PHARMACOLOGICAL PROFILE

II. PHARMACOLOGICAL PROFILE

A. Hepatoprotective Effects Early pharmacological studies have demonstrated the ability of Sch B and

other lignans in protecting against liver damage induced by a variety of chemicals, such as acetaminophen, carbon tetrachloride (CCl 4 ), thiacetamide, and immunlogical toxins, in rodents (11–18). Recent studies in our laboratory

protected against CCl 4 and tumor necrosis factor-a-induced hepatotoxicity in

a dose-dependent manner in mice (19,20). Preliminary structure-activity relationship study, using Sch B and its lignan analogs, namely, schisandrin

A (Sch A, Fig. 1b), schisandrin C (Sch C, Fig. 1c), and a synthetic interme- diate of Sch C, dimethyl diphenyl bicarboxylate (DDB, Fig. 1d), indicated that the methylenedioxy group as well as the cyclooctadiene ring structure of Sch B are important structural determinants in its hepatoprotective action (21,22). Petreating mice with Sch B at a daily dose of 0.125–0.5 mmol/kg or

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1 mmol/kg, respectively, for 3 days also reduced the extent of hepatic injury induced by tacrine/bis-tacrine or menadione (23,24). In addition, mice pre- treated with Sch B at an oral daily dose of 1 mmol/kg for 3 days showed protective effect against galactosamine/endotoxin-induced toxicity (25). One point worth noting is that since lignans can cause irreversible inhibition on

hepatic GPT (26), the inability of DDB to protect against CCl 4 hepatotox- icity, though decreasing plasma GPT activity, was evidenced by the negative histological assessment on hepatic damage (27). Paradoxically, DDB has been shown to improve liver functions of patients suffering from chronic hepatitis (28). The hepatoprotective effect of DDB may therefore be limited to certain

kinds of liver injury, probably not that produced by CCl 4 . Treating rats with gomisin A [Gom A, Fig. 1e, a hydroxyl group (C 7 )- containing structural analog of Sch B] at a daily dose of 30 or 100 mg/kg for

4 days could suppress the increase in serum transaminase activity and the appearance of histological changes induced in liver by CCl 4 or D -galactos- amine (29). Gom A–treated rats (10–100 mg/kg/day, p.o., for 4 days) showed an accelerated proliferation of hepatocytes and recovery of liver function, as well as an increased hepatic blood flow after partial hepatectomy (30). In addition, Gom A (10 or 30 mg/kg, p.o., for 3 or 6 weeks) suppressed the fibrosis proliferation and accelerated both the liver regeneration and the

recovery of liver function in CCl 4 -induced chronic liver injury in rats (31). The effect of Gom A treatment on immunologically induced liver injuries has been investigated (32–34). Gom A pretreatment (5–50 mg/kg, p.o., for 4 weeks) reduced the mortality in mice or occurrence of hepatic failure in guinea pigs subjected to immunological challenge. As for sponta- neous hepatitis developed in Long Evans Cinnamon rats, Gom A treatment did not change the death rate, but the survival time was increased by 7–10 weeks when compared with that of the control (35).

B. Cardioprotective Effects An early study in our laboratory has shown the myocardial protective effect of

Shengmai San (36), a TCM formula used for the treatment of coronary heart disease (37), and the lignans derived from FS were found to contribute to the cardioprotective action (36). Pretreatment with the lignan-enriched FS extract, which has recently been shown to produce Sch B-like in vivo antioxidant activity at an equivalent potency of f30% (w/w) (38), at an oral dose of 0.8 g/kg/day for 3 days, protected against isoproterenol-induced myocardial injury in rats and ischemia-reperfusion (IR)-induced injury in isolated perfused hearts prepared from the pretreated rats (36). The effects of Sch B treatment on myocardial IR injury in isolated rat hearts were subsequently investigated under both in vitro and ex vivo conditions (39).

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In vitro administration of liposome-entrapped Sch B to the isolated-perfused hearts was found to be unable to protect against myocardial IR injury, whereas ascorbic acid and Trolox, a water-soluble analog of a-tocopherol, supplemented perfusate-produced protective effect, as evidenced by the significant decrease in the extent of lactate dehydrogenase (LDH) leakage as well as improvement in contractile force recovery. However, pretreatment with Sch B (0.6–1.2 mmol/kg/day, p.o., for 3 days) protected against IR- induced myocardial damage in a dose-dependent manner. A preliminary structure-activity relationship study indicated that both the methylenedioxy group and the cycloctadiene ring structure of Sch B are important structural determinants in mediating the protection against myocardial IR injury (40). Investigation of the possible pharmacological preconditioning effect of low doses of Sch B on the myocardium is underway in our laboratory.

C. Neuroprotective Effects Sch B and schisanhenol (Sal, Fig. 1f, also a dibenzocyclooctadiene lignan)

have been shown to protect oxidative damage induced in aging and ischemic- reperfused rat brain (41). Incubation of 8-month-old rat brain mitochondria and membrane suspension with a mixture of Fe 2+ -cysteine resulted in the formation of malondialdehyde (MDA), an end product of lipid peroxidation, and a decrease in ATPase activity. Sch B and Sal (100 AM) completely

inhibited these peroxidative damages induced by Fe 2+ -cysteine in rat brain mitochondrial and membrane preparation [41]. Oral administration of Sch B

or Sal (150 mg/kg) caused the increase in cytosolic glutathione peroxidase activity in rat brain tissue under anoxia and reoxygenation condition, with the effect of Sal being more potent (41). A recent study in our laboratory has demonstrated that Sch B pretreatment (1–2 mmol/kg, p.o., for 3 days) could reduce the mortality rate in a dose-dependent manner in mice following an intracerebroventricular injection of tert-butylhydroperoxide (42). However, DDB, when being administered at a dose of 2 mmol/kg/day, did not produce any significant effect on tert-butylhydroperoxide-induced cerebrotoxicity (43).

The ability of lignans to antagonize the effect of CNS-suppressing sub- stances, such as barbiturates, chloral hydrate, and halothane, supports their CNS-activating activity (44). The lignan extract (1–5 mg/kg, i.p.) could inhibit the CNS suppressive effect of hexenal and chloral hydrate in rats. Moreover, the CNS-activating effect of lignans was antagonized by dopamine receptor

DA 2 blocker. However, schisandrol A (Sol A, Fig. 1g, another lignan present in the extract) was found to prolong the sleeping time induced by phenobar- bital and decrease the spontaneous motor activity in mice, a manifestation of CNS-depressing activity (45). Further investigation has revealed the signifi-

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cant elevations of dopamine and its metabolite DOPAC (in striatum) and DA (in hypothalamus) in mice after intraperitoneal administration of Sol A at a dose of 50 or 100 mg/kg (46). Furthermore, Sol A showed no affinity for

dopamine D 1 and D 2 receptor, serotonin receptors, and a 1 - and a 2 -adrenergic receptors, nor did it affect the binding of dopamine D 1 and D 2 receptors. The inhibition exerted by schisandrol A on CNS activity may be related to the modulatory effect on dopamine metabolism in the CNS (46).

The cholinergic system is also influenced by the lignans. The lignan extract (10–30 mg/kg, p.o.) decreased the convulsant threshold and potenti- ated the antidiuretic action of nicotine, and potentiated the excitatory effect of carbachol on rat intestine (47,48). However, the lignans potentiated the action of reserpine only at a higher dose of 1.5 g/kg (48). In fact, the lignans affect the cholinergic system in a biphasic manner. At a lower dose of 280 mg/kg (p.o.), an indirect nicotinomimetic action was produced, whereas at a higher dose of 840 mg/kg (p.o.), a cholinolytic effect was observed (49).

A recent study has shown that pretreating mice with Sch B (0.025–0.5 mmol/kg/day, p.o., for 5 days) could enhance the passive avoidance response in mice, an indication of enhancement in cognitive function (23). This is consistent with the finding that lignan treatment (5–10 mg/day, p.o.) could improve the intellectual activity in humans (49).

D. Anticarcinogenic Effects The effect of Gom A on hepatocarcinogenesis caused by 3V-methyl-4-di-

methylaminobenzene (3V-MeDAB) in male Donryu rats has been investigated (50). Gom A treatment (30 mg/kg/day, p.o., for 5 weeks) significantly inhibited both increases in the number and size of glutathione S-transferases placental form (GST-P)-positive foci, a marker enzyme of preneoplasm, and the population of diploid nuclei, an indicator of proliferative state of hepatocytes, in the liver from rats simultaneously treated with 3VMeDAB. Gom A treatment also decreased the number of other hepatic-altered foci, such as those of the clear cell and basophilic cell type in the early stages (51). While Gom A increased GST activity in the liver by raising the level of GST-1 and 2 isozymes, it was observed that the biliary excretion of 3VMeDAB-related aminoazo dyes was increased in Gom A–treated rats, with its content in the liver being decreased in rats fed with a 3V-MeDAB-containing diet (50). Further study indicated that Gom A treatment (0.03% in the diet for 10 weeks) could reverse the 3V-MeDAB-induced increase in the ratio of diploid nuclei to tetrapoid nuclei (52). Gom A may therefore inhibit the hepatocarcin- genesis induced by 3V-MeDAB by enhancing the excretion of the carcinogen from the liver as well as reversing the abnormal cytokinesis (50–53). When different types of tumor promoters such as phenobarbital (PB) and deoxy-

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cholic acid (DCA) were administered for 5 weeks after the initiation by 3V- MeDAB, preneoplastic alterations in the liver, as determined by GST-P, were markedly increased (54). Gom A (30 mg/kg/day, p.o., for 5 weeks) signifi- cantly inhibited the increases in the number and size of GST-P-positive foci, regardless of the presence of tumor promoters (54). Gom A treatment also suppressed the increase in serum bile acid concentration by DCA, but produced no effect on PB-treated animals. Since hepatocarcinogenesis has been reported to be promoted by exogenous administration of bile acids (55,56), the result suggests that the inhibitory effect of Gom A on the promotive action of DCA (but not PB) may be related to the decrease in bile acid production, presumably by improving bile acid metabolism (57).

Application (1 Ag) of 1 2-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, to mouse ear could induce inflammation, and local admin- istration of Gom A (0.6 mg/ear) inhibited TPA-induced inflammation (58). Furthermore, when administered at 5 Ag/mouse, Gom A markedly sup- pressed the promotion effect of TPA (2.5 Ag/mouse) on skin tumor formation in mice after initiation with 7,12-dimethylbenz[a]anthracene (50 Ag/mouse) (58). The results suggest that the inhibition of tumor promotion by Gom A may be due to its anti-inflammatory activity.

E. Physical-Performance-Enhancing Effect The effects of FS on counteracting fatigue, increasing endurance, and im-

proving physical performance of sportsmen have long been reported (59). Until the late 1980s, no controlled studies have been documented. In a series of studies using horses, a dried ethanolic extract of FS, presumably lignan- containing, was administered orally (12 or 50 g/horse, p.o.) to thoroughbred horses 30 min prior to an 800-m race at maximum speed, and to spring horses subjected to a 12-min gallop at a speed of 400 m/min or a 5-min gallop at a speed of 700 m/min (60,61). The respiratory frequency and cardiac rate were significantly reduced in the treated horses subjected to both types of exercise, with the latter type of varied intensity, as compared to the respective control group. While plasma glucose concentration increased significantly in both types of exercise in the treated horses, the plasma lactic acid level was lower in treated horses, with the degree of decrease being more prominent in racing horses. Interestingly, horses treated with the lignan-containing FS extract were able to complete the race at an average of 1.8 sec faster, indicating an improvement in physical performance.

Poorly performing sports horses, which were found to be associated with long-lasting high serum activities of g-glutamyltransferase (GGT), glutamate-oxaloacete transaminase (GOT), and creatine phosphokinase (CPK) (62), were orally administered with 3 g/day of the lignan-containing

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FS extract (63). During the 14-day course of treatment, serum GGT and GOT activities were decreased on the seventh and fourteenth day. The decrease in serum CPK activity suggested a healing effect on muscle damage previously present in these horses (63). In this connection, a recent study in our laboratory has shown that pretreatment with a lignan-enriched FS extract, which contains biologically active lignans at f30% (w/w) (vida infra), at a daily oral dose of 0.8 g/kg for 3 days protected against physical-exercise- induced muscle damage in rats (64).

F. Other Pharmacological Effects Sch C and some other lignans were found to inhibit the growth of human

immunodeficiency virus (HIV)-infected H9 cells at effective concentrations (EC) ranging from 0.006 to 1.2 Ag/mL (65). Recently, halogenated derivatives of gomisin J (Fig. 1g) have been shown to possess anti-HIV activities by in- hibiting reverse transcriptase activity as well as expressing cytoprotective action in HIV-infected H9 cells (66). In addition, the growth and clonogenicity as well as the topoisomerase II activity of hepatocarcinoma cells were

inhibited by DDB (67). Sch C, with EC 50 ranging from 0.36 to 7 Ag/mL, pro- duced cytotoxic effect on KB epidermoid carcinoma of nasopharynx, COLO- 205 colon carcinoma, HEPA hepatoma, and HELA cervix tumor cells (68).

Several lignans isolated from FS were found to inhibit rat liver acetyl- CoA: cholesterol acetyltransferase activity at IC 50 values of 25–200 mM, with gomisin N, a stereoisomer of Sch B, being most potent (69).