**Treatment of Herpes Simplex Virus with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula**

Blanca Silvia González López1, Masaji Yamamoto2 and Hiroshi Sakagami3 *1Laboratorio de Patología Bucal, Facultad de Odontología, Universidad Autónoma del Estado de México, 2Maruzen Pharmaceuticals Co., Ltd., 3Meikai University School of Dentistry, 1México 2,3Japan* 

#### **1. Introduction**

170 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

Zhou, B., Zhong, N. & Guan, Y. (2007). Treatment with convalescent plasma for influenza A

Herpes simplex virus type 1 (HSV-1) commonly infects the mucosa and skin epithelial cells, and the virus remains latent in sensory neurons mainly in the trigeminal ganglia. Once a patient has been infected, the infection continues for life (Hunt, 2011a). Differences in HSV-1 prevalence have been reported around the world. According to Smith & Robinson (2002), the incidence in lower socioeconomic countries is higher. Primary infection, occur mainly in infants and young children, infections are usually mild or subclinical. Acute gingivoestomatitis is characterized by the appearance of multiple vesicular and ulcerative painful lesions in oral mucosa, with inflammation and bleeding of the gums, may also be associated with systemic symptoms (Arduino & Porter, 2008). Once the clinical infection concludes, the virus reaches peripheral nerves which supply sensation to the skin, migrating along the nerve axon to the dorsal root ganglia of the trigeminal or facial nerves and goes into latency stage (Esmann, 2001)

Recurrences of HSV-1 can be triggered by internal and external factors. The reactivation mechanism is unknown, the virus begins to replicate within the ganglion and grows down the nerves and out into the skin or mucous membranes (Koelle & Corey, 2008). After a prodromal of tingling, warmth or itching, the clinical lesion appear (Fatahzadeh & Schwartz 2007). The recurrence of oral HSV- 1 is developed almost always in the vermilion border of the lips but lesions can appear elsewhere around perioral skin (Siegel, 2002).

Prevention of infection can be achieved by avoiding the physical contact, kissing when the lesions are present, touching or using the articles that the patient has used (eating or drinking utensils, glasses, or straws). However, in order to prevent the recurrences, the control of external factors is recommended; avoiding the exposure to wind burn and ultraviolet radiation, using labial protectants and controlling the emotional stress (Paterson & Kwong, 2008).

Treatment of Herpes Simplex Virus

**1.2.1 Some examples of traditional medicine** 

inactivated the clinical isolates of HSV *in vitro*.

cycle *in vitro*.

which amounts of HS exposed on the cell surface had been reduced.

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 173

remedies claimed to be beneficial in the treatment of herpes infection, arguing that alternative medicine could be beneficial in the treatment of herpes infection through enhancing the immune system. The interest in alternative drugs having antiviral effect is increasingly, since HSV-1, might develop resistance to commonly used antiviral agents.

Besides, despite the fact that some patients manifested side effects, the majority of the

There are a number of natural remedies used in the HSV-1 treatment; some of them have been the subject of scientific analysis, demonstrating *in vitro* and *in vivo* satisfactory results.

In an experimental-placebo study, the application of zinc oxide/glycine cream showed shorter duration of cold sore lesions and reduction in overall severity of signs and symptoms (Godfrey, et al., 2001). In a pilot study, Femiano, et al. (2005) reported similar results and a reduction of number of episodes of herpes labialis. Singh, et al. (2005) in a clinical trial, evaluated a combination of L-lysine with a mixture of botanicals and other nutrients, with satisfactory results. Since the early 90s, Kümel et al. (1990a, 1991b) explained that the zinc ions inactivate virus by inhibition of the virion glycoprotein's function after a nonspecific accumulation of zinc into many virion membrane components, thus inhibiting viral adsorption and penetration. Also, Arens & Travis (2000) demonstrated that zinc salts

Mårdberg et al., (2001), demonstrated that viruses with mutations at residues Arg129,130, Ile142, Arg143,145, Arg145,147, Arg151,155 and Arg155,160 had significantly impaired the heparan sulfate (HS) binding. Impairment of the HS-binding activity of glycoprotein C, by these mutations had profound consequences for virus attachment and infection of cells in

Recently Katsuyama et al., (2008) established that Butyroyl-arginine, an arginine derivative, strongly inactivates the enveloped virus, as HSV-1. The authors suggest that the ability of arginine to bind membranes may be responsible for the inactivation of viruses. Naito et al.,

Huleihel & Isanu (2002) reported that propolis could block the cell membrane receptors for HSV-1, blocking the penetration of viral particles into the cells and/or inducing the intracellular metabolic changes of host cells, which would in turn affect the viral replication

Ascorbic acid has been shown to inactivate HSV-1, prevent the virus reactivation, have antiinflammatory properties and to enhance the immune function (Gaby, 2006; Hovi, et al., 1995; Yoon et al., 2000). Supplementation with flavonoids further increases the effectiveness of vitamin C (Terezhalmy, et al., 1978). According to Narayana, et al., (2001), flavonoids showed strong antiviral activity against HSV-1. Essential oils of ginger, thyme hyssop and sandalwood have been demonstrated to inactivate HSV-1 before it enters into the cells, even in acyclovir resistant HSV-1 *(*Schnitzler, et al., 2007*).* Melissa officinalis (*lemon balm)* contains rosmarinic acid, phenolic acids and tannins; rosmarinic acid has been reported to show antiinflammatory and potent antioxidant action. Schnitzler, et al. (2008), demonstrated that balm oil affected the viruses *in vitro* before adsorption, although the mechanism of action is

(2009) also has demonstrated the inhibition of HSV-1 multiplication by ariginine.

natural remedies did not show a high prevalence of adverse or severe reactions.

On the other hand, internal factors that are related to the recurrence outbreaks such as, fever, illness, menstruation, gastrointestinal and respiratory infections, diseases as diabetes and hyperthyroidism, fatigue and factors that depress the immune system are difficult to control (Siegel, 2002; Paterson & Kwong, 2008).

Although different clinical assays have been developed in order to assess the efficacy of topical or oral antivirals, its effectiveness has not been demonstrated due to the immediate and complete termination of viral replication, the restoration of previously infected cells, and the inactivation of free virions (Hamuy & Berman 1998).

There is no treatment that can eradicate herpes virus, even though antiviral medications can reduce the frequency, duration, and severity of outbreaks (Emmert, 2006; Siegel, 2002; Sprurance, et al., 2003; Sprurance, et al., 2005)

#### **1.1 Conventional treatment**

According to Hunt (2011b), there are different phases of life cycle of virus; adsorption and penetration of the virus in the host cell, and early transcription, in which DNA polymerase, DNA binding proteins, thymidine kinase and ribonucleotide reductase are synthesized. These proteins are virally-coded, not host-coded enzymes, and therefore potentially weak in the virus life cycle, making them promising targets for anti-viral drugs.

The nucleoside analogues acyclovir, valacyclovir, are phosphorylated initially by viral thymidine kinase to eventually form a nucleoside triphosphate, and these molecules inhibit herpes simplex virus (HSV-1) polymerase, inhibiting replication of HSV-1 (Balzarini, 1994). The best anti-viral drugs are nucleoside analogs such as acyclovir (acycloguanosine). It gets into the cell across the plasma membrane as the nucleoside form and is then specifically phosphorylated inside the cell by herpes virus thymidine kinase to an active form. The advantages of nucleoside analogs are that they are only activated by the virus-infected cell and the activated form of the drug is rendered even more specific as a result of the viral DNA polymerase being more sensitive to the drug than the host enzyme (Hunt, 2001b)

In general, acyclovir compounds are safe and effective for treatment of HSV-1 reactivation and have good oral bioavailability (Chon & Elliott, 2007). However, topical administration of acyclovir at the acute stage of the lesion disease seems to be ineffective. On the other hand, its capability to avoid the recurrent episodes produces controversial efficacy (Elish, 2004; Spruance, et al., 2002). Emmert (2000) suggested that patients with mild and infrequent recurrences are not beneficiated with acyclovir treatment. There is general consensus that the therapy is most effective when started soon after symptoms occur.

Rare adverse effects include: coma, seizures, neutropenia, leukopenia, tremor, ataxia, encephalopathy, psychotic symptoms, crystalluria, anorexia, fatigue, hepatitis, Stevens-Johnson syndrome, toxic epidermal necrolysis and/or anaphylaxis (United States Food and Drug Administration, FDA, 2011). Also the appearance of virus strains resistant to frequently used anti-herpes virus drugs (Greco, et al., 2007 ; Stránská, et al., 2005; Ziyaeyan, et al, 2007)

#### **1.2 Alternative treatment**

It has been suggested that there are insufficient scientific evidences to support the use of alternative medicine in HSV-1 infection**.** Even though, anecdotal reports of alternative remedies claimed to be beneficial in the treatment of herpes infection, arguing that alternative medicine could be beneficial in the treatment of herpes infection through enhancing the immune system. The interest in alternative drugs having antiviral effect is increasingly, since HSV-1, might develop resistance to commonly used antiviral agents.

Besides, despite the fact that some patients manifested side effects, the majority of the natural remedies did not show a high prevalence of adverse or severe reactions.

There are a number of natural remedies used in the HSV-1 treatment; some of them have been the subject of scientific analysis, demonstrating *in vitro* and *in vivo* satisfactory results.

#### **1.2.1 Some examples of traditional medicine**

172 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

On the other hand, internal factors that are related to the recurrence outbreaks such as, fever, illness, menstruation, gastrointestinal and respiratory infections, diseases as diabetes and hyperthyroidism, fatigue and factors that depress the immune system are difficult to

Although different clinical assays have been developed in order to assess the efficacy of topical or oral antivirals, its effectiveness has not been demonstrated due to the immediate and complete termination of viral replication, the restoration of previously infected cells,

There is no treatment that can eradicate herpes virus, even though antiviral medications can reduce the frequency, duration, and severity of outbreaks (Emmert, 2006; Siegel, 2002;

According to Hunt (2011b), there are different phases of life cycle of virus; adsorption and penetration of the virus in the host cell, and early transcription, in which DNA polymerase, DNA binding proteins, thymidine kinase and ribonucleotide reductase are synthesized. These proteins are virally-coded, not host-coded enzymes, and therefore potentially weak in

The nucleoside analogues acyclovir, valacyclovir, are phosphorylated initially by viral thymidine kinase to eventually form a nucleoside triphosphate, and these molecules inhibit herpes simplex virus (HSV-1) polymerase, inhibiting replication of HSV-1 (Balzarini, 1994). The best anti-viral drugs are nucleoside analogs such as acyclovir (acycloguanosine). It gets into the cell across the plasma membrane as the nucleoside form and is then specifically phosphorylated inside the cell by herpes virus thymidine kinase to an active form. The advantages of nucleoside analogs are that they are only activated by the virus-infected cell and the activated form of the drug is rendered even more specific as a result of the viral DNA polymerase being more sensitive to the drug than the host enzyme (Hunt, 2001b)

In general, acyclovir compounds are safe and effective for treatment of HSV-1 reactivation and have good oral bioavailability (Chon & Elliott, 2007). However, topical administration of acyclovir at the acute stage of the lesion disease seems to be ineffective. On the other hand, its capability to avoid the recurrent episodes produces controversial efficacy (Elish, 2004; Spruance, et al., 2002). Emmert (2000) suggested that patients with mild and infrequent recurrences are not beneficiated with acyclovir treatment. There is general consensus that the therapy is most effective when started soon after symptoms occur.

Rare adverse effects include: coma, seizures, neutropenia, leukopenia, tremor, ataxia, encephalopathy, psychotic symptoms, crystalluria, anorexia, fatigue, hepatitis, Stevens-Johnson syndrome, toxic epidermal necrolysis and/or anaphylaxis (United States Food and Drug Administration, FDA, 2011). Also the appearance of virus strains resistant to frequently used anti-herpes virus drugs (Greco, et al., 2007 ; Stránská, et al., 2005; Ziyaeyan, et al, 2007)

It has been suggested that there are insufficient scientific evidences to support the use of alternative medicine in HSV-1 infection**.** Even though, anecdotal reports of alternative

control (Siegel, 2002; Paterson & Kwong, 2008).

Sprurance, et al., 2003; Sprurance, et al., 2005)

**1.1 Conventional treatment** 

**1.2 Alternative treatment** 

and the inactivation of free virions (Hamuy & Berman 1998).

the virus life cycle, making them promising targets for anti-viral drugs.

In an experimental-placebo study, the application of zinc oxide/glycine cream showed shorter duration of cold sore lesions and reduction in overall severity of signs and symptoms (Godfrey, et al., 2001). In a pilot study, Femiano, et al. (2005) reported similar results and a reduction of number of episodes of herpes labialis. Singh, et al. (2005) in a clinical trial, evaluated a combination of L-lysine with a mixture of botanicals and other nutrients, with satisfactory results. Since the early 90s, Kümel et al. (1990a, 1991b) explained that the zinc ions inactivate virus by inhibition of the virion glycoprotein's function after a nonspecific accumulation of zinc into many virion membrane components, thus inhibiting viral adsorption and penetration. Also, Arens & Travis (2000) demonstrated that zinc salts inactivated the clinical isolates of HSV *in vitro*.

Mårdberg et al., (2001), demonstrated that viruses with mutations at residues Arg129,130, Ile142, Arg143,145, Arg145,147, Arg151,155 and Arg155,160 had significantly impaired the heparan sulfate (HS) binding. Impairment of the HS-binding activity of glycoprotein C, by these mutations had profound consequences for virus attachment and infection of cells in which amounts of HS exposed on the cell surface had been reduced.

Recently Katsuyama et al., (2008) established that Butyroyl-arginine, an arginine derivative, strongly inactivates the enveloped virus, as HSV-1. The authors suggest that the ability of arginine to bind membranes may be responsible for the inactivation of viruses. Naito et al., (2009) also has demonstrated the inhibition of HSV-1 multiplication by ariginine.

Huleihel & Isanu (2002) reported that propolis could block the cell membrane receptors for HSV-1, blocking the penetration of viral particles into the cells and/or inducing the intracellular metabolic changes of host cells, which would in turn affect the viral replication cycle *in vitro*.

Ascorbic acid has been shown to inactivate HSV-1, prevent the virus reactivation, have antiinflammatory properties and to enhance the immune function (Gaby, 2006; Hovi, et al., 1995; Yoon et al., 2000). Supplementation with flavonoids further increases the effectiveness of vitamin C (Terezhalmy, et al., 1978). According to Narayana, et al., (2001), flavonoids showed strong antiviral activity against HSV-1. Essential oils of ginger, thyme hyssop and sandalwood have been demonstrated to inactivate HSV-1 before it enters into the cells, even in acyclovir resistant HSV-1 *(*Schnitzler, et al., 2007*).* Melissa officinalis (*lemon balm)* contains rosmarinic acid, phenolic acids and tannins; rosmarinic acid has been reported to show antiinflammatory and potent antioxidant action. Schnitzler, et al. (2008), demonstrated that balm oil affected the viruses *in vitro* before adsorption, although the mechanism of action is

Treatment of Herpes Simplex Virus

Tannin Hydrolysable Tannin

Condensed

Lignin carbohydrate complex (LCC)

tannin

OHOH

O H3C

**2. Functionality of LCC as alternative medicine**

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 175

Lignin (polymers of phenylpropenoids), tannin and flavonoid are three major polyphenols in the natural kingdom (Table 1). So far, thousands of tannin and flavonoid-related compounds have been isolated from the methanol extracts of various plants and their complete structures have been elucidated. In contrast, lignins, extracted with alkaline solution, have been bound to polysaccharides (composed of glucose, arabinose, mannose, galactose, fucose, or uronic acids) to form lignin-carbohydrate complex (LCC) (Fig. 2).

with glucose or related sugars

Flavonoid Oxygen containing cyclic structure between two benzene rings

polysaccharide

O

HO

HO

OH

MeO

HO

MeO O OMeO

OH MeO

OH MeO

OH

**LCC**

HO

Sinapyl alcohol

Coniferyl alcohol

OH

MeO

•Antiviral •Anti-oxidation

OH OH

**Polysaccharide** (Glucose, Mannose, Galactose, Arabinose) •Binding to dectin-2 or -1 •Immunopotentiation

•Anti-UV activity •Synergy with vitamin C

LignHi 3 nC

MeO

<sup>O</sup> <sup>O</sup>

O

OH

OMe

O

HO

Fig. 2. Structure and function of lignin-carbohydrate complex (LCC)

Table 1. Representative polyphenols present in the natural kingdom.

O

Lignin HO

OH OMe MeO O HO HO

Component unit MW (kDa)

0.5~4

0.3~2

0.3~1

10~200

Esters of gallic acid and its oxidative derivatives

Flavan oligomers or polymers where their constituent monomeric flavans are connected mainly by C-4 – C-8 or C-4 – C-6 linkages

Complex of phenylpropenoid polymers and

OH *p*-Coumaryl alcohol

unclear. They suggest that the balm oil could bind the viral proteins involved in the host adsorption and penetration, or damage the virions envelop. Carson, et al. (2006) have reported that tea tree oil of *Melaleuca alternifolia* showed the greatest effect on free virus.

Surveys of alternative treatment for HSV-1 are difficult to perform, since numerous patients are needed the regular contact for long period until the completion of the study. Even so, we have applied the vitamin C-supplemented tablet of lignin-carbohydrate complex (LCC) prepared from the pine cone of *Pinus parviflora* Sieb et Zucc., to a sample of HSV-1 patients, and investigated its clinical effect for the first time, with satisfactory results (González et al., 2009). The inhibitory effect of pine cone lignin and ascorbic combination treatment depend on antioxidant and immunopotentiating activities of lignin and ascorbic acid (Sakagami, et al., 1992).

The goal of both conventional and alternative treatment is promoting faster healing, reduction of symptoms, as well as decreasing the frequency of recurrent episodes. There are different phases in the viral cycle, to which the medicaments could be applied, according to the properties and mechanism of action of each medication (Fig. 1)

Fig. 1. A desirable effect of alternative treatment on HVS-1 virus cycle (Everett, 2006; Frick, 2003; Mettenleiter, et al., 2006; Newcomb, et al., 2007, Pinnoji, et al, 2007).

#### **2. Functionality of LCC as alternative medicine**

174 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

unclear. They suggest that the balm oil could bind the viral proteins involved in the host adsorption and penetration, or damage the virions envelop. Carson, et al. (2006) have reported that tea tree oil of *Melaleuca alternifolia* showed the greatest effect on free virus.

Surveys of alternative treatment for HSV-1 are difficult to perform, since numerous patients are needed the regular contact for long period until the completion of the study. Even so, we have applied the vitamin C-supplemented tablet of lignin-carbohydrate complex (LCC) prepared from the pine cone of *Pinus parviflora* Sieb et Zucc., to a sample of HSV-1 patients, and investigated its clinical effect for the first time, with satisfactory results (González et al., 2009). The inhibitory effect of pine cone lignin and ascorbic combination treatment depend on antioxidant and immunopotentiating activities of lignin and ascorbic acid (Sakagami, et

The goal of both conventional and alternative treatment is promoting faster healing, reduction of symptoms, as well as decreasing the frequency of recurrent episodes. There are different phases in the viral cycle, to which the medicaments could be applied, according to

> HSV-1 Nuclear replicating enveloped virus Transmitted Contact infectious area or saliva drops

of virus proteins Latent infection

Fig. 1. A desirable effect of alternative treatment on HVS-1 virus cycle (Everett, 2006; Frick,

2003; Mettenleiter, et al., 2006; Newcomb, et al., 2007, Pinnoji, et al, 2007).

Reactivation ICP4 is a transactivator of genes associated with lytic infection in HSV-1. Elements surrounding the gene for ICP4 bind a protein known as the human neuronal protein Neuronal Restrictive Silencing Factor (NRSF)

Immune evasion HSV evades the immune system through interference with MHC class I presentation of antigen on the cell surface.

Immune evasion HSV evades the immune system through interference with MHC class I presentation of antigen on the cell surface

Trigeminal Ganglia Latency Associated Transcript (LAT) is known to regulate the host cell genome and interferes with natural cell death mechanisms.

Interactions of glycoprotein on the surface of the enveloped virus, with receptors on the surface of the host cells

Prevention

Interference with viral attachment by binding viral cellular glycoprotein

After the viral capsid enters the cellular cytoplasm, it is transported to the cell nucleus. Once attached to the nucleus at a nuclear entry pore, the capsid ejects its DNA contents *via* the capsid portal.

the properties and mechanism of action of each medication (Fig. 1)

Replication & Transcription

Genetic inoculation

Cellular entry

Infection

Inhibition of viral DNA polymerase

Interference with viral attachment

Inhibition of helicase

Enhancement of immune system

al., 1992).

Lignin (polymers of phenylpropenoids), tannin and flavonoid are three major polyphenols in the natural kingdom (Table 1). So far, thousands of tannin and flavonoid-related compounds have been isolated from the methanol extracts of various plants and their complete structures have been elucidated. In contrast, lignins, extracted with alkaline solution, have been bound to polysaccharides (composed of glucose, arabinose, mannose, galactose, fucose, or uronic acids) to form lignin-carbohydrate complex (LCC) (Fig. 2).


Table 1. Representative polyphenols present in the natural kingdom.

Fig. 2. Structure and function of lignin-carbohydrate complex (LCC)

Treatment of Herpes Simplex Virus

systems (Table 2).

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 177

group (T/C%)] increased with the acidity: Fr. I (T/C=98%) < Fr. II (T/C=118%) < Fr. III (T/C=175%), Fr. IV (T/C=151 %) < Fr. V (T/C=172%) (Sakagami, et al., 1987). Higher antitumor activity was recovered by extraction with 1%NaOH, precipitated by acidification (pH

The most active acidic polysaccharide (IV) was subjected to spectral analysis, and identified as lignin-carbohydrate complex (LCC), based on the following evidences (Sakagami, et al., 1989). (i) UV absorption spectra:minimum absorption at 260 nm, maximum absorption at 280 nm, broad maximum absorption at 500 nm. (ii) IR spectra: hydroxyl group with hydrogen bonding (3400 cm-1), aliphatic C-H (2700 cm-1), carbonyl group conjugated to πelectron system (1700 ~ 1600 cm-1), aromatic double bond (1600, 1500 cm-1), C-O expansion and contraction (1400 ~ 1000 cm-1), no ester bonding.(iii) ESR: one strong signal at g=2.003 under solid state at room temperature. Signal intensity was significantly reduced by oxidation and reduction.(iv) 1H-NMR: When measured in 0.2%NaOD-D2O, the presence of hydrogens in aromatic CH (*δ*6.5~7.5 ppm), >C=C< (*δ*4.5~5.5 ppm) and –O-CH-CH (*δ*3.0 ~ 4.0 ppm) was suggested. When the sample was acetylated by pyridine-acetic acid anhydride and dissolved in CDCl3, the presence of acetyl group bound to phenolic OH (*δ* 2.3 ppm) or bound to alcoholic OH (*δ* 2.1 ppm) was confirmed. (v) Thin layer chromatography: Rf value of Fr. VI was the same with that of commercial alkali-lignin in various solvent

5) (Fr. VI) (T/C=227%), and also by ethanol precipitation (Fr. VII) (T/C=282%).

UV Absorption spectra absorption peak at 260, 280, 500 nm (broad) IR spectra 3400, 2700, 1700~1600, 1600, 1500, 1400~1000 cm-1

Elementary analysis C (43.21%), H (3.96%), N (2.61%), S (not detectable)

Composition of neutral sugar Gal (44.7%), Glc (26.9%), Man (19.0%), Fuc (9.4%)

Table 2. Identification of Fr. IV from pine cone of *Pinus parviflora* Sieb. et Zucc. as LCC, based on chemical analyses. Cited from Sakagami et al. (1987), with permission.

Frs. VI and VII, prepared from the pine cones of *Pinus parviflora* Sieb. and Zucc. (T/C=227, 247%) showed higher antitumor activity in mice, than those prepared from *Pinus densiflora* Sieb. et Zucc (T/C=155, 245%), *Pinus thunbergii* Parl. (T/C=218, 191%), *Pinus elliottii* var. Elliotti (T/C=170, 217%), *Pinus taeda* L. (T/C=196, 179%), *Pinus caribaea* var. Hondurenses (T/C=114, 147%), *Pinus sylvestris* L. (T/C=180, 135%), or the pine seed shells of *Pinus parviflora* Sieb. et Zucc. (T/C=194. 220%), and *Pinus armandii* Franch (T/C=125%). Furthermore, the yields of Frs. VI and VII, prepared from *Pinus parviflora* Sieb. et Zucc. (0.51, 0.91%) were much higher than those from other pine cone sources [0.19±0.18 (0.001~0.48), 0.31±0.16 (0.06~0.48) %] (Harada, et al., 1988). These data suggest that acidic polysaccharides (Frs. VI and VII) are responsible for the legendary antitumor potential of the pine cone of

Molecular weight 10 kDa on gel filtration chromatography

ESR spectra g=2.003 (strong signal) 1H-NMR spectra δ6.5~7.5, 4.5~5.5, 3.0~4.0 ppm TLC The same Rf value with alkali-lignin

Neutral sugar/uronic acid 11.0%/1.7%

**2.2 Distribution of LCC in the natural kingdom**

*Pinus parviflora* Sieb. et Zucc.

This structural complexity of LCC has made it difficult to elucidate its complete structure. Varying the ratio of polysaccharide to phenylpropenoid polymer produces heterogeneity in the acidity, water-solubility, ethanol-insolubility, and molecular weight of LCC that might strongly affect its antiviral potency (Sakagami, et al., 2005, 2010b). However, this possibility has not been tested yet by any investigators. LCC was recoverable at much higher yield from the alkaline solution, in contrast to tannins and flavonoids (Fig. 3). The higher yield of LCC is very convenient for the mass-production in the factory.

#### **2.1 Identification of LCC as an active antitumor principle of pine cone extract**

We have paid attention to the folklore that intake of the hot water extract of pine cone of *Pinus parviflora* Sieb. et Zucc is effective for gastroenterological tumors. We isolated various polysaccharide fractions (Fig. 3), and investigated their antitumor activity against ascites sarcoma-180 cells (Sakagami, et al., 1987).

Fig. 3. Fractional preparation of LCC fractions from *Pinus parviflora* Sieb. et Zucc. Cited from Sakagami et al. (1987), with permission.

Pine cone was treated with ethanol to remove the sticky resin that contains cytotoxic substances, and then extracted with hot-water and then alkaline solution (1% NaOH). Polysaccharides in the hot water extract were precipitated by 86% ethanol, and then applied to DEAE-cellulose column chromatography. Neutral polysaccharide fraction (Fr. I) passed through the column, and then acidic polysaccharide fractions (Fr. II and III) rich in uronic acid were eluted from DEAE cellulose column chromatography with 0.5 and 2 M NaCl, respectively. The most acidic polysaccharides (Fr. VI and Fr. V) were eluted with 0.15M NaOH. The anti-tumor activity [evaluated by the survival ratio of treated group to control

This structural complexity of LCC has made it difficult to elucidate its complete structure. Varying the ratio of polysaccharide to phenylpropenoid polymer produces heterogeneity in the acidity, water-solubility, ethanol-insolubility, and molecular weight of LCC that might strongly affect its antiviral potency (Sakagami, et al., 2005, 2010b). However, this possibility has not been tested yet by any investigators. LCC was recoverable at much higher yield from the alkaline solution, in contrast to tannins and flavonoids (Fig. 3). The higher yield of

We have paid attention to the folklore that intake of the hot water extract of pine cone of *Pinus parviflora* Sieb. et Zucc is effective for gastroenterological tumors. We isolated various polysaccharide fractions (Fig. 3), and investigated their antitumor activity against ascites

Fr. VI ×1 EtOH

ppt sup Fr. VIII ×5 EtOH

> Fr. IX (0.74%)

ppt sup

(0.11%)

(0.88%)

**2.1 Identification of LCC as an active antitumor principle of pine cone extract**

LCC is very convenient for the mass-production in the factory.

Pine cone (100%)

EtOH wash Hot water extraction

Extract Residue

EtOH precipitation 1%NaOH extraction DEAE cellulose column pH adjusted to 5

Flow through fr. Adsorbed fr. ppt sup

(0.10%) Fr. II Fr. III Fr. IV Fr. V ppt sup

(0.61%) (0.01%) (0.004%) (0.14%) Fr. VII ×2 EtOH

Fig. 3. Fractional preparation of LCC fractions from *Pinus parviflora* Sieb. et Zucc. Cited from

Pine cone was treated with ethanol to remove the sticky resin that contains cytotoxic substances, and then extracted with hot-water and then alkaline solution (1% NaOH). Polysaccharides in the hot water extract were precipitated by 86% ethanol, and then applied to DEAE-cellulose column chromatography. Neutral polysaccharide fraction (Fr. I) passed through the column, and then acidic polysaccharide fractions (Fr. II and III) rich in uronic acid were eluted from DEAE cellulose column chromatography with 0.5 and 2 M NaCl, respectively. The most acidic polysaccharides (Fr. VI and Fr. V) were eluted with 0.15M NaOH. The anti-tumor activity [evaluated by the survival ratio of treated group to control

Fr. I (0.14%)

Anti-tumor activity against mouse ascites tumor (T/C%): Fr. VII (282) > Fr. VI (227)> Fr. XI (198) > Fr. III (175) > Fr. V (172)

> Fr. VIII (166) > Fr. IV (151) > Fr. II (118) > Fr. I (96)

Sakagami et al. (1987), with permission.

sarcoma-180 cells (Sakagami, et al., 1987).

group (T/C%)] increased with the acidity: Fr. I (T/C=98%) < Fr. II (T/C=118%) < Fr. III (T/C=175%), Fr. IV (T/C=151 %) < Fr. V (T/C=172%) (Sakagami, et al., 1987). Higher antitumor activity was recovered by extraction with 1%NaOH, precipitated by acidification (pH 5) (Fr. VI) (T/C=227%), and also by ethanol precipitation (Fr. VII) (T/C=282%).

The most active acidic polysaccharide (IV) was subjected to spectral analysis, and identified as lignin-carbohydrate complex (LCC), based on the following evidences (Sakagami, et al., 1989). (i) UV absorption spectra:minimum absorption at 260 nm, maximum absorption at 280 nm, broad maximum absorption at 500 nm. (ii) IR spectra: hydroxyl group with hydrogen bonding (3400 cm-1), aliphatic C-H (2700 cm-1), carbonyl group conjugated to πelectron system (1700 ~ 1600 cm-1), aromatic double bond (1600, 1500 cm-1), C-O expansion and contraction (1400 ~ 1000 cm-1), no ester bonding.(iii) ESR: one strong signal at g=2.003 under solid state at room temperature. Signal intensity was significantly reduced by oxidation and reduction.(iv) 1H-NMR: When measured in 0.2%NaOD-D2O, the presence of hydrogens in aromatic CH (*δ*6.5~7.5 ppm), >C=C< (*δ*4.5~5.5 ppm) and –O-CH-CH (*δ*3.0 ~ 4.0 ppm) was suggested. When the sample was acetylated by pyridine-acetic acid anhydride and dissolved in CDCl3, the presence of acetyl group bound to phenolic OH (*δ* 2.3 ppm) or bound to alcoholic OH (*δ* 2.1 ppm) was confirmed. (v) Thin layer chromatography: Rf value of Fr. VI was the same with that of commercial alkali-lignin in various solvent systems (Table 2).


Table 2. Identification of Fr. IV from pine cone of *Pinus parviflora* Sieb. et Zucc. as LCC, based on chemical analyses. Cited from Sakagami et al. (1987), with permission.

#### **2.2 Distribution of LCC in the natural kingdom**

Frs. VI and VII, prepared from the pine cones of *Pinus parviflora* Sieb. and Zucc. (T/C=227, 247%) showed higher antitumor activity in mice, than those prepared from *Pinus densiflora* Sieb. et Zucc (T/C=155, 245%), *Pinus thunbergii* Parl. (T/C=218, 191%), *Pinus elliottii* var. Elliotti (T/C=170, 217%), *Pinus taeda* L. (T/C=196, 179%), *Pinus caribaea* var. Hondurenses (T/C=114, 147%), *Pinus sylvestris* L. (T/C=180, 135%), or the pine seed shells of *Pinus parviflora* Sieb. et Zucc. (T/C=194. 220%), and *Pinus armandii* Franch (T/C=125%). Furthermore, the yields of Frs. VI and VII, prepared from *Pinus parviflora* Sieb. et Zucc. (0.51, 0.91%) were much higher than those from other pine cone sources [0.19±0.18 (0.001~0.48), 0.31±0.16 (0.06~0.48) %] (Harada, et al., 1988). These data suggest that acidic polysaccharides (Frs. VI and VII) are responsible for the legendary antitumor potential of the pine cone of *Pinus parviflora* Sieb. et Zucc.

Treatment of Herpes Simplex Virus

Inhibition of plaque formation (%)

0 20

assays. Cited from Fukuchi, et al., (1989a), with permission.

Viable cell number (

**polysaccharides** 

×10-4 ml)

40 60

80 100

120

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 179

0 0.1 1 10

Concentration (g/ml)

01234

Time after addition (days)

Fig. 5. Effect of Fr. VI on the growth of CV-1 cells. Each value represents mean of triplicate

**2.3.2 Anti-HSV activity of LCCs from other plants, natural and chemically modified** 

Neutral polysaccharide (Fr. I) and uronic acid-rich polysaccharide (Fr. II) from pine cone extract of *Pinus parviflora* Sieb. et Zucc. had no anti-HSV activity at 10 g/ml. Similarly, popular antitumor polysaccharides, such as paramylon, PSK, Schizophyllan, and chemically modified glucans (*N,N*-dimethylaminoethylparamylon, sodium carboxymethylparamylon, sodium paramylon sulfate, carboxymethyl TAK) were all inactive at 10 g/ml. On the other hand, Fr. V tightly bound to DEAE-cellulose chromatography, and four LCC fractions (Frs.

Fig. 4. Inhibition of HSV-1 plaque formation by Fr. VI and Fr. VIb in various cultured cells. The indicated doses of Fr. VI were added to the CV-1 cells at the time of infection with HSV-1 (HF) (●) or HSV-1 (F) (○), to the Vero cells at the time of infection with herpes virus simplex type 2 (G) (■), or to the A549 cells at the time of infection with HSV-1 (HF) (□). The CV-1 cells were also infected with HSV-1 (HF) in the presence of the indicated doses of Fr. VIb (▲). After washing with DME, these infected cells were overlaid with agarose, further incubated for 2 days in the presence of the same amounts of Fr. VI or Fr. VIb, and the number of plaques was then determined. Each value represents mean of triplicate

determinations. Cited from Fukuchi, et al., (1989a), with permission.

C V -1-H S V -1(H F) C V -1-H S V -1(F) V ero-H S V -2(G ) A 549-H S V -1(H F) C V -1-H S V -1(H F)'

0 μg/m l Fr. V I

30 100 300

LCCs from pine cone from *Pinus parviflora* Sieb et Zucc., and *Pinus elliottii* var. Elliotti [SI (selectivity index for measuring anti-HIV activity) =14, 28), bark of *Erythroxylum catuaba* Arr. Cam. (SI=43) (Manabe, et al., 1992), husk and mass of *Theobroma cacao* (SI= 311, 46) (Sakagami et al., 2008, 2011) and cultured extract of *Lentinus edodes* mycelia (LEM) (SI=>94) (Kawano et al. 2010) and mulberry juice (SI=7) (Sakagami et al., 2007, 2010b; Sakagami & Watanabe, 2011) showed higher anti-HIV activity than lower molecular weight polyphenols, such as tannins (SI=1-11) (Nakashima, et al., 1992b) and flavonoids (SI=1) (Fukai, et al., 2000), and natural and chemically modified glucans [*N*,*N*-dimethylaminoethyl paramylon, *N*,*N*-diethylaminoethyl paramylon, 2-hydroxy-3-trimethylammoniopropyl paramylon, sodium caroboxymethyl paramylon, carboxymethyl-TAK) (SI=1) except for sulfated polysaccharide (such as paramylon sulfate and dextran sulfate) (Koizumi, et al., 1993). Limited digestion of lignin structure by NaClO2 resulted in significant loss of anti-HIV activity (from SI=14 to 3), whereas removal of the monosaccharide residues by acidcatalyzed hydrolysis did not significantly affect the anti-HIV activity (from SI= 14 to 13) (Lai et al., 1992) suggesting that that phenylpropenoid polymer, but not sugar moiety, is important for anti-HIV activity. This was confirmed by our finding that dehydrogenation polymers of phenylpropenoids without carbohydrate showed generally higher anti-HIV activity (SI=105) than LCCs (Nakashima, et al., 1992a). On the other hand, phenylpropenoid monomers (*p*-coumaric acid, ferulic acid, caffeic acid) were inactive, suggesting the importance of highly polymerized structure (Nakashima, et al., 1992a). The mechanism of anti-HIV activity induction has been suggested to be mediated by the inhibition of HIV adsorption to the cells (Nakashima, et al., 1992a). *In vitro*, LCCs have also been reported to inhibit the HIV-1 reverse transcriptase activity (Lai, et al., 1990, 1992) and HIV-1 protease activity (Ichimura, et al., 1999).

#### **2.3 Anti-HSV activity** *in vitro*

#### **2.3.1 Inhibition of HSV-1 infection by Fr. VI**

Inhibition of HSV infection was determined by plaque assay. Cells were inoculated with HSV-1 (200-400 plaques per well (3.5 cm diameter) 2 days after infection. Fr. VI showed potent anti-HSV-1 activity. Addition of Fr. VI during and after adsorption significantly reduced the number of plaques without affecting the morphology of the CV-1 cells. The plaque formation of HSV-1 was significantly inhibited by Fr. VI at a concentration of more than 0.1 g/ml and completely inhibited by Fr. VI at more than 10 g/ml (Fig. 4) (Fukuchi, et al., 1989a). Fr. VI inhibited the cytopathic effect of two different HSV-1 strains (HF and F) and HSV-2 strain G on two samples of cultured monkey kidney cells (CV-1 and Vero) and one sample of human adenocarcinoma cells (A-549). From the doseresponse curves, the doses of Fr. VI that inhibited plaque formation by 50% (50% effective dose) in these cells were calculated to be 0.1-0.3 g/ml (Fig. 4). When Fr. VI was adsorbed on and eluted from Sephadex LH-60, anti-HIV activity was slightly enhanced (Fr. VIb in Fig. 4).

Neither the growth rate nor the saturation density of CV-1 cells was significantly affected by up to 100 g/ml of Fr. VI (Fig. 5). This indicates that the anti-HSV-1 effect of Fr. VI was not merely due to toxicity for the host cell.

LCCs from pine cone from *Pinus parviflora* Sieb et Zucc., and *Pinus elliottii* var. Elliotti [SI (selectivity index for measuring anti-HIV activity) =14, 28), bark of *Erythroxylum catuaba* Arr. Cam. (SI=43) (Manabe, et al., 1992), husk and mass of *Theobroma cacao* (SI= 311, 46) (Sakagami et al., 2008, 2011) and cultured extract of *Lentinus edodes* mycelia (LEM) (SI=>94) (Kawano et al. 2010) and mulberry juice (SI=7) (Sakagami et al., 2007, 2010b; Sakagami & Watanabe, 2011) showed higher anti-HIV activity than lower molecular weight polyphenols, such as tannins (SI=1-11) (Nakashima, et al., 1992b) and flavonoids (SI=1) (Fukai, et al., 2000), and natural and chemically modified glucans [*N*,*N*-dimethylaminoethyl paramylon, *N*,*N*-diethylaminoethyl paramylon, 2-hydroxy-3-trimethylammoniopropyl paramylon, sodium caroboxymethyl paramylon, carboxymethyl-TAK) (SI=1) except for sulfated polysaccharide (such as paramylon sulfate and dextran sulfate) (Koizumi, et al., 1993). Limited digestion of lignin structure by NaClO2 resulted in significant loss of anti-HIV activity (from SI=14 to 3), whereas removal of the monosaccharide residues by acidcatalyzed hydrolysis did not significantly affect the anti-HIV activity (from SI= 14 to 13) (Lai et al., 1992) suggesting that that phenylpropenoid polymer, but not sugar moiety, is important for anti-HIV activity. This was confirmed by our finding that dehydrogenation polymers of phenylpropenoids without carbohydrate showed generally higher anti-HIV activity (SI=105) than LCCs (Nakashima, et al., 1992a). On the other hand, phenylpropenoid monomers (*p*-coumaric acid, ferulic acid, caffeic acid) were inactive, suggesting the importance of highly polymerized structure (Nakashima, et al., 1992a). The mechanism of anti-HIV activity induction has been suggested to be mediated by the inhibition of HIV adsorption to the cells (Nakashima, et al., 1992a). *In vitro*, LCCs have also been reported to inhibit the HIV-1 reverse transcriptase activity (Lai, et al., 1990, 1992) and HIV-1 protease

Inhibition of HSV infection was determined by plaque assay. Cells were inoculated with HSV-1 (200-400 plaques per well (3.5 cm diameter) 2 days after infection. Fr. VI showed potent anti-HSV-1 activity. Addition of Fr. VI during and after adsorption significantly reduced the number of plaques without affecting the morphology of the CV-1 cells. The plaque formation of HSV-1 was significantly inhibited by Fr. VI at a concentration of more than 0.1 g/ml and completely inhibited by Fr. VI at more than 10 g/ml (Fig. 4) (Fukuchi, et al., 1989a). Fr. VI inhibited the cytopathic effect of two different HSV-1 strains (HF and F) and HSV-2 strain G on two samples of cultured monkey kidney cells (CV-1 and Vero) and one sample of human adenocarcinoma cells (A-549). From the doseresponse curves, the doses of Fr. VI that inhibited plaque formation by 50% (50% effective dose) in these cells were calculated to be 0.1-0.3 g/ml (Fig. 4). When Fr. VI was adsorbed on and eluted from Sephadex LH-60, anti-HIV activity was slightly enhanced (Fr. VIb in

Neither the growth rate nor the saturation density of CV-1 cells was significantly affected by up to 100 g/ml of Fr. VI (Fig. 5). This indicates that the anti-HSV-1 effect of Fr. VI was not

activity (Ichimura, et al., 1999).

**2.3 Anti-HSV activity** *in vitro* 

Fig. 4).

**2.3.1 Inhibition of HSV-1 infection by Fr. VI**

merely due to toxicity for the host cell.

Fig. 4. Inhibition of HSV-1 plaque formation by Fr. VI and Fr. VIb in various cultured cells. The indicated doses of Fr. VI were added to the CV-1 cells at the time of infection with HSV-1 (HF) (●) or HSV-1 (F) (○), to the Vero cells at the time of infection with herpes virus simplex type 2 (G) (■), or to the A549 cells at the time of infection with HSV-1 (HF) (□). The CV-1 cells were also infected with HSV-1 (HF) in the presence of the indicated doses of Fr. VIb (▲). After washing with DME, these infected cells were overlaid with agarose, further incubated for 2 days in the presence of the same amounts of Fr. VI or Fr. VIb, and the number of plaques was then determined. Each value represents mean of triplicate determinations. Cited from Fukuchi, et al., (1989a), with permission.

Fig. 5. Effect of Fr. VI on the growth of CV-1 cells. Each value represents mean of triplicate assays. Cited from Fukuchi, et al., (1989a), with permission.

#### **2.3.2 Anti-HSV activity of LCCs from other plants, natural and chemically modified polysaccharides**

Neutral polysaccharide (Fr. I) and uronic acid-rich polysaccharide (Fr. II) from pine cone extract of *Pinus parviflora* Sieb. et Zucc. had no anti-HSV activity at 10 g/ml. Similarly, popular antitumor polysaccharides, such as paramylon, PSK, Schizophyllan, and chemically modified glucans (*N,N*-dimethylaminoethylparamylon, sodium carboxymethylparamylon, sodium paramylon sulfate, carboxymethyl TAK) were all inactive at 10 g/ml. On the other hand, Fr. V tightly bound to DEAE-cellulose chromatography, and four LCC fractions (Frs.

Treatment of Herpes Simplex Virus

Fr. VI (g/ml)

permission.

**2.3.3 Interference by Fr. VI of HSV-1 cellular adsorption** 

was slightly lower than that of Fr. VI (Table 5).

Cell-bound radioactivity

Fr. VI 0 3007168 10 438230 85 Lignin 0 5367184 10 2774491 48 100 19353 96

triplicate assays. Cited from Fukuchi, et al., (1989a), with permission.

Sample Dose (g/ml) cpm % of inhibition

Table 5. Inhibition of radiolabeled virus adsorption by Fr. VI and lignin. CV-1 cells were incubated for 1 hour at 37C with the radioactive virus particle equivalent to 60,000 cpm (20,000 PFU/well) in the absence or presence of the indicated amounts of Fr. VI or lignin, and the cell-bound radioactivity was then determined. Each value represents mean S.D. of

We next investigated the effect of Fr. VI on virus penetration. Cells were first adsorbed for 1~2 hours with virus (200-400 PFU/well) at 4C, a condition that allows virus adsorption but not virus penetration. The cells were treated with Fr. VI and the temperature was then raised to 37C to initiate virus penetration. Fr. VI (1 g/ml) did not inhibit plaque formation after completion of virus adsorption (data not shown). From the results, it was concluded that Fr. VI inhibits virus adsorption on target cells, but does not inhibit virus penetration.

of the cells with Fr. VI for 6 days did not decrease the number of plaques.

0 0 0 229 10 (6 days) 0 0 204 11 0 10 0 0 100 0 0 10 230 0 10 (6 days) 0 10 188 18

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 181

To determine the point of inhibition of HSV-1 infection, cells were treated with Fr. VI at various times before and after HSV-1 infection. Table 4 shows that: (i) no protective effect was observed when Fr. VI was not present in the adsorption medium, and (ii) pretreatment

Before adsorption During adsorption After adsorption No. of plaques % of inhibition

Table 4. Dependence of anti-HSV activity induction by Fr. VI on treatment schedule. CV-1 cells were infected with HSV-1 strain HF. Fr. VI was added at the indicated stages. Each value represents mean of three separate assays. Cited from Fukuchi, et al., (1989a), with

The results suggest that the protective effect of Fr. VI might be caused by its inhibition of virus adsorption. To test this possibility, the CV-1 cells were incubated with higher concentrations of radiolabeled virus particles (20,000 PFU) in the presence of Fr. VI, and the cell-bound radioactivity was measured. Table 5 shows Fr. VI at 10 g/ml significantly inhibited the binding of the radiolabeled virus particles, even when the cells were incubated with higher concentrations of virus. Lignin similarly inhibited virus adsorption, but its effect

VI-IX) almost completely inhibited the HSV-infection at 10 g/ml (Table 3). LCC fractions (Frs. VI, VII) obtained from cones of other Japanese pine trees (*Pinus densiflora* Sieb. et Zucc., *Pinus thunbergii* Parl.), three Brazilian pine trees (*Pinus elliottii* var. Elliottii, *Pinus taeda* L., *Pinus caribaea* var. Hondurensus) and one Finnish pine tree (*Pinus sylvestris* L.), and from the seed shells of *Pinus parviflora* Sieb. et Zucc., and *Pinus armandii* Franch, also showed potent anti-HSV activity (Table 3).


Table 3. Anti-HSV activity of LCCs, natural and synthetic polysaccharides, added at 10 g/ml. Each value represents mean of triplicate assays. Cited from Fukuchi, et al., (1989a), with permission.

#### **2.3.3 Interference by Fr. VI of HSV-1 cellular adsorption**

180 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

VI-IX) almost completely inhibited the HSV-infection at 10 g/ml (Table 3). LCC fractions (Frs. VI, VII) obtained from cones of other Japanese pine trees (*Pinus densiflora* Sieb. et Zucc., *Pinus thunbergii* Parl.), three Brazilian pine trees (*Pinus elliottii* var. Elliottii, *Pinus taeda* L., *Pinus caribaea* var. Hondurensus) and one Finnish pine tree (*Pinus sylvestris* L.), and from the seed shells of *Pinus parviflora* Sieb. et Zucc., and *Pinus armandii* Franch, also showed potent

plaques

% of inhibition

Sample Fr. No. of

Neutral polysaccharide from cone of *Pinus parviflora* Sieb et Zucc. I 310 0 Acidic polysaccharide from cone of *Pinus parviflora* Sieb et Zucc. II 321 0 LCCs from pine cone of *Pinus parviflora* Sieb et Zucc. V 0 100 VI 0 100 VII 33 89 VIII 0 100 IX 0 100 LCCs from cone of *Pinus densiflora* Sieb. et Zucc. VI 0 100 VII 9 97 LCCs from cone of *Pinus thunbergii* Parl. VI 0 100 VII 0 100 LCCs from cone of *Pinus elliottii* var. Elliottii VI 2 99 VII 0 100 LCCs from cone of *Pinus taeda* L. VI 0 100 VII 12 96 LCCs from cone of *Pinus caribaea* var. Hondurenses VI 0 100 VII 0 100 LCCs from cone of *Pinus sylvestris* L. VI 0 100 VII 0 100 LCCs from seed shell of *Pinus parviflora* Sieb et. Zucc. VI 0 100 VII 0 100 LCC from seed shell of *Pinus armandii* Franch VI 0 100 Paramylon 310 0 PSK 320 0 Schizophyllan 325 0 *N,N*-dimethyaminoethylparamylon 306 0 Sodium carboxymethylparamylon 312 0 Sodium paramylon sulfate 299 2 Carboxymethyl-TAK 320 0 Alkali-lignin 0 100 Tannic acid 0 100 Saline (control) 305 Table 3. Anti-HSV activity of LCCs, natural and synthetic polysaccharides, added at 10 g/ml. Each value represents mean of triplicate assays. Cited from Fukuchi, et al., (1989a),

anti-HSV activity (Table 3).

with permission.

To determine the point of inhibition of HSV-1 infection, cells were treated with Fr. VI at various times before and after HSV-1 infection. Table 4 shows that: (i) no protective effect was observed when Fr. VI was not present in the adsorption medium, and (ii) pretreatment of the cells with Fr. VI for 6 days did not decrease the number of plaques.


Table 4. Dependence of anti-HSV activity induction by Fr. VI on treatment schedule. CV-1 cells were infected with HSV-1 strain HF. Fr. VI was added at the indicated stages. Each value represents mean of three separate assays. Cited from Fukuchi, et al., (1989a), with permission.

The results suggest that the protective effect of Fr. VI might be caused by its inhibition of virus adsorption. To test this possibility, the CV-1 cells were incubated with higher concentrations of radiolabeled virus particles (20,000 PFU) in the presence of Fr. VI, and the cell-bound radioactivity was measured. Table 5 shows Fr. VI at 10 g/ml significantly inhibited the binding of the radiolabeled virus particles, even when the cells were incubated with higher concentrations of virus. Lignin similarly inhibited virus adsorption, but its effect was slightly lower than that of Fr. VI (Table 5).


Table 5. Inhibition of radiolabeled virus adsorption by Fr. VI and lignin. CV-1 cells were incubated for 1 hour at 37C with the radioactive virus particle equivalent to 60,000 cpm (20,000 PFU/well) in the absence or presence of the indicated amounts of Fr. VI or lignin, and the cell-bound radioactivity was then determined. Each value represents mean S.D. of triplicate assays. Cited from Fukuchi, et al., (1989a), with permission.

We next investigated the effect of Fr. VI on virus penetration. Cells were first adsorbed for 1~2 hours with virus (200-400 PFU/well) at 4C, a condition that allows virus adsorption but not virus penetration. The cells were treated with Fr. VI and the temperature was then raised to 37C to initiate virus penetration. Fr. VI (1 g/ml) did not inhibit plaque formation after completion of virus adsorption (data not shown). From the results, it was concluded that Fr. VI inhibits virus adsorption on target cells, but does not inhibit virus penetration.

Treatment of Herpes Simplex Virus

González et al. (2009) with permission.

González, et al., (2009) with permission.

**No of patients**

**No of patients**

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 183

took part in the study. The patients were classified into the prodromic (16 patients), erytema (11 patients), papule edema (1 patient), vesicle/pustule (13 patients) and ulcer stages (7 patients). One mg of LCC-ascorbic acid tablet or solution was orally administered three times daily for a month. Clinical evaluations were made at least three times a week during the two first weeks after the onset and every six months during the subsequent year to identify recurrence episodes. The patients who began the LCC-ascorbic acid treatment within the first 48 hours did not develop HSV-1 characteristic lesions, whereas those patients who began the treatment later experienced a shorter duration of cold sore lesions and a decrease in the symptoms compared with previous episodes. The majority of the patients reported a reduction in the severity of symptoms and a reduction in the recurrence episodes after the LCC-ascorbic acid treatment compared with previous episodes, suggesting its possible

> 1 3 4 5 6 7 8 9 10 12 14 15 **Duration of syptoms (days)**

**Duration of symptoms (days)** 

Usual duration Duration after treatment

12345679 **Initial day of treatment**

Decreases in symptoms No decrease in symptoms

Fig. 8. Symptoms reduction according to the day of starting pine cone lignin and ascorbic acid complex treatment after onset. Symptoms were reduced notoriously when the treatment was taken in the first 48 h. after onset Kendall's Tau-b 0.456 p= 0.001. Cited from

Fig. 7. Previous and new duration of lesions after pine cone ascorbic acid treatment. A significant difference in the duration of lesions was found between before and after treatment. Usual duration is based on patient report. Student's t 4.202 p =0.001. Cited from

applicability for the prevention and treatment of HSV-1 infection. Figs. 7, 8 and 9.

We have previously reported that LCC also inhibited the adsorption of HIV to the cells (Nakashima, et al., 1992a, 1992b).

Recently, carboxylated lignins, synthesized using 4-hydroxy cinnamic acid scaffold by enzymatic oxidative coupling inhibited the entry of HSV-1 entry into the cells (Thakkar et al., 2010). Sulfated LCL (PPS-2b) (MW8500) also showed anti-HSV activity possibly by inhibiting the viral binding and penetration into host cells. Prunella cream formulated with a semi-purified fraction significantly reduced the skin lesion and mortality induced by HSV-1 infection in Guinea pigs (Zhang et al., 2007) The anti-HSV activity of sulfated lignins depended on their molecular weight, with the maximum at 39.4 kDa (Raghuraman et al. 2007).

#### **2.4 Clinical effect of LCC-ascorbic acid tablet**

The combination of alternative products can provide an effective therapy. To evaluate anti-HSV-1 activity of a pine cone LCC and ascorbic acid treatment, a clinical pilot study was carried out. We have modified the extraction method of LCC to achieve the mass production at the factory level (Fig. 6). Each LCC-ascorbic acid tablet contained a mixture of 50 mg pine cone extract powder JS, 50 mg ascorbic acid, 83 mg maltitol, 13 mg potato starch and 13 mg calcium stearate.

Fig. 6. Modified method for mass production of LCC at the factory level.

A pilot clinical study with pine cone lignin and ascorbic acid complex treatment against HSV-1-patients was carried out to evaluate the reduction of the duration with lesions, and the decrease of symptoms. A convenience sample of forty eight healthy patients of both genders between 4 and 61 years old (mean: 31±16.12 years), with active lesions of HSV-1,

We have previously reported that LCC also inhibited the adsorption of HIV to the cells

Recently, carboxylated lignins, synthesized using 4-hydroxy cinnamic acid scaffold by enzymatic oxidative coupling inhibited the entry of HSV-1 entry into the cells (Thakkar et al., 2010). Sulfated LCL (PPS-2b) (MW8500) also showed anti-HSV activity possibly by inhibiting the viral binding and penetration into host cells. Prunella cream formulated with a semi-purified fraction significantly reduced the skin lesion and mortality induced by HSV-1 infection in Guinea pigs (Zhang et al., 2007) The anti-HSV activity of sulfated lignins depended on their molecular weight, with the maximum at 39.4 kDa (Raghuraman et al.

The combination of alternative products can provide an effective therapy. To evaluate anti-HSV-1 activity of a pine cone LCC and ascorbic acid treatment, a clinical pilot study was carried out. We have modified the extraction method of LCC to achieve the mass production at the factory level (Fig. 6). Each LCC-ascorbic acid tablet contained a mixture of 50 mg pine cone extract powder JS, 50 mg ascorbic acid, 83 mg maltitol, 13 mg potato starch and 13 mg

> Pine cone (100%) Hot water wash 1% NaOH extraction

Neutralized with HCl

EtOH treatment ppt

Concentrated Mixed with dextrin Dried Crude LCC fraction (50%)

Fig. 6. Modified method for mass production of LCC at the factory level.

Concentrated with reduced pressure

Dissolved in hot water to remove EtOH

A pilot clinical study with pine cone lignin and ascorbic acid complex treatment against HSV-1-patients was carried out to evaluate the reduction of the duration with lesions, and the decrease of symptoms. A convenience sample of forty eight healthy patients of both genders between 4 and 61 years old (mean: 31±16.12 years), with active lesions of HSV-1,

Extract Residue

(Nakashima, et al., 1992a, 1992b).

**2.4 Clinical effect of LCC-ascorbic acid tablet** 

2007).

calcium stearate.

took part in the study. The patients were classified into the prodromic (16 patients), erytema (11 patients), papule edema (1 patient), vesicle/pustule (13 patients) and ulcer stages (7 patients). One mg of LCC-ascorbic acid tablet or solution was orally administered three times daily for a month. Clinical evaluations were made at least three times a week during the two first weeks after the onset and every six months during the subsequent year to identify recurrence episodes. The patients who began the LCC-ascorbic acid treatment within the first 48 hours did not develop HSV-1 characteristic lesions, whereas those patients who began the treatment later experienced a shorter duration of cold sore lesions and a decrease in the symptoms compared with previous episodes. The majority of the patients reported a reduction in the severity of symptoms and a reduction in the recurrence episodes after the LCC-ascorbic acid treatment compared with previous episodes, suggesting its possible applicability for the prevention and treatment of HSV-1 infection. Figs. 7, 8 and 9.

Fig. 7. Previous and new duration of lesions after pine cone ascorbic acid treatment. A significant difference in the duration of lesions was found between before and after treatment. Usual duration is based on patient report. Student's t 4.202 p =0.001. Cited from González et al. (2009) with permission.

Fig. 8. Symptoms reduction according to the day of starting pine cone lignin and ascorbic acid complex treatment after onset. Symptoms were reduced notoriously when the treatment was taken in the first 48 h. after onset Kendall's Tau-b 0.456 p= 0.001. Cited from González, et al., (2009) with permission.

Treatment of Herpes Simplex Virus

Patient Date of

<sup>49</sup>Mar,

<sup>23</sup>Mar,

<sup>21</sup>July,

<sup>53</sup>Feb,

<sup>57</sup>Jan

24\* Mar,

vitamin C.

Female Male

initiation of treatment

31

01

10

02

10

29

\*The patient presented a light skin rash.

Decrease of

Apr, 03

Mar,

July,

Feb, 02

Jan

Mar, 30

Fig. 10. Male 21 years old, a) Vesicles, b) Scab early stage

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 185

The patients will be seen as soon as possible after the start of the outbreaks. The tablets will be delivered to patients with the instruction of taking a tablet 3 times a day for 30 days. The patients will be seen daily or at least three times during the episode until the complete healing had occurred. Any possible adverse effects will be recorded. When a recurrence occurs, the patients will repeat the treatment as previously described. The placebo group

Until now we have identified 18 patients with a history of infection by HSV-1, who accepted to participate in the study, they signed the informed consent and answered the questionnaire. Among them only six patients have attended the clinic at the early stage of the disease, we carried out a clinical follow-up of patients to register the evolution of the disease (Table 6). In general the patients reported a reduction of the duration of the lesions

One patient presented a light rash at the tenth day of LCC treatment. This disappeared without treatment, once the patient suspended the treatment. At present, it is not clear whether the reaction could be related with the LCC treatment. Alternatively, this may suggest that the combination of LCC and vitamin C is necessary to reduce such incidence.

symptoms Gender/Age Edema Eritema Vesicle Erosion Seudomebrane Scab

11

Feb, 03

Mar, 31

Table 6. Evolution of the lesions of HSV-1 in five patients treated with LCC tablet without

31 - - - Apr,

01 - Mar,

July, 12

Feb, 04

12

Apr, 01

**a b** 

08

July , 12

Feb, 04

Jan 12

Apr, 01

06

Mar, 09

July, 13

Feb, 05

Jan 13

Apr, 02

Apr, 06

Mar, 09

July, 13

Feb, 05

Jan 14

Apr, 02

treatment protocol will be the same, but tablets without LCC will be administrated.

and of the symptoms, according to their previous experience (Fig. 10-14).

Mar,

02 - Mar,

10 - July,

Feb, 02

Mar, 29

10 - - Jan

Fig. 9. Recurrence rate after pine cone lignin ascorbic acid complex treatment. Cited from González et al. (2009) with permission.

Majority of the patients reported the reduction in the severity of symptoms and in the recurrent episodes. This pilot study suggests possible applicability of LCC-vitamin C tablet for the prevention and treatment of HSV-1 infection (González, et al., 2009). However, it is not clear whether this clinical effect is due to the antiviral action of LCC itself or due to the combination effect of LCC + vitamin C.

We found significant differences between the usual and current duration after the pine cone lignin and ascorbic acid combination treatment and differences in the reduction of symptoms, taking into account the historical data provided by the patients. However, the small number of patients and the lack of a control group limited our ability to generalize the results to a larger population. To evaluate the effectiveness of pine cone lignin and ascorbic acid combination treatment, subsequent double-blind randomized controlled clinical trials must be done in a representative sample of patients who suffer from recurrent HSV-1.

#### **2.5 Clinical effect of LCC tablet without vitamin C**

In order to evaluate the effect of LCC obtained from pine " *Pinus parviflora* Sieb et Zucc." without ascorbic acid, on the treatment of infection by HSV type I, we already started a cross-sectional study on a sample of the patients, from the School of Dentistry of the Mexico State University. It has been planned to develop a double blind randomized clinical study in a larger, heterogeneous sample of captive population, incorporating students, professors and working personal of the Schools of Health Sciences of the University, the study will be carried out during three years.

Patients aged 18 or more who gave informed consent will enter into the study; they will be initially identified through an interview, and they will be asked to answer a questionnaire that includes signs and symptoms, the triggers and the treatments used. The patients will be randomized for treatment or placebo. No other antiviral drugs will be permitted during the study

No recurrence 4 w eeks 5 to 10 w eeks

Fig. 9. Recurrence rate after pine cone lignin ascorbic acid complex treatment. Cited from

Majority of the patients reported the reduction in the severity of symptoms and in the recurrent episodes. This pilot study suggests possible applicability of LCC-vitamin C tablet for the prevention and treatment of HSV-1 infection (González, et al., 2009). However, it is not clear whether this clinical effect is due to the antiviral action of LCC itself or due to the

We found significant differences between the usual and current duration after the pine cone lignin and ascorbic acid combination treatment and differences in the reduction of symptoms, taking into account the historical data provided by the patients. However, the small number of patients and the lack of a control group limited our ability to generalize the results to a larger population. To evaluate the effectiveness of pine cone lignin and ascorbic acid combination treatment, subsequent double-blind randomized controlled clinical trials must be done in a representative sample of patients who suffer from recurrent HSV-1.

In order to evaluate the effect of LCC obtained from pine " *Pinus parviflora* Sieb et Zucc." without ascorbic acid, on the treatment of infection by HSV type I, we already started a cross-sectional study on a sample of the patients, from the School of Dentistry of the Mexico State University. It has been planned to develop a double blind randomized clinical study in a larger, heterogeneous sample of captive population, incorporating students, professors and working personal of the Schools of Health Sciences of the University, the study will be

Patients aged 18 or more who gave informed consent will enter into the study; they will be initially identified through an interview, and they will be asked to answer a questionnaire that includes signs and symptoms, the triggers and the treatments used. The patients will be randomized for treatment or placebo. No other antiviral drugs will be permitted during the

11 to 20 w eeks More than 20 w eeks

González et al. (2009) with permission.

combination effect of LCC + vitamin C.

carried out during three years.

study

**2.5 Clinical effect of LCC tablet without vitamin C**

**2%2% 2% 4%**

**90%**

The patients will be seen as soon as possible after the start of the outbreaks. The tablets will be delivered to patients with the instruction of taking a tablet 3 times a day for 30 days. The patients will be seen daily or at least three times during the episode until the complete healing had occurred. Any possible adverse effects will be recorded. When a recurrence occurs, the patients will repeat the treatment as previously described. The placebo group treatment protocol will be the same, but tablets without LCC will be administrated.

Until now we have identified 18 patients with a history of infection by HSV-1, who accepted to participate in the study, they signed the informed consent and answered the questionnaire. Among them only six patients have attended the clinic at the early stage of the disease, we carried out a clinical follow-up of patients to register the evolution of the disease (Table 6). In general the patients reported a reduction of the duration of the lesions and of the symptoms, according to their previous experience (Fig. 10-14).

One patient presented a light rash at the tenth day of LCC treatment. This disappeared without treatment, once the patient suspended the treatment. At present, it is not clear whether the reaction could be related with the LCC treatment. Alternatively, this may suggest that the combination of LCC and vitamin C is necessary to reduce such incidence.


\*The patient presented a light skin rash.

Table 6. Evolution of the lesions of HSV-1 in five patients treated with LCC tablet without vitamin C.

Fig. 10. Male 21 years old, a) Vesicles, b) Scab early stage

Treatment of Herpes Simplex Virus

over expression experiments.

research.

**3. Conclusion** 

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 187

We have demonstrated that LCC from pine cone of *Pinus parviflora* Sieb. Zucc. showed anti-HSV activity *in vitro*, by inhibiting the viral adsorption to the cells. LCC shows broad antiviral spectrum. LCC exhibited high affinity with influenza virus, and in contact to LCC, influenza virus rapidly lose the virulence (Sakagami, et al., 1992). LCC showed much higher anti-HIV activity than tannins (Nakashima, et al., 1992a, 1992b), whereas both LCC and tannins showed potent anti-HSV activity (Fukuchi, et al., 1989a, 1989b). Since virus is one of major risk factor of oral cavity cancer (Sakagami, 2010a), anti-viral action of LCC may

LCC shows also immunopotentiating activity. Administration with LCC induced antitumor, antimicrobial and anti-parasite activity, and enhanced the endogenous TNF production. At present, the receptors for LCC have not been identified. Recently, we have found LCC fraction isolated from *Lentinus edodes* mycelia extract (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) associated protein kinase 70kDa (Zap70), Janus tyrosine kinase (Jak)2 (1.0- to 1.2-fold), nuclear factor (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 (Kushida, et al., 2011). Identification of dectin-2 as LCC receptor awaits further confirmation with siRNA and gene

The other intriguing property of LCC is the synergy with vitamin C. Ascorbate derivatives that produced the doublet signal of ascorbate radical (sodium-L-ascorbate, L-ascorbic acid, Disoascorbic acid, 6-β-D-galactosyl-L-ascorbate, sodium 5,6-benzylidene-L-ascorbate) induced apoptosis in HL-60 cells, whereas ascorbate derivatives that did not produce radicals (Lascorbic acid-2-phosphate magnesium salt, L-ascorbic acid 2-sulfate and dehydroascorbic acid) did not induce apoptosis (Sakagami, et al., 1996a, 1996b). High concentrations of LCC 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 enhanced the radical intensity and cytotoxicity of sodium ascorbate. On the other hand,

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of sodium ascorbate (Sakagami et al., 2000, 2005, 2008) . This suggests the possible application of LCC as stimulator of vitamin C action, especially in the field of UV protection and anti-aging

Solvent fractionation of alkaline extract of the leaves of *Sasa senanensis* Rehder (SE) demonstrated that (i) chlorophyllin in SE was recovered from the water layer, that contains majority of compounds (more than 81%) and inhibited the NO production by macrophages more potently than other *n*-hexane, diethyl ether and ethylacetate layers (Sakagami, et al., 2010c). Three-dimensional HPLC analysis demonstrated that the majority of SE components are recovered from one major peak. Furthermore, LCC isolated from SE showed the unique greenish color of chlorophyllin (absorption maximum = 452 nm) (Sakagami, et al., 2010c).

), hydroxyl radical and

reduce the incidence of virus-triggered diseases such as cancer.

lower concentrations of LCC stimulated the superoxide anion (O2-

Fig. 11. Male 24 years old, a) Ulcerative stage, c) Seudomembrane stage, d) Skin rash

Fig. 12. Male, 25 years old. a) Erosion stage, b) Late scab stage, c) Complete healthy stage

Fig. 13. Female 57 years old, a) Ulcerative stage, b) Scab early stage

Fig. 14. Female 53 years old, a) Vesicle stage, b) Ulcerative stage, b) Seudomembrane stage

Fig. 10-14. Stages in the evolution of HSV-1 lesions in patients who received treatment with LCC. Only representative photographs were shown.

#### **3. Conclusion**

186 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

**b c** 

Fig. 11. Male 24 years old, a) Ulcerative stage, c) Seudomembrane stage, d) Skin rash

**a** 

Fig. 12. Male, 25 years old. a) Erosion stage, b) Late scab stage, c) Complete healthy stage

**a b c** 

**<sup>a</sup><sup>b</sup>**

**c** 

Fig. 14. Female 53 years old, a) Vesicle stage, b) Ulcerative stage, b) Seudomembrane stage

Fig. 10-14. Stages in the evolution of HSV-1 lesions in patients who received treatment with

Fig. 13. Female 57 years old, a) Ulcerative stage, b) Scab early stage

**b** 

LCC. Only representative photographs were shown.

**a**

We have demonstrated that LCC from pine cone of *Pinus parviflora* Sieb. Zucc. showed anti-HSV activity *in vitro*, by inhibiting the viral adsorption to the cells. LCC shows broad antiviral spectrum. LCC exhibited high affinity with influenza virus, and in contact to LCC, influenza virus rapidly lose the virulence (Sakagami, et al., 1992). LCC showed much higher anti-HIV activity than tannins (Nakashima, et al., 1992a, 1992b), whereas both LCC and tannins showed potent anti-HSV activity (Fukuchi, et al., 1989a, 1989b). Since virus is one of major risk factor of oral cavity cancer (Sakagami, 2010a), anti-viral action of LCC may reduce the incidence of virus-triggered diseases such as cancer.

LCC shows also immunopotentiating activity. Administration with LCC induced antitumor, antimicrobial and anti-parasite activity, and enhanced the endogenous TNF production. At present, the receptors for LCC have not been identified. Recently, we have found LCC fraction isolated from *Lentinus edodes* mycelia extract (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) associated protein kinase 70kDa (Zap70), Janus tyrosine kinase (Jak)2 (1.0- to 1.2-fold), nuclear factor (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 (Kushida, et al., 2011). Identification of dectin-2 as LCC receptor awaits further confirmation with siRNA and gene over expression experiments.

The other intriguing property of LCC is the synergy with vitamin C. Ascorbate derivatives that produced the doublet signal of ascorbate radical (sodium-L-ascorbate, L-ascorbic acid, Disoascorbic acid, 6-β-D-galactosyl-L-ascorbate, sodium 5,6-benzylidene-L-ascorbate) induced apoptosis in HL-60 cells, whereas ascorbate derivatives that did not produce radicals (Lascorbic acid-2-phosphate magnesium salt, L-ascorbic acid 2-sulfate and dehydroascorbic acid) did not induce apoptosis (Sakagami, et al., 1996a, 1996b). High concentrations of LCC 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 enhanced the radical intensity and cytotoxicity of sodium ascorbate. On the other hand, lower concentrations of LCC stimulated the superoxide anion (O2-), hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of sodium ascorbate (Sakagami et al., 2000, 2005, 2008) . This suggests the possible application of LCC as stimulator of vitamin C action, especially in the field of UV protection and anti-aging research.

Solvent fractionation of alkaline extract of the leaves of *Sasa senanensis* Rehder (SE) demonstrated that (i) chlorophyllin in SE was recovered from the water layer, that contains majority of compounds (more than 81%) and inhibited the NO production by macrophages more potently than other *n*-hexane, diethyl ether and ethylacetate layers (Sakagami, et al., 2010c). Three-dimensional HPLC analysis demonstrated that the majority of SE components are recovered from one major peak. Furthermore, LCC isolated from SE showed the unique greenish color of chlorophyllin (absorption maximum = 452 nm) (Sakagami, et al., 2010c).

Treatment of Herpes Simplex Virus

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 189

Fukai, T., Sakagami, H.; Toguchi, M.; Takayama, F.; Iwakura, I.; Atsumi, T.; Ueha, T.;

Fukuchi, K; Sakagami, H.; Ikeda, M.; Kawazoe, Y.; Oh-hara, T.; Konno, K.; Ichikawa, S.;

Fukuchi, K; Sakagami, H.; Okuda, T.; Hatano, T.; Tanuma, S.; Kitajima, K.; Inoue, Y.; Inoue

Gaby, AR. Natural Remedies for Herpes Simplex. (June, 2006). *Alternative Medicine Review*,

González, BS.; Yamamoto, MA.; Utsumi, K.; Aratsu, C. & Sakagami, H. (Nov-Dec, 2009).

Godfrey, HR.; Godfrey, NJ.; Godfrey, JC. & Riley, D. (May-June, 2001). A randomized

Hamuy, R. & Berman, B. (July-Aug, 1998). Treatment of herpes simplex virus infections with

Harada, H.; Sakagami, H.; Konno, K.; Sato T.; Osawa, N., Fujimaki, M. & Komatsu, N. (July-

Huleihel, M. & Isanu, V. Nov, 2002) Anti-herpes simplex virus effect of an aqueous extract of propolis. *Israel Medical Association Journal, 4 (suppl):* 923-927 ISSN: 1565-1088. Hunt, R. (July, 2011a). *Microbiology and immunology On-Line*. Medical Microbiology Virology

Hunt, R. (July,2011b). *Microbiology and immunology On-Line*. Medical Microbiology Virology -

Hovi, T.; Hirvimies, A.; Stenvik, M.; Vuola, E. & Pippuri, R. (June,1995). Topical treatment of

Ichimura, T.; Otake, T.; Mori, H. & Maruyama, S. (Dec, 1999). HIV-1 protease inhibition and

Retrieved from http://pathmicro.med.sc.edu/virol/herpes.htm

from http://pathmicro.med.sc.edu/virol/herpes.htm

*Ressearch*, Vol, 27, No.3, pp.: 263-270, ISSN: 0166-3542

Vol.20, No.4, pp. 2525-2536. ISSN: 0250-7005.

Vol.9, No.2, pp. 313-317. ISSN: 0250-7005.

Vol.11, No.(5-6), pp. 285-298. ISSN: 0166-3542.

No.1, pp. 11-18. ISSN: 1871-5265

ISSN: 1167-1122.

581-587. ISSN: 0250-7005.

http://www.encyclopedia.com/doc/1G1-148424510.html

virus. *In Vivo*, Vol.23, No.6, pp. 1011-1016. ISSN: 0258-851X.

Nakashima, H. & Nomura, T. (July-Aug, 2000). Cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines. *Anticancer Research,*

Hata, N.; Kondo, H.; & Nonoyama, M. (Mar-Apr, 1989a). Inhibition of herpes simplex virus infection by pine cone antitumor substances. *Anticancer Research,*

,S; Ichikawa, S.; Nonoyama, M. & Konno, K. (June-July, 1989b). Inhibition of herpes simplex virus infection by tannins and related compounds. *Antiviral Research,*

Vol.11,No.2,pp. 93-101. ISSN: 1089-5159. Retrieved from:

Clinical Pilot study of lignin-ascorbic acid combination treatment of herpes simplex

clinical trial on the treatment of oral herpes with topical zinc oxide/glycine. *Alternative Therapies in Health and Medicine*, Vol.7, No.3, pp. 49-56. ISSN: 1078-6791. Greco, A.; Diaz, JJ.; Thouvenot, D. & Morfin, F. (Mar, 2007). Novel targets for the

development of anti-herpes compounds. *Infectious Disorders - Drug Targets,* Vol.7,

topical antiviral agents. *European Journal of Dermatology,* Vol.8, No.5, pp. 310–319.

Aug, 1988). Induction of antimicrobial activity by antitumor substances from pine cone extract of *Pinus parviflora* Sieb. et Zucc. *Anticancer Research,* Vol.8, No.4, pp.


Chapter Nine Anti-viral Chemotherapy. (5th Edition), Murray PR, Rosenthal KS, Pfaller Michael AMD, ISBN: 978-0-323-05470-6, University of South Carolina.

recurrent mucocutaneous herpes with ascorbic acid containing solution. *Antiviral* 

anti-HIV effect of natural and synthetic water-soluble lignin-like substances.

These data strongly suggest the possible association of chlorophyllin with LCC in the native state or during extraction with alkaline solutions. Biological significance of such association remains to be investigated.

#### **4. Acknowledgment**

This study was supported in part by Universidad Autónoma del Estado de México (González FE04/2009) and a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (Sakagami No.14370607, 2002-2004, Sakagami No. 19592156, 2007- 2009).

#### **5. References**


These data strongly suggest the possible association of chlorophyllin with LCC in the native state or during extraction with alkaline solutions. Biological significance of such association

This study was supported in part by Universidad Autónoma del Estado de México (González FE04/2009) and a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (Sakagami No.14370607, 2002-2004, Sakagami No. 19592156, 2007-

Arduino, PG. & Porter, SR. (Feb, 2008). Herpes Simplex Virus Type 1 infection: overview on

Arens, M. & Travis, S. (May, 2000). Zinc salts Inactivate Clinical isolates of herpes simplex

Balzarini, J. (1994). Metabolism and mechanism of antiretroviral action of purine and

Bsoul, SA & Terezhalmy, GT. (May, 2004). Vitamin C in health and disease. Journal *Contemporary Dental Practice*, Vol.5, No.2, pp. 001-013 ISSN:1526-3711 Carson CF.; Hammer KA. & Riley, TV. (Jan 2006) Melaleuca alternifolia (Tea Tree) Oil: a

Chon, T.; Nguyen, L. & Elliott, TC. (July, 2007). Clinical inquiries. What are the best

Elish, D.; Singh, F. & Weinberg, JM. (July, 2004). Therapeutic options for herpes labialis, II:

Esmann, J. (Feb, 2001). The many challenges of facial herpes simplex virus infection. *Journal of Antimicrobial Chemotherapy*, VOL. 47 Topic T1*,* pp.17-27, ISSN 0305-7453. Emmert, DH. (Mar, 2000). Treatment of Common Cutaneous Herpes Simplex Virus

Everett, RD. (Aug, 2000). ICP0, a regulator of herpes simplex virus during lytic and latent

Fatahzadeh, M. & Schwartz, RA. (Nov, 2007). Human herpes simplex labialis. *Journal Clinical and Experimental Dermatology*, Vol.32, No.6, pp. 625-630. ISSN: 1365-2230 Femiano, F.; Gombos, F, & Scully, C. (Aug, 2005). Recurrent herpes labialis: a pilot study of

Frick DN. Helicases as antiviral drug target. (July-Aug, 2003) *Drug News and Perspectives*,

Topical agents. *Cutis,* Vol.74, No.1, pp. 5-40. ISSN: 0011-4162.

infection. *Bioessays,* Vol.22, No.8, pp. 761–70. ISSN: 0265-9247.

*Reviews,* Vol.19, No.1, pp. 50-62. ISSN: 0893-8512.

1708. ISSN: 0002-838X. Retrieved from http://www.aafp.org/afp/20000315/1697.html

Vol.16, No.6, pp. 352- 62 ISSN:0214-0934

423-425. ISSN: 0904-2512.

relevant clinic-pathological features. *Journal Oral Pathology Medicine*, Vol.37, No.2,

virus in vitro*. Journal of clinical Microbiology,* Vol. 38, No.5, pp. 1758-1762.

pyrimidine derivatives. *Pharmacy World & Science*, Vol.15, No.16, pp. 113-26. ISSN:

review on antimicrobial and other medicinal properties. *Clinical Microbiology* 

treatments for herpes labialis?. *Journal of Family Practice,* Vol.56, No.2, pp. 576–8.

Infections. *American Academy of Family Physician*, Vol.61, No.6, pp. 1697-704, 1705-6.

the efficacy of zinc therapy. *Journal of Oral Pathology & Medicine,* Vol. 34, No.7, pp*.* 

remains to be investigated.

**4. Acknowledgment** 

2009).

**5. References** 

pp. 107-21. ISSN: 1600-0714.

ISSN:0095-1137

ISSN: 0094-3509.

0928-1231.


Treatment of Herpes Simplex Virus

91-103. ISSN: 0166-3542.

ISSN: 1525-7797

*Pharmacology*, Vol 33, pp. 2-16 ISSN:0253-7613

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 191

Naito, T.; Irie, H.;Tsujimoto, K.; Ikeda, K ; Arakawa, T. & Koyama, AH. (Apr., 2009).

Nakashima, H.; Murakami, T.; Yamamoto, N.; Naoe, T.; Kawazoe, Y.; Konno, K. &

Narayana, KRAJ.; Sripal, M. & Chaluvadi, NR. (2001). Bioflavonoids classification,

Newcomb, WW.; Booy, FP. & Brown, JC. (July, 2007). "Uncoating the herpes simplex virus genome". *Journal of Molecular Biology.* Vol.370,No.4: 633–42. ISSN: 1089-8638. Paterson, J. & Kwong, M. (Apr, 2008). Recurrent Herpes Labialis, Assessment and Non-

*of Molecular Medicine*, Vol.23, No 4, pp. 495-499. ISSN: 1107-3756

*Pharmaceutical Bulletin*, Vol.40, No.8, pp. 2102-2105. ISSN: 1347-5223. Nakashima, H.; Murakami, T.; Yamamoto, N.; Sakagami, H.; Tanuma, S.; Hatano, T.;

Antiviral effect of arginine against herpes simplex virus type 1. *International Journal* 

Sakagami, H. (Aug, 1992a). Lignified materials as medicinal resources. V. Anti-HIV (human immunodeficiency virus) activity of some synthetic lignins. *Chemical and* 

Yoshida, T. & Okuda, T. (May, 1992b). Inhibition of human immunodeficiency viral replication by tannins and related compounds. *Antiviral Research,* Vol.18, No.1, pp.

pharmacological, biochemical effects and therapeutic potential. *Indian Journal of* 

Prescription Treatment. *Glaxo Smithkline Consumer Healthcare,* ISSN: 1079-2082. Retrived from http://www.pharmacyresource.ca/recurrent\_herpes\_labialis.pdf Pinnoji, RC.; Bedadala, GR.; George, B.; Holland, TC.; Hill, JM.; Hsia, SC. (June, 2007).

"Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification". *Journal of Virology .* Vol. 4, No.1, pp. 8149–56 56. ISSN: 0022-538X. Raghuraman, A.; Tiwari, V.; Zhao, Q.; Shukla, D.; Debnath, AK. & Desai, UR. (May, 2007).

Viral inhibition studies on sulfated lignin, a chemically modified biopolymer and a potential mimic of heparin sulfate. *Biomacromolecules*, Vol.8, No.5. pp. 1759-1763.

classification, pharmacological, biochemical effects and therapeutic potential. *Indian* 

Komatsu, N.; & Konno, K. (Nov-Dec, 1987). Antitumor activity of polysaccharide fractions from pine cone extract of *Pinus parviflora* Sieb. et Zucc. *Anticancer Research*,

1989). Molecular species of the antitumor and antiviral fraction from pine cone

immunological functions of extracts from the cone of Japanese white pine, Pinus parviflora Sieb. et Zucc. *Advances in Experimental Medicine and Biology,* Vol 319, No,

Ohata, H.; Momose, K.; & Takeda M. (Mar, 1996a). The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death. *Life Sciences*, Vol.58, No.14, pp. 1131-1138. ISSN: 0024-3205.

Raj Narayana, NK.; Spiral, RM.; Chaluvadi, MR.; & Krishna DR. (June, 2000). Bioflavonoids

Sakagami, H.; Ikeda, M.; Unten, S.; Takeda, K.; Murayama, J.; Hamada, A.; Kimura, K.;

Sakagami, H.; Oh-hara, T.; Kaiya T.; Kawazoe, Y.; Nonoyama, M.; & Konno, K. (Nov-Dec,

Sakagami, H.; Kuribayashi, N.; Iida, M.; Hagiwara, T.; Takahashi, H.; Yoshida, H.; Shiota, F.;

extract. *Anticancer Research*, Vol.9, No.6, pp. 1593-1598. ISSN: 0250-7005. Sakagami, H.; Konno, K.; Kawazoe, Y.; Lai, P.; & Nonoyama, M. (1992) Multiple

*Journal of Pharmacology,* Vol.33, pp. 2-16. ISSN: ISSN 0253-7613.

Vol.7, No.6, pp. 1153-1160. ISSN: 0250-7005.

pp.331-335. ISSN: 0065-2598

*Bioscience Biotechnology & Biochemistry*, Vol.63, No.12, pp. 2202-2024. ISSN: 0916- 8451.


Katsuyama, Y.; Yamasaki, H.; Tsujimoto, K.; Koyama HA., Ejima, D. & Arakawa, T. (Sept,

Kawano, M.; Sakagami, H.; Satoh, K.; Shioda, S.; Kanamoto, T; Terakubo, S.; Nakashima, H.

Koelle, DM. & Corey, L. (Feb, 2008). Herpes simplex: insights on pathogenesis and possible vaccines. *Annual Review of Medicine*, Vol.59, pp. 381–395. ISSN: 0066-4219. Koizumi, N.; Sakagami, H.; Utsumi, A.; Fujinaga, S.; Takeda, M.; Asano, K.; Sugawara, I.;

Kushida, T.; Makino, T.; Tomomura, M.; Tomomura, A.; & Sakagami, H. (Apr, 2011).

Kümel, G.; Schrader, S.; Zentgraf, H. & Brendel, M. (July, 1991) Therapy of banal HSV

Lai, PK.; Donovan, J.; Takayama, H.; Sakagami, H.; Tanaka, A.; Konno, K. & Nonoyama, M.

Lai, PK.; Oh-hara, T.; Tamura, Y.; Kawazoe, Y.; Konno, K.; Sakagami, H.; Tanaka, A. &

Manabe, H.; Sakagami, H.; Ishizone, H.; Kusano, H.; Fujimaki, M.; Wada, C.; Komatsu, N.;

Mårdberg, K; Trybala, E.; Glorioso, JC. & Bergström, T.(Aug, 2001). Mutational analysis of

*Journal of General Virology*, Vol. 82, No.8, pp. 1941-1950 ISSN:14652099 Mettenleiter, TC.; Klupp, BG. & Granzow, H. (Aug , 2006). "Herpesvirus assembly: a tale of

42, No. 7, pp.439-45. (Summary, Article in Germany) ISSN:0017-8470 Kümel, G.; Schrader, S.; Zentgraf, H.; Daus, H, & Brendel, M. (Dec 1990) The mechanism of

*Pharmaceutics,* Vol. 362, No.1-2, pp. 92-98 ISSN: 0378-5173

(LEM). *In Vivo*, Vol.24, No.4, pp. 543-552. ISSN : 0258-851X.

*Research,* Vol.31, No.4, pp. 1241-1248. ISSN: 0250-7005.

No.1, pp. 1-14. ISSN: 0166-3542.

pp 2989-2997. ISSN: 0022-1317

No.4, pp. 303-323. ISSN: 0022-1260.

1931-8405.

0258-851X.

1369-5274

8451.

*Bioscience Biotechnology & Biochemistry*, Vol.63, No.12, pp. 2202-2024. ISSN: 0916-

2008). Butyroyl-arginine as a potent virus inactivation agent. *International Journal of* 

& Makino, T. (July-Aug,, 2010). Lignin-like activity of *Lentinus edodes mycelia* Extract

Ichikawa, S.; Kondo, H.; Mori, S.; Miyatake, K.; Nakano, Y.; Nakashima, H.; Murakami, T.; Miyano, N. & Yamamoto,, N. (May, 1993). Anti-HIV (human immunodeficiency virus) activity of sulfated paramylon. *Antiviral Research*, Vol.21,

Enhancement of dectin-2 gene expression by lignin-carbohydrate complex from *Lentinus edodes* extract (LEM) in mouse macrophage-like cell line. *Anticancer* 

lesions: molecular mechanisms of the antiviral activity of zinc sulfate. *Hautarzt*. Vol.

the antiherpetic activity of zinc sulphate. *Journal of General Virology,* Vol. 71 No. 12,

(Feb, 1990). Modification of human immunodeficiency viral replication by pine cone extracts. *AIDS Research and Human Retroviruses,* Vol.6, No.2, pp. 205-217. ISSN:

Nonoyama, M. (1992). Polymeric phenylpropenoids are the active components in the pine cone extract that inhibit the replication of type-1 human immunodeficiency virus *in vitro*. *Journal of General and Applied Microbiology*, Vol.38,

Nakashima, H.; Murakami, T.; & Yamamoto N. (Mar-Apr, 1992). Effects of Catuaba extracts on microbial and HIV infection. *In Vivo*, Vol.6, No.2, pp. 161-166. ISSN:

the major heparin sulfate-binding domain of herpes simplex type 1 glycoprotein C.

two membranes". *Current Opinion in Microbiology,* Vol.9, No.4, pp. 423–9. ISSN:


Treatment of Herpes Simplex Virus

8177.

40. ISSN: 0944-7113.

with Lignin-Carbohydrate Complex Tablet, an Alternative Therapeutic Formula 193

Schnitzler, P.; Schuhmacher, A.; Astani, A.; & Reichling, J. (Sep, 2008). Melissa officinalis oil

Siegel, MA. (Sep, 002) Diagnosis and management of recurrent herpes simplex infections

Singh, BB.; Udani J.; Vinjamury S.; Der-Martirosian C.; Gandhi S.; Khorsan R.; Nanjegowda

Smith, JS. & Robinson, NJ. (Oct, 2002). Age-specific prevalence of infection with herpes

Spruance, SL.; Jones, T.; Blatter, MM.; Vargas, CM.; Barber, J.; Hill, J.; Goldstein, D. &

Spruance, SL.; Nett, R.; Mabury, T. Wolff, R. Johnson, J. & Spaulding, T. for the acyclovir

Spruance, SL. (Dec, 2005). Herpes Simplex Virus. Prophylactic chemotherapy with acyclovir

Stránská, R.; Schuurman, R.; Nienhuis, E.; Goedegebuure, IW.; Polman, M.; Weel, JF.;

Thakkar, JN.; Tiwari, V. & Dessai, UR. (Sep, 2010). Nonsulfated, cinnamic acid-based lignins

Terezhalmy, G.T.; Bottomley, WK, & Pelleu, GB (1978). The use of water-soluble

United States Food and Drug Administration [FDA], (June, 2011), Retrieved from

Yoon, JC.; Cho, JJ.; Yoo, SM. & Ha, YM. (Feb, 2000). Antiviral activity of ascorbic acid against

Zhang, Y., But, P.P., Ooi, V.E., Xu, H.X., Delaney, G.D., Lee, S.H. & Lee, S.F. (Sep. 2007).

http://www.drugs.com/sfx/acyclovir-side-effects.html

retrieve from http://www.highbeam.com. ISSN: 1089-5159.

Vol.15, No.186, Suppl 1:S3-28. ISSN: 1537-6613.

No.3, pp. 1072-1080. ISSN: 1098-6596.

ISSN: 0066-4804

ISSN: 0146-6615.

ISSN: 0368-3494.

pp. 1412-1416. ISSN: 1525-7797

No.1, pp. 56-62, ISSN1528-395X.

affects infectivity of enveloped herpesviruses. *Phytomedicine,* Vol 15, No.9, pp 734-

*Journal of the American Dental Association,* Vol 133, No 9, pp.,1245-1249 ISSN: 0002-

D. & Singh, V. (June, 2005) Safety and effectiveness of an L-lysine, zinc, and herbalbased product on the treatment of facial and circumoral herpes.(Original Research: Lysine / Herpes)." *Alternative Medicine Review. Thorne Research Inc.*. *HighBeam* 

simplex virus types 2 and 1: a global review*. The Journal of Infectious Diseases*,

Schultz, M. (Mar, 2003). High-Dose, Short Duration, Early Valaciclovir Therapy for Episodic Treatment of Cold Sores: Results of Two Randomized, Placebo-Controlled, Multicenter Studies. *Antimicrobial Agents and Chemotherapy*, Vol.47,

cream study group. (July 2002) Acyclovir cream for treatment of herpes simplex labialis: Results of two randomized, double bind, vehicle-controlled, multicenter clinical trials. *Antimicrobial Agents and Chemotherapy,* Vol. 46, No.7, pp. 2238-2243,

for recurrent herpes simplex. *Journal of Medical Virology,* Vol.41, No.1, pp. 27-32.

Wertheim-Van Dillen, PM.; Berkhout, RJ. & van Loon, AM. (Jan, 2005). Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization. *Journal of Clinical Virology,* Vol.32, No.1, pp. 7-18. ISSN: 1386-6532.

are potent antagonists of HSV-1 entry into cells. *Biomacromolecules*, Vol.11, No. 5,

bioflavonoid ascorbic acid complex in the treatment of recurrent herpes labialis. *Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontic,* Vol.45,

herpes simplex virus. *Journal Korean Society Microbiology,* Vol.35*,* No.1, pp. 1-8.

Chemical properties, mode of action, and in vivo anti-herpes activities of a lignin-


Sakagami, H.; Satoh, K.; Ohata, H.; Takahashi, H.; Yoshida, H.; Iida, M.; Kuribayashi, N.;

Sakagami, H.; Satoh, K.; Hakeda, Y. & Kumegawa, M. (Feb, 2000). Apoptosis-inducing

Sakagami, H.; Hashimoto, K.; Suzuki, F.; Ogiwara, T.; Satoh, K.; Ito H.; Hatano, T.; Yoshida,

Sakagami, H.; Asano, K.; Satoh, K.; Takahashi, K.; Kobayashi, M.; Koga, N.; Takahashi, H.;

mulberry juice fractions. *In Vivo*, Vol.21, No.3, pp. 499-506. ISSN: 0258-851X. Sakagami, H.; Satoh, K.; Fukamachi, H.; Ikarashi, T.; Simizu, A.; Yano, K.; Kanamoto, T.;

Sakagami, H. (2010a). Chapter 5.1.8. Oral Cavity Cancer. *Cancer Report 2010,* Tuncer AM,

Sakagami, H.; Zhou Li.; Kawano, M.; Thet, MM.; Takana, S.; Machino, M.; Amano, S.;

Sakagami, H.; Kawano, M.; May Maw, T.; Hashimoto, K.; Satoh, K.; Kanamoto, T.;

carbohydrate complex. *In Vivo,* Vol.25, No.2, pp. 229-236. ISSN: 0258-851X. Sakagami, H. & Watanabe S. (2011). Beneficial effects of mulberry on human health.

Schnitzler, P.; Koch C. & Reichling, J. (2007) Susceptibility of drug–resistant clinical herpes

Vol.16, No.5A, pp. 2635-2644. ISSN: 0250-7005.

No.17, pp. 2108-2120. ISSN: 0031-9422.

143. ISSN: 1939-4586.

327-33. ISSN: 0258-851X.

ISSN: 0163-7258.

196-4.

0066-4804.

735-744. ISSN: 0258-851X.

Sakagami, T.; Momose K. & Takeda, M. (Sep-Oct, 1996b). Relationship between ascorbyl radical intensity and apoptosis-inducing activity. *Anticancer Research*,

activity of vitamin C and vitamin K. *Cell Molecular Biology,* Vol.46, No.1, pp. 129-

T. & Fujisawa S. (Sep, 2005). Molecular requirements of lignin-carbohydrate complexes for expression of unique biological activities. *Phytochemistry,* Vol.66,

Tachikawa, R.; Tashiro, T.; Hasegawa, A.; Kurihara, K.; Ikarashi, T.; Kanamoto, T.; Terakubo, S.; Nakashima, H.; Watanabe, S. & Nakamura, W. (May-June, 2007). Anti-stress, anti-HIV and vitamin C-synergized radical scavenging activity of

Terakubo, S.; Nakashima, H.; Hasegawa, H.; Nomura, A.; Utsumi, K.; Yamamoto, M.; Maeda, Y. & Osawa, K. (May-June, 2008). Anti-HIV and vitamin C-synergized radical scavenging activity of cacao husk lignin fractions. *In Vivo*, Vol.22, No.3, pp.

Moore M, Qiao YL, Yoo K-Y, Tajima K, Ozgul N, Gultekin M, pp. 222-226, MN Medical & Nobel Publishing Company, Ankara, Turkey.ISBN:978-975-567-058-4. Sakagami, H.; Kushida, T.; Oizumi, T.; Nakashima, H. & Makino, T. (Oct, 2010b).

Distribution of lignin carbohydrate complex in plant kingdom and its functionality as alternative medicine. *Pharmacology & Therapeutics,* Vol.128, No.1, pp. 91-105.

Kuroshita, R.; Watanabe, S.; Chu, Q.; Wang, QT.; Kanamoto, T.; Terakubo, S.; Nakashima, H.; Sekine, K.; Shirataki, Y.; Hao, ZC.; Uesawa, Y.; Mohri, K.; Kitajima, M.; Oizumi, H. & Oizumi, T. (Sep-Oct, 2010c). Multiple Biological complex of alkaline extract of the leaves of *Sasa senanensis* Rehder. *In Vivo,* Vol.24, No.1, pp.

Terakubo, S.; Nakashima, H.; Haishima, Y.; Maeda, Y. & Sakurai, K. (Mar-Apr, 2011). Anti-HIV and immunomodulation activities of cacao mass lignin

*Phytotherapeutics and Human Health: Pharmacological and Molecular Aspects,* Farooqui AA, pp. 257-273, Nova Science Publishers, Inc, Hauppauge, NY. ISBN: 978-1-61761-

simplex virus type 1 strains to essential oils of ginger, thyme, hyssop, and sandalwood. *Antimicrobial Agents and Chemotherapy,* Vol *51,* pp. 1859-1862. ISSN


carbohydrate complex from Prunella vulgaris. *Antiviral Research*, Vol. 75, No. 3, pp. 242-249. ISSN: 0166-3542

Ziyaeyan, M.; Alborzi, A.; Japoni, A.; Kadivar, M.; Davarpanah, MA.; Pourabbas, B. & Abassian, A. (Dec, 2007). Frequency of acyclovir-resistant herpes simplex viruses isolated from the general immunocompetent population and patients with acquired immunodeficiency syndrome. *International Journal of Dermatology,* Vol.46, No.12, pp. 1263-6. ISSN: 0011-9059.

Ziyaeyan, M.; Alborzi, A.; Japoni, A.; Kadivar, M.; Davarpanah, MA.; Pourabbas, B. &

242-249. ISSN: 0166-3542

No.12, pp. 1263-6. ISSN: 0011-9059.

carbohydrate complex from Prunella vulgaris. *Antiviral Research*, Vol. 75, No. 3, pp.

Abassian, A. (Dec, 2007). Frequency of acyclovir-resistant herpes simplex viruses isolated from the general immunocompetent population and patients with acquired immunodeficiency syndrome. *International Journal of Dermatology,* Vol.46,

### *Edited by Patrick Arbuthnot*

The articles that appear in Antiviral Drugs - Aspects of Clinical Use and Recent Advances cover several topics that reflect the varied mechanisms of viral disease pathogenesis and treatment. Clinical management and new developments in the treatment of virus-related diseases are the two main sections of the book. The first part reviews the treatment of hepatitis C virus infection, the management of virus-related acute retinal necrosis, the use of leflunomide therapy in renal transplant patients, and mathematical modeling of HIV-1 treatment responses. Basic research topics are dealt with in the second half of the book. New developments in the treatment of the influenza virus, the use of animal models for HIV-1 drug development, the use of single chain camelid antibodies against negative strand RNA viruses, countering norovirus infection, and the use of plant extracts to treat herpes simplex virus infection are described. The content of the book is not intended to be comprehensive, but aims to provide the reader with insights into selected aspects of established and new viral therapies.

Antiviral Drugs - Aspects of Clinical Use and Recent Advances

Antiviral Drugs

Aspects of Clinical Use and Recent Advances

*Edited by Patrick Arbuthnot*

Photo by MLiberra / iStock