O elution) and preparative HPLC (Shimadzu LC-10AD pump, Shimadzu SPD-M10AVP photodiode array detector, separation column: Inatsil ODS-3, eluted with H2O : acetonitrile : formic acid (90:10:0.1), and proposed the putative structures

proposed the putative structures as *p*-coumaric acid derivative(s) (Figure 4) [31].

H

H

**1 2 4 5**

H

**7**

**<sup>8</sup> <sup>9</sup>**

HO R2

26 Figure 4. Identification of anti-UV substance(s) as *p*-coumaric acid derivative(s).Cited from [31]

**Figure 4.** Identification of anti-UV substance(s) as *p*-coumaric acid derivative(s). Cited from [31] with permission.

R3

H

O R1

18 of *Sasa senanensis* Rehder. Cited from [32], with permission.

Compound **1** Compound **2** Compound **3**

elute with 60% MeOH

Sephadex LH-20

Fr. 1 - 5 Fr. 6 Fr. 7 Fr. 8 HPLC HPLC

Leaves of *S. senanensis* MeOH extract

Water portion EtOAc portion

24 as *p*-coumaric acid derivative(s) (Figure 4) [31].

A B

13C <sup>1</sup> H

2,6 131.0 7.44 (d, *J*=8.6Hz) 3,5 116.8 6.80 (d, *J*=8.6Hz)

C D

7 146.2 7.58 (d, *J*=15.5Hz) 8 115.8 6.29 (br.d, *J*=14.7Hz)

1 127.4

4 161.0

9 159.5

19

with permission.

176 Alternative Medicine

25

28

27 with permission.

Anti-human immunodeficiency virus (HIV) activity was assessed quantitatively by a selec‐ tivity index (SI=CC50/EC50, where CC50 is the 50% cytotoxic concentration against mock-in‐ fected MT-4 cells, and EC50 is the 50% effective concentration against HIV-infected cells). Products A, B and C all effectively and dose-dependently reduced the cytopathic effect of HIV infection (closed symbols in Figure 5), although their anti-HIV activity was much lower than that of positive controls [dextran sulfate (SI=1378), curdlan sulfate (SI=5606), azidothy‐ midine (SI=17746), 2',3'-dideoxycytidine (SI=5123)] (Table 4). The potency of anti-HIV activi‐ ty was in the order of product A (Sasa-Health®, SE) (SI=607) > product C (SI=117) > product B (SI=111) (Exp. I, Table 4) [4]. A granulated powder of *Sasa senanensis* Rehder leaf extract (SE-10) (Figure 1D) (SI=54) showed slightly higher anti-HIV activity than SE (SI=45) (Exp. 2, Table 4) [19]. Among the components of SE, LCC fractions prepared as described in Figure 3 (SI=37~62) showed comparable or slightly higher activity anti-HIV activity than unfractio‐ nated SE (SI=36) (Exp. 3, Table 4) [28]. Luteolin glycosides, luteolin 6-*C*-β-D-glucoside, luteo‐ lin 7-*O*-β-D-glucoside, luteolin 6-*C*-α-L-arabinoside and tricin from *Sasa senanensis* Rehder leaf extract showed somewhat lower anti-HIV activity (SI=2~24) (Exp. 4, Table 4) [32]. The anti-HIV activity of LCCs isolated from SE was comparable with that of LCCs from pine cone, catuaba bark [33], cacao husk [34], cacao mass [35], cultured extract of *Lentinus edodes* mycelia extract [36] and mulberry juice [37, 38], and synthetic lignin (dehydrogenation poly‐ mers of phenylpropanoids) [39], and was generally higher than that of tannins [40], flavo‐ noids [41], gallic acid, (-)-epigallocatechin 3-*O*-gallate (EGCG), curcumin, and chemically modified glucans [42] (Exp. 5, Table 4) and Kampo medicines and its constituent plant ex‐ tracts [43] (Exp. 6, Table 4).

SE also protected the MDCK cells from the cytopathic effect of influenza virus infection (CC50=0.67%, EC50=0.060%, SI=11) (Figure 6). Tricin showed potent anti-human cytomegalo‐ virus activity [24].

#### **3.2. 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 nuclea‐ tum* and *Prevotella intermedia* (Figure 7A, 7B). The MIC50 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 H2S and CH3SH, but not (CH3)2H. SE more efficiently reduced the production of H2S in *Fusobacterium nucleatum*, with a 50% inhibitory concentration (IC50) of 0.04% (Figure 7C). On the other hand, SE more effi‐ ciently reduced the production of CH3SH in *Prevotella intermedia*, with an IC50 of 0.16% (Fig‐ ure 7D). A higher concentration of SE (2.5%) completely eliminated both H2S and CH3SH [2].


Cited from [4], with permission.

Viable cell number (%)

HIV+ HIV-

Product **B**

Product **C**

Product **A**

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=117

Concentration (%)

**Figure 5.** Figure 1. Anti-HIV activity of three commercial products of *Sasa senanensis* Rehder extract. HIV-1IIIB-infected (HIV+) and mock-infected (HIV–) MT-4 cells were incubated for 5 days with the indicated concentrations of products A (upper panel), B (center panel) and C (lower panel) and the viable cell number was determined by the MTT assay and expressed as a percentage that of the control. Data represent the mean±standard deviation from triplicate assays. It should be noted that product C exhibited a weak cytostatic effect at lower concentrations (indicated by dotted circle).

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=111

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=607

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**Table 4.** Anti-HIV activity of polyphenols.

**S I SI**

Hydrolyzable tannins dimer (n=39) <1

Curcumin <1

**Exp. 1 (Alkaline extract**) **Exp. 5 (other plant extracts)**

SE 45

178 Alternative Medicine

LCC Fr II (acid precipitation ×2)) 58

**Table 4.** Anti-HIV activity of polyphenols.

SE 40

Product A (SE) 607 LCC from pine trees (n=2) 27 Product B 111 LCC from pine seed shell 12 Product C 117 LCC from catuaba bark 43 Dextran sulfate 1378 LCC from cacao husk 311 Curdlan sulfate 5606 LCC from cacao mass 46 AZT 17746 LCC from cultured LEM 94 ddC 5123 LCC from mulberry juice 7

**Exp. 2 (SE product)** Phenylpropenoid polymers (n=23) 105

Exp. 3 (SE component) Hydrolyzable tannins trimer (n=4) 3

LCC Fr I (acid precipitation) 37 Condensed tannins (n=8) <1

LCC Fr III (acid precipitation × 2) 62 Flavonoids (n=160) <1 Polysaccharide fraction Fr IV ><1 Gallic acid <1

Butanol extract <1 (-)-Epigallocatechin 3-*O*-gallate <1

Exp. 4 (SE component) Chlorophyllin 5

Luteolin 7-*O*-β-D-glucoside [**2**] 7 Kampo medicines (n=10) <1.0 Luteolin 6-*C*-α-L-arabinoside [**3**] >7 Constituent plant extracts (n=25) 1.3

Tricin [**4**] 24 AZT 17850

Luteolin 6-*C*-β-D-glucoside [**1**] >2 **Exp. 6 (Plant extracts)**

SE 36 Hydrolyzable tannins tetramer (n=3) 11

SE-10 54 Neutral polysaccharide from pine cone 1 Dextran sulfate 160 *N*,*N*-dimethylaminoethyl paramylon <1 Curdlan sulfate 781 *N*,*N*-diethylaminoethyl paramylon <1 AZT 6931 *N*,*N*-dimethylaminoethyl curdlan <1 ddC 905 Hydrolyzable tannins monomer (n=21) <1

**Figure 5.** Figure 1. Anti-HIV activity of three commercial products of *Sasa senanensis* Rehder extract. HIV-1IIIB-infected (HIV+) and mock-infected (HIV–) MT-4 cells were incubated for 5 days with the indicated concentrations of products A (upper panel), B (center panel) and C (lower panel) and the viable cell number was determined by the MTT assay and expressed as a percentage that of the control. Data represent the mean±standard deviation from triplicate assays. It should be noted that product C exhibited a weak cytostatic effect at lower concentrations (indicated by dotted circle). Cited from [4], with permission.

38

<sup>33</sup>from [4], with permission. <sup>34</sup>

Viable cell number (%)

HIV+ HIV-

Product **B**

Product **C**

Product **A**

37 activity [24].

42 with permission.

1 2 3

Running Title <sup>7</sup>

Figure 5. Figure 1. Anti-HIV activity of three commercial products of *Sasa senanensis* Rehder extract. HIV-1IIIB-infected (HIV+) and mock-infected (HIV–) MT-4 cells were incubated for 5 days with the indicated concentrations of products **A** (upper panel), **B** (center panel) and **C** (lower panel) and the viable cell number was determined by the MTT assay and expressed as a percentage that of the control. Data represent the mean±standard deviation from triplicate assays. It should be noted that product **C** exhibited a weak cytostatic effect at lower concentrations (indicated by dotted circle). Cited

SI=117

0 0.00064 0.0032 0.016 0.08 0.4 2 10

Concentration (%)

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=111

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=607

36 (CC50=0.67%, EC50=0.060%, SI=11) (Figure 6). Tricin showed potent anti-human cytomegalovirus

**3.3. Antitumor activity**

model [26].

to be investigated [2].

**3.4. Membrane stabilizing activity**

the GTP release (Figure 8B) [13].

chlorophyll present in Fr. 3, 4 and 5.

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 angiogene‐ sis in another mouse model of human breast cancer, transgenic FVB-Her2/NeuN mouse

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SE showed slightly higher cytotoxicity against the human squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, NA) (mean CC50=6.22%, 3.62 mg/mL) and the human glio‐ blastoma cell lines (T98G, U-87MG) (mean CC50=5.43%, 3.16 mg/mL), as compared with the human oral normal cells [gingival fibroblast (HGF), pulp cell (HPC), periodontal ligament fibroblast (HPLF)] (mean CC50=6.90%, 4.01 mg/mL), and was more cytotoxic to the human myelogenous leukemic cell lines (HL-60, ML-1, KG-1) (CC50=1.18%, 0.68 mg/mL) and the hu‐ man T-cell leukemia cell line (MT-4) (CC50=1.41%, 0.82 mg/mL), with an approximate tumor specificity index of 1.62 (Table 5). Although SE did not show high tumor-specific cytotoxici‐ ty, 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

In order to investigate whether SE contains membrane -stabilizing activity, SE was defatted with hexane, and fractionated on Silica gel chromatography, according to the polarity, into Fr. 1 (eluted with *n*-hexane: CH2Cl2), Fr. 2 (CH2Cl2), Fr. 3 (acetone), Frs 4 and 5 (methanol) and Fr. 6 (residue). SE inhibited the hemolysis of rat red blood cells in hypotinic buffer by 13%. Frs. 3, 4 and 5 ihibited the homolysis approximately 35, 20 and 35%, respectively [12],

Membrane stability can be evaluated by the extracellular leakage of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transamiase (GPT) from the hepatocytes. Control hepatocytes released 85.1± 5.4 (mean±SD) K.U./ml GOT into the culture medium. SE (1~5 μi=60~300 μg/ml) significantly inhibited the release of GOT. Among the SE fractions, Frs. 1, 4 and 6 showed the inhibitory effects (Figure 8A). Control hepatocytes released 37.0 ± 3.6K.U./ml GPT inoto the culture medium. SE (1~2 μl=60, 120 μg/ml) significantly inhibited

SE, Fr. 3 and Fr. 5 showed the surfactant action by reducing the surface tension. These sub‐ stances did not significantly affect the phase-transtion temperature of dipalmitoyl phospha‐ tidylcholine (DPPC)-liposome bilayer nor the membrane-fluidity. These data suggest that the membrane-stabilizing activity of SE may be generated by polysaccharide, lignin, or

suggesting the membrane-stabilization activity of SE and its fractions.

39 Figure 6. Anti-influenza virus activity of SE. Influenza virus-infected or mock-infected MDCK cells 40 were incubated for 3 days with the indicated concentrations of SE, and the viable cell number was 41 determined by MTT method. Each value represents a mean from triplicate assays. Cited from [2] **Figure 6.** Anti-influenza virus activity of SE. Influenza virus-infected or mock-infected MDCK cells were incubated for 3 days with the indicated concentrations of SE, and the viable cell number was determined by MTT method. Each value represents a mean from triplicate assays. Cited from [2] with permission.

**Figure 7.** Antibacterial activity of SE. *Fusobacterium nucleatum* (A, C) and *Prevotella intermedia* (B, D) were cultured anaerobically for 24 hours at 37oC with the indicated concentrations of SE in capped 15-cm centrifugation tubes. The VSC released into the culture medium (black bar: H2S, white bar, CH3SH) was quantified by gas chromatography (C, D). Bacterial growth was measured by recording the absorbance at 620 nm, using a microplate reader (A, B). (A, B) Bacter‐ iostatic activity of SE. Each point represents mean ±S.D. from triplicate assays. (C, D) Effect on VSC. Each bar represents mean ±S.D. from triplicate assays. Without bar (0.02 and 0.04%SE (C), and 0.08 and 0.16% SE (D)) means the value from a single assay. Cited from [2] with permission.

#### **3.3. Antitumor activity**

Running Title <sup>7</sup>

Figure 5. Figure 1. Anti-HIV activity of three commercial products of *Sasa senanensis* Rehder extract. HIV-1IIIB-infected (HIV+) and mock-infected (HIV–) MT-4 cells were incubated for 5 days with the indicated concentrations of products **A** (upper panel), **B** (center panel) and **C** (lower panel) and the viable cell number was determined by the MTT assay and expressed as a percentage that of the control. Data represent the mean±standard deviation from triplicate assays. It should be noted that product **C** exhibited a weak cytostatic effect at lower concentrations (indicated by dotted circle). Cited

SI=117

0 0.00064 0.0032 0.016 0.08 0.4 2 10

Concentration (%)

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=111

0 0.00064 0.0032 0.016 0.08 0.4 2 10

SI=607

35 SE also protected the MDCK cells from the cytopathic effect of influenza virus infection 36 (CC50=0.67%, EC50=0.060%, SI=11) (Figure 6). Tricin showed potent anti-human cytomegalovirus

0.00 0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.50

D

0 0.08 0.16 0.31 0.63 1.25 2.5

A B

0 0.005 0.01 0.02 0.039 0.078 0.16 0.31 0.63 SE (%)

> 0.0 0.2 0.4 0.6 0.8 1.0

150

SE (%)

**Figure 7.** Antibacterial activity of SE. *Fusobacterium nucleatum* (A, C) and *Prevotella intermedia* (B, D) were cultured anaerobically for 24 hours at 37oC with the indicated concentrations of SE in capped 15-cm centrifugation tubes. The VSC released into the culture medium (black bar: H2S, white bar, CH3SH) was quantified by gas chromatography (C, D). Bacterial growth was measured by recording the absorbance at 620 nm, using a microplate reader (A, B). (A, B) Bacter‐ iostatic activity of SE. Each point represents mean ±S.D. from triplicate assays. (C, D) Effect on VSC. Each bar represents mean ±S.D. from triplicate assays. Without bar (0.02 and 0.04%SE (C), and 0.08 and 0.16% SE (D)) means the value

0

50

100

C

**Figure 6.** Anti-influenza virus activity of SE. Influenza virus-infected or mock-infected MDCK cells were incubated for 3 days with the indicated concentrations of SE, and the viable cell number was determined by MTT method. Each value

*Fusobacterium nucleatum Prevotella intermedia*

<sup>33</sup>from [4], with permission. <sup>34</sup>

Viable cell number (%)

HIV+ HIV-

Product **B**

Product **C**

Product **A**

Infected Non-infected

37 activity [24].

0

42 with permission.

represents a mean from triplicate assays. Cited from [2] with permission.

0.00 0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.50

0 0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5

from a single assay. Cited from [2] with permission.

20

40

60

Viable cell number (%)

38

180 Alternative Medicine

0.0 0.1 0.2 0.3 0.4 0.5

150

Absorbance at 620 nm

VSC (%)

0

50

100

80

100

120

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 angiogene‐ sis in another mouse model of human breast cancer, transgenic FVB-Her2/NeuN mouse model [26].

39 Figure 6. Anti-influenza virus activity of SE. Influenza virus-infected or mock-infected MDCK cells 40 were incubated for 3 days with the indicated concentrations of SE, and the viable cell number was 41 determined by MTT method. Each value represents a mean from triplicate assays. Cited from [2] SE showed slightly higher cytotoxicity against the human squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, NA) (mean CC50=6.22%, 3.62 mg/mL) and the human glio‐ blastoma cell lines (T98G, U-87MG) (mean CC50=5.43%, 3.16 mg/mL), as compared with the human oral normal cells [gingival fibroblast (HGF), pulp cell (HPC), periodontal ligament fibroblast (HPLF)] (mean CC50=6.90%, 4.01 mg/mL), and was more cytotoxic to the human myelogenous leukemic cell lines (HL-60, ML-1, KG-1) (CC50=1.18%, 0.68 mg/mL) and the hu‐ man T-cell leukemia cell line (MT-4) (CC50=1.41%, 0.82 mg/mL), with an approximate tumor specificity index of 1.62 (Table 5). Although SE did not show high tumor-specific cytotoxici‐ ty, 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].

#### **3.4. Membrane stabilizing activity**

In order to investigate whether SE contains membrane -stabilizing activity, SE was defatted with hexane, and fractionated on Silica gel chromatography, according to the polarity, into Fr. 1 (eluted with *n*-hexane: CH2Cl2), Fr. 2 (CH2Cl2), Fr. 3 (acetone), Frs 4 and 5 (methanol) and Fr. 6 (residue). SE inhibited the hemolysis of rat red blood cells in hypotinic buffer by 13%. Frs. 3, 4 and 5 ihibited the homolysis approximately 35, 20 and 35%, respectively [12], suggesting the membrane-stabilization activity of SE and its fractions.

Membrane stability can be evaluated by the extracellular leakage of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transamiase (GPT) from the hepatocytes. Control hepatocytes released 85.1± 5.4 (mean±SD) K.U./ml GOT into the culture medium. SE (1~5 μi=60~300 μg/ml) significantly inhibited the release of GOT. Among the SE fractions, Frs. 1, 4 and 6 showed the inhibitory effects (Figure 8A). Control hepatocytes released 37.0 ± 3.6K.U./ml GPT inoto the culture medium. SE (1~2 μl=60, 120 μg/ml) significantly inhibited the GTP release (Figure 8B) [13].

SE, Fr. 3 and Fr. 5 showed the surfactant action by reducing the surface tension. These sub‐ stances did not significantly affect the phase-transtion temperature of dipalmitoyl phospha‐ tidylcholine (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.


**Figure 8.** Effect of SE and each fraction on the leakage of GOT (A) and GPT (B) from rat cultured hepatocytes. Hepato‐ cytes were suspended in Williams' E medium for 24 h at 37ºC and culture was continued in the same medium with SE or each fraction for 24 h at 37ºC. Each value was mean±S.D. of five experiments, and expressed as Karmen Units/ml of medium. Values are significant: ∗*p*<0.05, ∗∗*p*<0.01, ∗∗∗*p*<0.001; control *vs* experiments (students *t*-test). Cited

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We next compared the hepatocyte protective effect of SE, other Herbal extracts and tinctures (Aloe, Gambir, Swertiae, Plantaginis, Geranii, Houttuyniae extracts). Aloe extract rather en‐ hanced 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

Oral administration of hot water extract of leaves of bamboo of genus *Sasa* spp (HSBE) inhibited the carrageenan-induced edema and 12-*O*-tetradecanoylphorbol-13-acetate-in‐ duced ear swelling in mice, possibly by inhibiting the production of proinflammatory substances [prostaglandin E2 (PGE2), serotonin) and expression of 5-lipoxygenase, cycoox‐ ygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10. Al‐ though the anti-inflammatory activity of HSBE was much less than that of dexamethasone, the major activity was concentrated into lower molecular weight, dialyz‐

Single oral administration of SE (10~20 ml =0.6~1.2 mg/kg) slightly reduced the vasopermea‐ bility 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-in‐ duced 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 en‐ hanced formation of collagen fiber. This suggests that SE may stimulate the regeneration of

normal tissue during the restoration process of inflammatory tissues [12].

from [13] with permission.

other herbal extracts [13].

**3.5. Anti-inflammatory activity**

able and methanol eluted fraction [16].

The tumor-specificity index (TS) was measured by the following equation: TS= [CC50 (HGF) + CC50 (HPC) + CC50 (HPLF)] / [CC50 (HSC-2) + CC50 (HSC-3) + CC50 (HSC-4) + CC50 (Ca9-22) + CC50 (NA) + CC50 (T98G) + CC50 (U87MG) + CC50 (HL-60) + CC50 (ML-1) + CC50 (KG-1) + CC50 (MT-4)] x (11/3). Cited from [2] with permission.

**Table 5.** Cytotoxic activity of SE against human normal and tumor cells.

**Figure 8.** Effect of SE and each fraction on the leakage of GOT (A) and GPT (B) from rat cultured hepatocytes. Hepato‐ cytes were suspended in Williams' E medium for 24 h at 37ºC and culture was continued in the same medium with SE or each fraction for 24 h at 37ºC. Each value was mean±S.D. of five experiments, and expressed as Karmen Units/ml of medium. Values are significant: ∗*p*<0.05, ∗∗*p*<0.01, ∗∗∗*p*<0.001; control *vs* experiments (students *t*-test). Cited from [13] with permission.

We next compared the hepatocyte protective effect of SE, other Herbal extracts and tinctures (Aloe, Gambir, Swertiae, Plantaginis, Geranii, Houttuyniae extracts). Aloe extract rather en‐ hanced 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].

#### **3.5. Anti-inflammatory activity**

**CC50** % (v/v) mg/mL

Human normal cells

182 Alternative Medicine

Human oral squamous cell carcinoma cell lines

Human glioblastoma cell lines

Human myelogenous leukemia cell lines

Human T-cell leukemia cell line

CC50 (ML-1) + CC50 (KG-1) + CC50 (MT-4)] x (11/3). Cited from [2] with permission.

**Table 5.** Cytotoxic activity of SE against human normal and tumor cells.

Gingival fibroblast (HGF) 6.96 4.05

Periodontal ligament fibroblast (HPLF) 6.19 3.60

Pulp cell (HPC) 7.54 4.38

(mean) 6.90 4.01

HSC-2 8.49 4.94 HSC-3 5.99 3.49 HSC-4 5.69 3.31 Ca9-22 4.20 2.44 NA 6.71 3.91 (mean) 6.22 3.62

T98G 6.92 4.03 U87MG 3.94 2.29 (mean) 5.43 3.16

HL-60 1.14 0.66 ML-1 0.39 0.23 KG-1 2.00 1.16 (mean) 1.18 0.68

MT-4 1.41 0.82

TS value 1.62

The tumor-specificity index (TS) was measured by the following equation: TS= [CC50 (HGF) + CC50 (HPC) + CC50 (HPLF)] / [CC50 (HSC-2) + CC50 (HSC-3) + CC50 (HSC-4) + CC50 (Ca9-22) + CC50 (NA) + CC50 (T98G) + CC50 (U87MG) + CC50 (HL-60) +

Oral administration of hot water extract of leaves of bamboo of genus *Sasa* spp (HSBE) inhibited the carrageenan-induced edema and 12-*O*-tetradecanoylphorbol-13-acetate-in‐ duced ear swelling in mice, possibly by inhibiting the production of proinflammatory substances [prostaglandin E2 (PGE2), serotonin) and expression of 5-lipoxygenase, cycoox‐ ygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10. Al‐ though the anti-inflammatory activity of HSBE was much less than that of dexamethasone, the major activity was concentrated into lower molecular weight, dialyz‐ able and methanol eluted fraction [16].

Single oral administration of SE (10~20 ml =0.6~1.2 mg/kg) slightly reduced the vasopermea‐ bility 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-in‐ duced 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 en‐ hanced 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 clear‐ ance 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.

was comparable with each other (IC50=2.1 and 1.9 mg/ml, respectively), but 4-fold higher

\*

\*

SE (g/ml)

**Figure 10.** SE inhibited the IL-1β-stimulated IL-8 production by HGF cells. HGF cells were incubated for 48 h with the indicated concentrations of SE in the presence of IL-1β (1 ng/ml), and the extracellular IL-8 concentration (A) and via‐

Product **A** 0.69% (0.46 mg/ml) 3.2% (2.1 mg/ml) Product **B** 0.67% (0.52 mg/ml) 10.3% (8.0 mg/ml) Product **C** 1.9% (0.54 mg/ml) 6.6% (1.9 mg/ml)

**Table 6.** Radical scavenging activity of three commercial products of *Sasa senanensis* Rehder extract. Data was cited

ble cell number (B) were determined. mean± S.D (n=3). \*<0.01. Cited from [15] with permission.

0 5 50 100 200 400 800 1164

**–Scavenging activity (IC50) ·OH–Scavenging activity (IC50)**

0 5 50 100 200 400 800 1164

\* \* <sup>A</sup>

\*

\*

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than that of product B (IC50=8.0 mg/ml) (Table 6) [4].

IL-8 (

0

0.5

1

1.5

2

2.5

Viable cell number (% of control)

from [2], with permission.

0

B

**O2 –**

40

80

120

160

n

g/ml)

**Figure 9.** Effect of SE on carrageenin (A) and formalin (B)-induced hind paw edema in rats.Each point represents the mean of eight rats with S.D. ●---● : control, ●―――● : SE 1ml/kg, ■―――■ : SE 5 ml/kg,▲―――▲ : SE 10 ml/kg,. ∗,∗∗ : significantly different from the control value with *p*<0.05, *p*<0.01 (Student's t-test). Cited from [12] with per‐ mission.

SE also inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) 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].

IL-1β induced one to two order higher production of proinflammatory substances (PGE2, IL-6, IL-8, MCP-1), but not NO and TNFα by human gingival fibroblast (HGF). SE also in‐ hibited the production of IL-8 production by IL-1β-stimulated human gingival fibroblast (Figure 10)[15].

#### **3.6. Radical scavenging activity**

ESR spectroscopy showed that SE (50%=29.1 mg/mL) did not produce any detectable ESR signal at pH 7.4 (radical intensity (RI)<0.089) and pH 10.0 (RI<0.11). At pH 13.0, a weak broad peak, similar to that of typical lignin [28], appeared (RI=0.14) [2].

Products A, B and C dose-dependently reduced the intensity of superoxide anion (O2 – ) (de‐ tected as DMPO-OOH) generated by hypoxanthine and xanthine oxidase reaction. The po‐ tency of O2 – scavenging activity of the three products was comparable: product A (IC50=0.46 mg/ml), product B (IC50=0.52 mg/ml) and product C (IC50=0.54 mg/ml) (Table 6) [4].

Products A, B and C dose-dependently reduced the intensity of hydroxyl radical ( OH) (de‐ tected as DMPO-OH) generated by the Fenton reaction. The potency of products A and C was comparable with each other (IC50=2.1 and 1.9 mg/ml, respectively), but 4-fold higher than that of product B (IC50=8.0 mg/ml) (Table 6) [4].

Oral administration of SE slightly increased the phagocytic index (assessed by carbon clear‐ ance method) after 3~5 h, but did not affect the phagocytic index at 7 days, suggesting that

**Figure 9.** Effect of SE on carrageenin (A) and formalin (B)-induced hind paw edema in rats.Each point represents the mean of eight rats with S.D. ●---● : control, ●―――● : SE 1ml/kg, ■―――■ : SE 5 ml/kg,▲―――▲ : SE 10 ml/kg,. ∗,∗∗ : significantly different from the control value with *p*<0.05, *p*<0.01 (Student's t-test). Cited from [12] with per‐

SE also inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) from the LPS-activated mouse macrophage-like cells RAW264.7 *via* inhibition of the expression of

IL-1β induced one to two order higher production of proinflammatory substances (PGE2, IL-6, IL-8, MCP-1), but not NO and TNFα by human gingival fibroblast (HGF). SE also in‐ hibited the production of IL-8 production by IL-1β-stimulated human gingival fibroblast

ESR spectroscopy showed that SE (50%=29.1 mg/mL) did not produce any detectable ESR signal at pH 7.4 (radical intensity (RI)<0.089) and pH 10.0 (RI<0.11). At pH 13.0, a weak

tected as DMPO-OOH) generated by hypoxanthine and xanthine oxidase reaction. The po‐

Products A, B and C dose-dependently reduced the intensity of hydroxyl radical ( OH) (de‐ tected as DMPO-OH) generated by the Fenton reaction. The potency of products A and C

scavenging activity of the three products was comparable: product A (IC50=0.46

– ) (de‐

Products A, B and C dose-dependently reduced the intensity of superoxide anion (O2

mg/ml), product B (IC50=0.52 mg/ml) and product C (IC50=0.54 mg/ml) (Table 6) [4].

broad peak, similar to that of typical lignin [28], appeared (RI=0.14) [2].

SE does not reduce the function of reticuloendothelial system.

iNOS and COX-2 at protein and mRNA levels [14].

mission.

184 Alternative Medicine

(Figure 10)[15].

tency of O2

–

**3.6. Radical scavenging activity**

A B

**Figure 10.** SE inhibited the IL-1β-stimulated IL-8 production by HGF cells. HGF cells were incubated for 48 h with the indicated concentrations of SE in the presence of IL-1β (1 ng/ml), and the extracellular IL-8 concentration (A) and via‐ ble cell number (B) were determined. mean± S.D (n=3). \*<0.01. Cited from [15] with permission.


**Table 6.** Radical scavenging activity of three commercial products of *Sasa senanensis* Rehder extract. Data was cited from [2], with permission.

#### **3.7. Anti-UV activity**

UV irradiation (6 J/m2 /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-in‐ duced 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 cytotoxici‐ ty 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 (=EC50). From the dose-response curve without UV irradia‐ tion, CC50 of SE was calculated to be 22.24 mg/ml. From these values, selectivity index SI (CC50/EC50) was calculated to be 41.96. Similar experiments were repeated three times to yield the mean value of SI=19.7±15.1 (mean of four independent experiments) (Exp. I, Ta‐ ble 7). The ant-UV activity of SE was slightly less than that of sodium ascorbate (SI=30.2±13.4) (mean of five independent experiments), but higher than that of luteolin 6- *C*-β-D-glucoside [1] (SI>8), luteolin 7-*O*-β-D-glucoside [2] (SI>6), luteolin 6-*C*-α-L-arabino‐ side [3] (SI>6), Tricin [4] (TS>3), gallic acid (SI=17.1), EGCG (SI=7.7), chlorophyllin (SI=0.53) and chlorophyll a (SI<0.24) (Exp. I, Table 7) [3].

LCCs from pine cones, pine seed and cultured LEM (SI=26-42) showed comparable anti-UV activity with SE (SI=39). Lignin precursor, vanillin, showed higher anti-UV activity compa‐ rable with that of sodium ascorbate (SI=64) (Exp. 3, Table 7) [44, 45]. On the other hand, chemically-modified glucans, such as *N*,*N*-dimethylaminoethyllaminarin, *N*,*N*-dimethylami‐ noethylpullulan, *N*,*N*-dimethylaminoethyldextran and paramylon sulfate (SI<1) [45] (Exp. 4, Table 7), hot water extract (Kampo medicines and constituent plant extracts) [43] (SI=1~2) (Exp. 5, Table 7) and tea extracts (green tea, black tea, oolong tea, burley tea, Jasmine tea) [44] (Exp. 6, Table 7) were also inactive (SI=1~2). These data suggests the alkaline extracts (such as SE and LCCs) show higher anti-UV activity than hot-water extracts (such as Kampo

Exp. 1 Exp. 3 (LCCs)

Sodium ascorbate 30 Exp. 4 (polysaccharides)

Catalase <1 Exp. 5 (Plant extracts)

Product B (BLE) 4 Exp. 6 (Tea extract)

Exp. 2 (SE products) Constituent plant extracts (n=25) 1

Product C (KS) 13 Green tea 3 Sodium acorbate 33 Black tea <1

SE 39 Burley tea <1 SE-10 129 Jasmine tea <1 Sodium ascorbate 90 Sodium ascorbate 30

*N*-Acetyl-L-cysteine <1

Product A (SE) 20

**Table 7.** Anti-UV activity of various natural products.

SE 20 LCC from pine cones (n=3) 33

Tricin [**4**] >3 Vanilline 64 Chlorophyllin <1 Sulfated lignin (n=2) >8 Chlorophyl a <1 Sodium acorbate 64

Gallic acid 17 *N*,*N*-Dimethylaminoethyllaminarin <1 EGCG 8 *N*,*N*-Dimethylaminoethylpullulan <1 Curcumin <1 *N*,*N*-Dimethylaminoethyldextran <1 Ar-turmerone <1 Paramylon sulfate <1

Luteolin 6-*C*-β-D-glucoside [**1**] >8 LCC from pine seed 26 Luteolin 7-*O*-β-D-glucoside [**2**] >6 LCC from cultured LEM 42 Luteolin 6-*C*-α-L-arabinoside [**3**] >6 SE 39

**S I SI**

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

http://dx.doi.org/10.5772/52187

187

Sodium ascorbate 89

Kampo medicines (n=10) 2

Oolong tea <1

medicines, tea extracts).

**Figure 11.** Anti-UV activity of SE. HSC-2 cells were treated without (○) or with UV (●) irradiation (6 J/m<sup>2</sup>/min) for 1 min in PBS(–) containing SE. Viable cell number determined by MTT method 48 h after irradiation, and expressed as percent of control (without UV irradiation). EC50: 50% effective concentration, CC50: 50% cytotoxic concentra‐ tion.Mean ± S.D. of triplicate determinations. Cited from [3] with permission.

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 ex‐ tract 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], sug‐ gesting some synergistic effect of SE and other components present in SE-10.

LCCs from pine cones, pine seed and cultured LEM (SI=26-42) showed comparable anti-UV activity with SE (SI=39). Lignin precursor, vanillin, showed higher anti-UV activity compa‐ rable with that of sodium ascorbate (SI=64) (Exp. 3, Table 7) [44, 45]. On the other hand, chemically-modified glucans, such as *N*,*N*-dimethylaminoethyllaminarin, *N*,*N*-dimethylami‐ noethylpullulan, *N*,*N*-dimethylaminoethyldextran and paramylon sulfate (SI<1) [45] (Exp. 4, Table 7), hot water extract (Kampo medicines and constituent plant extracts) [43] (SI=1~2) (Exp. 5, Table 7) and tea extracts (green tea, black tea, oolong tea, burley tea, Jasmine tea) [44] (Exp. 6, Table 7) were also inactive (SI=1~2). These data suggests the alkaline extracts (such as SE and LCCs) show higher anti-UV activity than hot-water extracts (such as Kampo medicines, tea extracts).


**Table 7.** Anti-UV activity of various natural products.

**3.7. Anti-UV activity**

186 Alternative Medicine

UV irradiation (6 J/m2

/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-in‐ duced 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 cytotoxici‐ ty 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 (=EC50). From the dose-response curve without UV irradia‐ tion, CC50 of SE was calculated to be 22.24 mg/ml. From these values, selectivity index SI (CC50/EC50) was calculated to be 41.96. Similar experiments were repeated three times to yield the mean value of SI=19.7±15.1 (mean of four independent experiments) (Exp. I, Ta‐ ble 7). The ant-UV activity of SE was slightly less than that of sodium ascorbate (SI=30.2±13.4) (mean of five independent experiments), but higher than that of luteolin 6- *C*-β-D-glucoside [1] (SI>8), luteolin 7-*O*-β-D-glucoside [2] (SI>6), luteolin 6-*C*-α-L-arabino‐ side [3] (SI>6), Tricin [4] (TS>3), gallic acid (SI=17.1), EGCG (SI=7.7), chlorophyllin

0 0.024 0.048 0.098 0.2 0.39 0.78 1.56 3.13 6.25 12.5 25 50

**Figure 11.** Anti-UV activity of SE. HSC-2 cells were treated without (○) or with UV (●) irradiation (6 J/m<sup>2</sup>/min) for 1 min in PBS(–) containing SE. Viable cell number determined by MTT method 48 h after irradiation, and expressed as percent of control (without UV irradiation). EC50: 50% effective concentration, CC50: 50% cytotoxic concentra‐

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 ex‐ tract 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], sug‐

gesting some synergistic effect of SE and other components present in SE-10.

SE (mg/ml)

EC50=0.53

**SI (CC50/EC50) =41.96**

CC50=22.24

(SI=0.53) and chlorophyll a (SI<0.24) (Exp. I, Table 7) [3].

tion.Mean ± S.D. of triplicate determinations. Cited from [3] with permission.

0

25

50

75

Viable cell number (% of control)

100

125

#### **3.8. Synergistic action with vitamin C**

Vitamin C exhibited either antioxidant or prooxidant activity, depending on the concentra‐ tion [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 internu‐ cleosomal DNA fragmentation and an increase in the intracellular Ca2+ concentration) in HL-60 cells. On the other hand, ascorbate derivatives that did not produce radicals (L-ascor‐ bic 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.

**3.9. Inhibition of CYP3A4 activity**

23

25

14 to the stimulated induction of hypoxia.

22 intensity of DMPO-OH radical) (Table 6) [2].

achieved by grapefruit juice (Figure 12) [4].

tive to the control (0%). Cited from [4] with permission.

CYP3A4-metabolizable drugs more potently.

**3.10. Possibility of complex formation between the components**

Residual activity (% of control)

26 **(i) 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 testoster‐ one, generally used for the assay of CYP3A4 activity. Product C exhibited the highest CYP3A4–inhibitory activity (IC50=58 μg/ml), followed by product B (IC50=124 μg/ml) and then product A (IC50=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

\*

\*

**Figure 12.** CYP3A4 inhibitory activity of products **A**, **B** and **C**. One millilitre of products **A**, **B** and **C** was freeze dried to produce the powder (66.1, 77.6 and 28.5 mg, respectively).Each value represents the mean ± S.D. (n=3). \*p<0.05 rela‐

SE-10 and SE dose-dependently inhibited the β-hydroxylation of testosterone, generally used for the assay of CYP3A4 activity. SE-10 (IC50= 0.516 μg SE equivalent/ml) had an ap‐ proximately 16% lower CYP3A4-inhibitory activity (IC50= 0.445 μg/ml) [19].Combined with our recent report [4], CYP3A4 inhibitory activity increases in the following order (from low‐ er 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

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

\*

(% of control)

*decandra* (Griff.) Ding Hou. and cacao husk scavenged O2

19 enhanced the radical scavenging activity of sodium ascorbate [27, 34].

DMPO-OOH radical intensity

10 M vitamin C 72.4 109.1

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

14 Book Title

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

15 Lower concentration of LCC (pine cone Fr. VI) and sodium ascorbate showed radical



20 Similarly, SE and vitamin C synergistically enhanced the activity that scavenging superoxide 21 anion radical (determined by the intensity of DMPO-OOH) and hydroxyl radical (determined by the

0.5% VC 0.25% + 5 M VC 1% VC 0.5% + 5 M VC

http://dx.doi.org/10.5772/52187

SE 48.3 47.1 < 60.4 [(48.3+72.4)/2] 65.4 75.5 < 87.3 [(65.4+109.1)/2]

24 Table 6. Synergistic radical scavenging activity of SE and vitamin C. Cited from [2] with permission.

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 (IC50=58 g/ml), followed by product **B** (IC50=124 g/ml) and then product **A** (IC50=403 g/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].

44 Figure 12. CYP3A4 inhibitory activity of products **A**, **B** and **C**. One millilitre of products **A**, **B** and **C** 45 was freeze dried to produce the powder (66.1, 77.6 and 28.5 mg, respectively).Each value represents

\*

● Product **A** □ Product **B** Δ Product **C**

46 the mean ± S.D. (n=3). \*p<0.05 relative to the control (0%). Cited from [4] with permission.

0 0.01 0.1 0.5 1 2

Concentration (%)

\*

DMPO-OH radical intensity

189

(% of control)

scavenging activity. LCC further stimulated the superoxide anion (O2

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 rad‐ ical 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 in‐ tensity and cytotoxic activity of sodium ascorbate [49].

Sodium ascorbate rapidly reduced the oxygen concentration in the culture medium, possi‐ bly 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.

Lower concentration of LCC (pine cone Fr. VI) and sodium ascorbate showed radical scav‐ enging activity. LCC further stimulated the superoxide anion (O2 - ) and 1,1-diphenyl-2-pic‐ rylhydrazyl (DPPH) radical scavenging activity of sodium ascorbate. LCCs from *Ceriops decandra* (Griff.) Ding Hou. and cacao husk scavenged O2 - and hydroxyl radical, and synerg‐ istically enhanced the radical scavenging activity of sodium ascorbate [27, 34].

Similarly, SE and vitamin C synergistically enhanced the activity that scavenging superox‐ ide anion radical (determined by the intensity of DMPO-OOH) and hydroxyl radical (deter‐ mined by the intensity of DMPO-OH radical) (Table 8) [2].


**Table 8.** Synergistic radical scavenging activity of SE and vitamin C. Cited from [2] with permission.

32 product **A** inhibited CYP3A4 to lower extent than that achieved by grapefruit juice (Figure 12) [4].

14 Book Title

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

15 Lower concentration of LCC (pine cone Fr. VI) and sodium ascorbate showed radical



20 Similarly, SE and vitamin C synergistically enhanced the activity that scavenging superoxide 21 anion radical (determined by the intensity of DMPO-OOH) and hydroxyl radical (determined by the

DMPO-OH radical intensity

(% of control)

scavenging activity. LCC further stimulated the superoxide anion (O2

#### **3.9. Inhibition of CYP3A4 activity** 24 Table 6. Synergistic radical scavenging activity of SE and vitamin C. Cited from [2] with permission. 25

23

14 to the stimulated induction of hypoxia.

22 intensity of DMPO-OH radical) (Table 6) [2].

**3.8. Synergistic action with vitamin C**

188 Alternative Medicine

Vitamin C exhibited either antioxidant or prooxidant activity, depending on the concentra‐ tion [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 internu‐ cleosomal DNA fragmentation and an increase in the intracellular Ca2+ concentration) in HL-60 cells. On the other hand, ascorbate derivatives that did not produce radicals (L-ascor‐ bic 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

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 rad‐ ical 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 in‐

Sodium ascorbate rapidly reduced the oxygen concentration in the culture medium, possi‐ bly 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

Lower concentration of LCC (pine cone Fr. VI) and sodium ascorbate showed radical scav‐

rylhydrazyl (DPPH) radical scavenging activity of sodium ascorbate. LCCs from *Ceriops*

Similarly, SE and vitamin C synergistically enhanced the activity that scavenging superox‐ ide anion radical (determined by the intensity of DMPO-OOH) and hydroxyl radical (deter‐


**control) DMPO-OH radical intensity (% of control)**

0.5% VC 0.25% + 5 μM VC 1% VC 0.5% + 5 μM VC SE 48.3 47.1 < 60.4 [(48.3+72.4)/2] 65.4 75.5 < 87.3 [(65.4+109.1)/2]

) and 1,1-diphenyl-2-pic‐


radical in apoptosis-induction by ascorbic acid-related compounds.

tensity and cytotoxic activity of sodium ascorbate [49].

due at least in part to the stimulated induction of hypoxia.

*decandra* (Griff.) Ding Hou. and cacao husk scavenged O2

mined by the intensity of DMPO-OH radical) (Table 8) [2].

enging activity. LCC further stimulated the superoxide anion (O2

**DMPO-OOH radical intensity (% of**

10 μM vitamin C 72.4 109.1

**Table 8.** Synergistic radical scavenging activity of SE and vitamin C. Cited from [2] with permission.

istically enhanced the radical scavenging activity of sodium ascorbate [27, 34].

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 testoster‐ one, generally used for the assay of CYP3A4 activity. Product C exhibited the highest CYP3A4–inhibitory activity (IC50=58 μg/ml), followed by product B (IC50=124 μg/ml) and then product A (IC50=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]. 26 **(i) Inhibition of CYP3A4 activity:**  27 CYP3A4 activity was measured by β-hydroxylation of testosterone in human recombinant CYP3A4. 28 Products **A**, **B** and **C** dose-dependently inhibited the β-hydroxylation of testosterone, generally used 29 for the assay of CYP3A4 activity. Product **C** exhibited the highest CYP3A4–inhibitory activity 30 (IC50=58 g/ml), followed by product **B** (IC50=124 g/ml) and then product **A** (IC50=403 g/ml). 31 Product **B** inhibited the CYP3A4 to an extent similar to that attained by Cu (II)- chlorophyllin;

*decandra* (Griff.) Ding Hou. and cacao husk scavenged O2

(% of control)

19 enhanced the radical scavenging activity of sodium ascorbate [27, 34].

DMPO-OOH radical intensity

44 Figure 12. CYP3A4 inhibitory activity of products **A**, **B** and **C**. One millilitre of products **A**, **B** and **C** 45 was freeze dried to produce the powder (66.1, 77.6 and 28.5 mg, respectively).Each value represents 46 the mean ± S.D. (n=3). \*p<0.05 relative to the control (0%). Cited from [4] with permission. **Figure 12.** CYP3A4 inhibitory activity of products **A**, **B** and **C**. One millilitre of products **A**, **B** and **C** was freeze dried to produce the powder (66.1, 77.6 and 28.5 mg, respectively).Each value represents the mean ± S.D. (n=3). \*p<0.05 rela‐ tive to the control (0%). Cited from [4] with permission.

SE-10 and SE dose-dependently inhibited the β-hydroxylation of testosterone, generally used for the assay of CYP3A4 activity. SE-10 (IC50= 0.516 μg SE equivalent/ml) had an ap‐ proximately 16% lower CYP3A4-inhibitory activity (IC50= 0.445 μg/ml) [19].Combined with our recent report [4], CYP3A4 inhibitory activity increases in the following order (from low‐ er 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.

#### **3.10. 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.

(Figure 15). Among these biological activities, antiviral, anti-UV and synergism with vita‐

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191

**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.

min C are unique properties to SE as well as LCC (Figure 15).

#### **3.11. 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 in‐ duced gingivitis patients [9].

Lichen planus is a chronic mucocutaneous disease that affects the skin, tongue, and oral mu‐ cosa. The most common presentation of oral lichen planus is the reticular form that mani‐ fests 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 rash‐ es 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 tis‐ sue 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 squa‐ mous cell carcinoma in comparison with healthy oral epithelium [54].

Potent antiviral, antibacterial, and anti-inflammatory activity of SE prompted us to investi‐ gate whether SE is effective on oral lichenoid dysplasia and osteoclastogenesis. A male pa‐ tient 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 experi‐ mental 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 im‐ proved 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 differ‐ entiation of mouse macrophage-like RAW264.7 cells towards osteoclasts (evaluated by TRAP-positive multinuclear cell formation). These pilot clinical study suggests the thera‐ peutic potentiality of SE against oral diseases [55].

## **4. Conclusion**

SE (Sasa Health®), alkaline extract of *Sasa senanensis* Rehder extract has shown diverse bio‐ logical activities including membrane stabilizing, anti-leukemia, anti-inflammatory, radical scavenging, anti-UV, bacteriostatic, antiviral, anti-stomatitis, and anti-lichen planus activity (Figure 15). Among these biological activities, antiviral, anti-UV and synergism with vita‐ min C are unique properties to SE as well as LCC (Figure 15).

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

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 in‐

Lichen planus is a chronic mucocutaneous disease that affects the skin, tongue, and oral mu‐ cosa. The most common presentation of oral lichen planus is the reticular form that mani‐ fests 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 rash‐ es 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 tis‐ sue 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 squa‐

Potent antiviral, antibacterial, and anti-inflammatory activity of SE prompted us to investi‐ gate whether SE is effective on oral lichenoid dysplasia and osteoclastogenesis. A male pa‐ tient 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 experi‐ mental 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 im‐ proved 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 differ‐ entiation of mouse macrophage-like RAW264.7 cells towards osteoclasts (evaluated by TRAP-positive multinuclear cell formation). These pilot clinical study suggests the thera‐

SE (Sasa Health®), alkaline extract of *Sasa senanensis* Rehder extract has shown diverse bio‐ logical activities including membrane stabilizing, anti-leukemia, anti-inflammatory, radical scavenging, anti-UV, bacteriostatic, antiviral, anti-stomatitis, and anti-lichen planus activity

**3.11. Clinical application for the treatment of oral diseases**

mous cell carcinoma in comparison with healthy oral epithelium [54].

peutic potentiality of SE against oral diseases [55].

**4. Conclusion**

macrophages.

190 Alternative Medicine

duced gingivitis patients [9].

**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.

SE as well as LCCs, which are efficiently extracted with alkaline solution, showed higher an‐ ti-HIV and anti-UV activity, as compared with hot water extract of many plant species in‐ cluding 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

Hot water extract Alkaline extract

We have reported broad antiviral spectrum of LCC ranging from HIV [57-59], influenza vi‐ rus [60-62], herpes simplex virus [63-65]. Oral administration of LCC from pine cone extract significantly improved the symptom of HSV-infected patients [66, 67], and lichenoid dyspla‐ sia patient [55]. These data suggest the possible application of SE to virally-induced diseas‐ es. 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

LCC is composed of two major components: polysaccharide and phenylpropanoide polymer [29, 30, 69, 70]. Limited digestion study demonstrated that anti-viral activity of LCC is gen‐ erated by its phenylpropanoid portion [58, 61], and immnopotentiation activity possibly by polysaccharide. Using DNA microarray analysis, we have recently reported that treatment of mouse macrophage-like J774.1 cells with LCC fractions isolated from LEM (Fr4) enhanced the expression of dectin-2 (4.2-fold) and toll-like receptor (TLR)-2 (2.5-fold) prominently, but only slightly modified the expression of dectin-1 (0.8-fold), complement receptor 3 (0.9-fold), TLR1, 3, 4, 9 and 13 (0.8- to 1.7-fold), spleen tyrosine kinase (Syk)b, zeta-chain (TCR) associ‐ ated protein kinase 70kDa (Zap70), Janus tyrosine kinase (Jak)2 (1.0- to 1.2-fold), nuclear fac‐

Product A (SE)

Product C (BLE) (LCC depleted)

Anti-tumor activity Anti-HIV activity Anti-UV activity

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193

Acidity

LCC

against certain types of diseases.

component of toothpaste.

Product C (KS)

**Figure 16.** Comparison of several biological activities between hot water and alkaline extracts.

Kampo medicines

**Figure 14.** Effect of SE on saliva inflammatory cytokine level. One lichenoid dysplasia patient was treated with SE for the indicated periods, and the salivary IL-6 and IL-8 concentrations were determined by ELISA. Each value represents mean±S.D. of triplicate assays. ○: Control, ●:SE treatment. Cited from [55] with permission.

**Figure 15.** Diverse biological activity of SE..

SE as well as LCCs, which are efficiently extracted with alkaline solution, showed higher an‐ ti-HIV and anti-UV activity, as compared with hot water extract of many plant species in‐ cluding 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.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

192 Alternative Medicine

Bacteriostatic

**Figure 15.** Diverse biological activity of SE..

**IL-8 (ng/ ml)**

**IL-6 (ng/ ml)**

IL-8 (ng/ml) IL-6 (ng/ml)

03/28/11 05/17/11 07/06/11 08/25/11 10/14/11 12/03/11 01/22/12 03/12/12

28/03/2011 17/05/2011 06/07/2011 25/08/2011 14/10/2011 03/12/2011 22/01/2012 12/03/2012

IL-6

**Day**

Date

03/28/11 05/17/11 07/06/11 08/25/11 10/14/11 12/03/11 01/22/12 03/12/12

28/03/2011 17/05/2011 06/07/2011 25/08/2011 14/10/2011 03/12/2011 22/01/2012 12/03/2012

IL-8

**Day**

**Figure 14.** Effect of SE on saliva inflammatory cytokine level. One lichenoid dysplasia patient was treated with SE for the indicated periods, and the salivary IL-6 and IL-8 concentrations were determined by ELISA. Each value represents

> **Sasa Health® (SE)**

Membrane stabilizing Anti-inflammatory

Anti-leukemia

**Antiviral activity Synergism with vitamin C (Characteristic to SE as well as LCCs)**

Anti-stomatitis Anti-lichen planus

Radical scavenging

**Anti-UV**

mean±S.D. of triplicate assays. ○: Control, ●:SE treatment. Cited from [55] with permission.

Date

**Figure 16.** Comparison of several biological activities between hot water and alkaline extracts.

We have reported broad antiviral spectrum of LCC ranging from HIV [57-59], influenza vi‐ rus [60-62], herpes simplex virus [63-65]. Oral administration of LCC from pine cone extract significantly improved the symptom of HSV-infected patients [66, 67], and lichenoid dyspla‐ sia patient [55]. These data suggest the possible application of SE to virally-induced diseas‐ es. 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.

LCC is composed of two major components: polysaccharide and phenylpropanoide polymer [29, 30, 69, 70]. Limited digestion study demonstrated that anti-viral activity of LCC is gen‐ erated by its phenylpropanoid portion [58, 61], and immnopotentiation activity possibly by polysaccharide. Using DNA microarray analysis, we have recently reported that treatment of mouse macrophage-like J774.1 cells with LCC fractions isolated from LEM (Fr4) enhanced the expression of dectin-2 (4.2-fold) and toll-like receptor (TLR)-2 (2.5-fold) prominently, but only slightly modified the expression of dectin-1 (0.8-fold), complement receptor 3 (0.9-fold), TLR1, 3, 4, 9 and 13 (0.8- to 1.7-fold), spleen tyrosine kinase (Syk)b, zeta-chain (TCR) associ‐ ated protein kinase 70kDa (Zap70), Janus tyrosine kinase (Jak)2 (1.0- to 1.2-fold), nuclear fac‐ tor (Nf)кb1, NFкb2, reticuloendotheliosis viral oncogene homolog (Rel)a, Relb (1.0- to 1.6 fold), Nfкbia, Nfкbib, Nfкbie, Nfкbi12 Nfкbiz (0.8- to 2.3-fold). On the other hand, LPS did not affect the expression of dectin-2 nor TLR-2. These data suggest the significant role of the activation of the dectin-2 signaling pathway in the action of LCC on macrophages [71]. It is generally accepted that dectin- 2 is the receptor for mannan, whereas dectin-1 is that for glu‐ can [72-76]. It remains to be investigated the signaling pathway of LCC via dectin-2.

[6] Tomioka H, Kpya S, Satake F, Nakamura T, Kurashige S. The effect of *in vitro* stimu‐ lation with Shojusen on the cytokine production of mouse peritoneal macrophages.

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

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195

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[14] Zhou L, Hashimoto K, Satoh K, Yokote Y, Kitajima M, Oizumi T, Oizumi H, Sakaga‐ mi H. Effect of *Sasa senanensis* Rehder extract on NO and PGE2 production by activat‐

[15] Ono M, Kantoh K, Ueki J, Shimada A, Wakabayashi H, Matsuta T, Sakagami H, Ku‐ mada H, Hamada N, Kitajima M, Oizumi H, Oizumi T. Quest for anti-inflammatory substances using IL-1β-stimulated gingival fibroblasts. In Vivo 2011;25(5) 763-768. [16] Akazaki N, Sasaki Y, Takeda, H, Hosokawa T, Takeshita K, Kanamori M, Tsuboi M, Nagumo S. Anti-inflammatory effects of Kumazasa water extract. Pharmacometrics

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## **Author details**

Hiroshi Sakagami1\*, Tomohiko Matsuta1 , Toshikazu Yasui1 , Oguchi Katsuji2 , Madoka Kitajima3 , Tomoko Sugiura3 , Hiroshi Oizumi3 and Takaaki Oizumi3

\*Address all correspondence to: sakagami@dent.meikai.ac.jp

1 Meikai University School of Dentistry, Sakado, Saitama, Japan

2 School of Medicine, Showa University, Tokyo, Japan

3 Daiwa Biological Research Institute Co., Ltd., Kanagawa, Japan

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194 Alternative Medicine

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**Section 4**

**Action Mechanism and Future Direction**


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**Chapter 9**

**Promotion of Blood Fluidity Using Electroacupuncture**

Acupuncture is an alternative medicine originating in ancient China that treats patients by manipulating thin, solid needles that have been inserted into acupuncture points in the skin. Current several scientific research supports acupuncture's efficacy in the relief of certain

Acupuncture's greatest effectiveness appears to be in symptomatic control of pain and nau‐ sea. The World Health Organization and the United States' National Institutes of Health (NIH) have stated that acupuncture can be effective in the treatment of neurological condi‐ tions and pain [3]. Moreover, it is thought that acupuncture regulates various biological functions. It was presented that acupuncture stimulus influences the cytokine level, hor‐

Blood roles in organisms include waste product removal, body temperature adjustment, as well as oxygen and nutritive supply. Physiologically the first priority driving force of blood

Blood flow is determined by co-action of the cardiovascular system and blood fluidity. However, blood flow is also controlled by a blood hydrodynamic characteristic. It is estab‐ lished that changes in the cardiovascular system will cause changes in blood properties as well [6-9]. Changes of blood cell composition and plasma components may influence blood fluidity in the long term [7], and blood cell activity, such as red blood cell agglutination, leu‐

It is believed that variations in blood fluidity result in disorders of the circulatory system such as arterial sclerosis or embolism, damage to vascular endothelium cells by hyperten‐

and reproduction in any medium, provided the original work is properly cited.

© 2012 Ishikawa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Stimulation**

Tadashi Hisamitsu

**1. Introduction**

http://dx.doi.org/10.5772/53036

circulation is cardiac pressure.

Shintaro Ishikawa, Kazuhito Asano and

types of pain and post-operative nausea [1,2].

mone level [4] and leukocyte number [5] as effects for blood.

kocyte adherence, and platelet aggregation, in the short term [8,9].

Additional information is available at the end of the chapter
