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Herbal remedies for the liver: Myths, proofs, and treatments
1250 SELECTED SUMMARIES
HERBAL REMEDIES FOR THE LIVER: MYTHS, PROOFS, AND TREATMENTS Shimizu I, Ma Y-R, Mizobuchi Y, Liu F, Miura T, Nakai Y, Yasuda M, Shiba M, Horie T, Amagaya S, Kawada N, Hori H, Ito S (Second Department of Internal Medicine, School of Medicine, University of Tokushima, Tokushima, Japan). Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology 1999;29:149–160. Shimizu et al. assayed the preventive and therapeutic effects of Sho-saiko-to, or TJ-9, on experimental hepatic fibrosis induced in male Wistar rats by a single intraperitoneal injection of dimethylnitrosamine or 0.5 mL pig serum twice weekly for 10 weeks. The rats were fed either a basic diet or the diet plus 1.5% TJ-9 for 2 weeks before induction of hepatic fibrosis (or for the last 2 weeks of treatment). The authors also studied the effect of TJ-9 and its bioactive ingredients on rat stellate cells and hepatocytes in primary culture and assessed the antioxidative activities. TJ-9 suppressed the induction of fibrous septa, reduced the deposition of type I collagen on microscopy, and lowered hepatic collagen concentrations and restored hepatic retinyl palmitate concentrations. The livers of rats fed TJ-9 showed markedly reduced numbers of smooth muscle actin (SMA)-positive stellate cells compared with control rats. This was confirmed using cultured rat stellate cells in which the addition of TJ-9 to the media inhibited staining of microfilaments with antibody to SMA and reduced the expression of SMA protein on Western blot, as well as attenuated cell spreading and induced cytoplasmic fat droplet deposition. In the fibrotic rat livers, TJ-9 reduced the hepatic levels of malondialdehyde, a cumulative end product of lipid peroxidation, compared with control rats. TJ-9 suppressed lipid peroxidation in the mitochondria of cultured rat hepatocytes that were oxidatively stressed, inhibited Fe2⫹/adenosine 58-diphosphate–induced lipid peroxidation in a dose-dependent manner, and showed apparent reactive oxygen species scavenging activity. Finally, the investigators isolated and identified the active components of TJ-9 as 2 flavonoids, baicalin and baicalein. They showed that these 2 compounds caused identical effects to the parent compound in terms of the antioxidant activity when added to cultured rat stellate cells and hepatocytes. Comment. Herbal remedies have been used to treat patients, including those with liver disease, in the Far East and the subcontinent of India for more than 4000 years. Apart from some interest in the sixteenth century, the Western medical establishment appeared almost completely unmoved by Eastern practices until recently despite the fact that the West had very little therapeutically to offer patients with liver diseases, apart from steroids, perhaps azathioprine, and lactulose. This might explain why, despite the introduction of -blockers, ursodeoxycholate, originally used in Japan, and antivirals during the past 20 years, more than 30% of patients in liver clinics were also taking herbal remedies (Am J Gastroenterol 1996;91:2654– 2655). It was with this background that the Western establishment began to be interested again in the Eastern herbal remedies. One of the
GASTROENTEROLOGY Vol. 117, No. 5
herbal preparations that has received most attention is silymarin, a standardized extract from milk thistle, the principal active component of which is the flavonoid silybinin, representing 60%–70% of silymarin. Among other actions, silybinin has been shown to be a scavenger of reactive oxygen species (Biochem Pharmacol 1994;48:753– 759); its antioxidant activity prevents or limits the extent of lipid peroxidation as well as preserves the lipid profile of hepatocyte membranes (Gastroenterology 1995;109:1941–1949). Silybinin has been shown to inhibit the proinflammatory leukotriene B4 in Kupffer cells (Hepatology 1996;23:749–754) and the oxidation of low-density lipoproteins. The compound also possesses antifibrotic properties, because it prevents the proliferation of stellate cells in vitro and retards collagen accumulation in chronic bile duct–ligated rats (Hepatology 1987;26:643–649). There have been numerous clinical trials involving silymarin. However, after some positive clinical studies (Scand J Gastroenterol 1982;17:517–522, J Hepatol 1989;9:105–113), the latest results in a multicenter, double-blind, randomized, controlled trial of silymarin vs. placebo in patients with alcoholic cirrhosis showed no effect on survival or the clinical course of the patients ( J Hepatol 1998;28:615–621). In the present study on TJ-9, the investigators found that the active components were baicalin and balicalein, both flavonoids with chemical structures very similar to that of silybinin. Each molecule contains a 2-phenyl-1-benzopyrane-4-one (flavone) structure (Hepatology 1999;29:282–284). Thus, Shimizu et al. confirm previous reports that TJ-9 prevents fibrosis by the inhibition of stellate cells previously shown in a different animal model, the choline-deficient rat ( J Hepatol 1998;28:298–306), as well as in isolated stellate cells ( J Hepatol 1998;29:642–649). They also provide very strong evidence for at least one mechanism for fibrosis by the prevention of the effects of oxidative stress, which stimulates stellate cell transformation, via the release of tumor necrosis factor ␣ ( J Hepatol 1998;29:836–847) in the same way as the related molecule, silybinin. Technically this study is thorough, well conceived, and well executed. But there are still some points to consider. In the in vitro studies, the investigators used stellate cells, hepatocytes, and mitochondria isolated from healthy livers that were oxidatively stressed by various stimuli. We feel it would have been more relevant and informative to perform these studies on cells isolated from sick or diseased livers. Second, small sample sizes of 3 or 4 were used for some assays, giving a potential type 1 error. Because oxidative stress has been hypothesized as a trigger for fibrogenesis, we are surprised that the authors did not examine the effects of TJ-9 on Kupffer cells, a primary source of reactive oxygen species in the liver. The effect of TJ-9 and its the bioactive fractions on superoxide anion generation, using xanthine-xanthine oxidase assay, was measured by continuously monitoring the changes in absorbance of cytochrome C over 30 minutes at 37°C. There is a potential problem in presenting and interpreting the data because the reaction is enzymatic and temperature dependent. After 30 minutes, all the cytochrome C will be consumed, irrespective of the presence of TJ-9. Hence, it is preferable to perform the experiment at lower temperatures and compare the data at different time points up to 30 minutes to show a different pattern of cytochrome C consumption in the 2 groups. The time points at which the investigators did observe differences between the 2 groups were not reported. Again, the authors performed and reported an effect of TJ-9 on the basal rate of superoxide release from healthy stellate cells and hepatocytes. Because we do not know the basal rates in cells from diseased livers, we have to be cautious in any extrapolation of the possible beneficial effects of TJ-9 and bioactive ingredients in patients.
November 1999
SELECTED SUMMARIES 1251
What can we learn from this information? Certainly, the studies so far point to the possibility that this group of flavonoids have antifibrotic properties both in vitro and in animal models of liver fibrosis, and that one mechanism at least is via their antioxidant effect. However, with the explosion of the use of herbal remedies by patients with liver disease, it is of the utmost importance that the medical establishment continues in a cautious and scientific way to study these properties in a clinical setting. It must be emphasized that at the same time the medical profession continues to take the lead in this area, as urged by a recent editorial (N Engl J Med 1998;339:839–841). We must, however, remain cautious, because natural product does not necessarily mean harmless product, and we must therefore be aware of possible harmful side effects of herbal remedies (Gastroenterology 1997;113:1408–1412). Nevertheless, in this instance, the long history of use of TJ-9 in Japan is reassuring. Because there have been disappointments in the past, rigorously randomized controlled clinical studies must be carried out to verify this encouraging in vitro and animal data before extrapolation to patients. Finally, even if the results of the clinical studies with TJ-9 are disappointing, the work of Shimizu et al. and previous studies on this group of flavonoids indicate that this group of compounds should be a fruitful area to explore for new antifibrotic agents. LAURIE BLENDIS, M.D. ARIEH BOMZON, Ph.D. FLORENCE WONG, M.D.
Editor’s Note: The following is a Reply to a Selected Summary that appeared in the July 1999 issue of GASTROENTEROLOGY (117;270– 272), H. pylori and Nonulcer Dyspepsia: Not Guilty as Charged (by M. L. Schubert). Reply. Dr. Schubert noted the unusually low placebo response in the study by McColl et al. (N Engl J Med 1998;339:1869–1874) as a possible explanation for their observation of a statistically significant, albeit weak, beneficial effect of Helicobacter treatment on dyspeptic symptoms. In contrast, we (N Engl J Med 1998;339:1875–1881) observed a similar degree of symptomatic improvement after Helico-
bacter treatment but no significant difference from placebo. We would like to propose some explanations for our differing conclusions. McColl et al. may have inadvertently included many ulcer patients who are known to benefit from Helicobacter treatment (Gut 1997;41: 43–48, Aliment Pharmacol Ther 1993;7:429–442). They conducted their study in Scotland, an area known for its high ulcer prevalence, (Aliment Pharmacol Ther 1993;7:429–442), and they performed diagnostic endoscopy with a 2-week delay after initial assessment; many ulcers initially present would be expected to heal during this period (Scand J Gastroenterol 1986;121:46–52). There were other major differences between our studies. First, routine repeated endoscopies were not part of the McColl protocol. Only 3 of their 32 antibiotic-treated patients and 6 of their 39 control patients with severe recurrent dyspepsia underwent reendoscopy in the second part of the study (ⱖ10 points on the Glasgow Dyspepsia Severity Score, corresponding to severely symptomatic ulcer patients [Eur J Gastroenterol Hepatol 1996:8:967–971]). Four of the 6 control patients had an ulcer at reendoscopy, indicating that many more patients in this study had dyspepsia due to ulcer disease. In our study, routine endoscopy was performed at 3 and 12 months. The annual ulcer incidence was 1% in the antibiotic-treated group and 4% in the control group. Blinding in the McColl study may have been compromised by 2 weeks of high-dose treatment with metronidazole, which is known to produce more frequent side effects than 1-week treatment (Gastroenterology 1997;113:S131–S148). Bias may also have arisen from the unduly long recall period of 6 months for dyspepsia-related events at the final interview (Health Serv Res 1997;32:367–384, J Clin Epidemiol 1998;51:237–244). The cutoff value between success and failure could be one single day with dyspeptic symptoms over a 6-month period, which the patient was supposed to remember at the end of the study. Finally, Helicobacter treatment did not improve the quality of life, the mean Glasgow Dyspepsia Severity Index, or the risk of suffering from severe dyspepsia in the McColl study. Therefore, we are not convinced that McColl et al. have shown a favorable effect of Helicobacter treatment; their data rather confirm our observation of a lack of effectiveness. ANDRE´ L. BLUM, M.D.
HERBAL REMEDIES FOR THE LIVER: MYTHS, PROOFS, AND TREATMENTS Shimizu I, Ma Y-R, Mizobuchi Y, Liu F, Miura T, Nakai Y, Yasuda M, Shiba M, Horie T, Amagaya S, Kawada N, Hori H, Ito S (Second Department of Internal Medicine, School of Medicine, University of Tokushima, Tokushima, Japan). Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology 1999;29:149–160. Shimizu et al. assayed the preventive and therapeutic effects of Sho-saiko-to, or TJ-9, on experimental hepatic fibrosis induced in male Wistar rats by a single intraperitoneal injection of dimethylnitrosamine or 0.5 mL pig serum twice weekly for 10 weeks. The rats were fed either a basic diet or the diet plus 1.5% TJ-9 for 2 weeks before induction of hepatic fibrosis (or for the last 2 weeks of treatment). The authors also studied the effect of TJ-9 and its bioactive ingredients on rat stellate cells and hepatocytes in primary culture and assessed the antioxidative activities. TJ-9 suppressed the induction of fibrous septa, reduced the deposition of type I collagen on microscopy, and lowered hepatic collagen concentrations and restored hepatic retinyl palmitate concentrations. The livers of rats fed TJ-9 showed markedly reduced numbers of smooth muscle actin (SMA)-positive stellate cells compared with control rats. This was confirmed using cultured rat stellate cells in which the addition of TJ-9 to the media inhibited staining of microfilaments with antibody to SMA and reduced the expression of SMA protein on Western blot, as well as attenuated cell spreading and induced cytoplasmic fat droplet deposition. In the fibrotic rat livers, TJ-9 reduced the hepatic levels of malondialdehyde, a cumulative end product of lipid peroxidation, compared with control rats. TJ-9 suppressed lipid peroxidation in the mitochondria of cultured rat hepatocytes that were oxidatively stressed, inhibited Fe2⫹/adenosine 58-diphosphate–induced lipid peroxidation in a dose-dependent manner, and showed apparent reactive oxygen species scavenging activity. Finally, the investigators isolated and identified the active components of TJ-9 as 2 flavonoids, baicalin and baicalein. They showed that these 2 compounds caused identical effects to the parent compound in terms of the antioxidant activity when added to cultured rat stellate cells and hepatocytes. Comment. Herbal remedies have been used to treat patients, including those with liver disease, in the Far East and the subcontinent of India for more than 4000 years. Apart from some interest in the sixteenth century, the Western medical establishment appeared almost completely unmoved by Eastern practices until recently despite the fact that the West had very little therapeutically to offer patients with liver diseases, apart from steroids, perhaps azathioprine, and lactulose. This might explain why, despite the introduction of -blockers, ursodeoxycholate, originally used in Japan, and antivirals during the past 20 years, more than 30% of patients in liver clinics were also taking herbal remedies (Am J Gastroenterol 1996;91:2654– 2655). It was with this background that the Western establishment began to be interested again in the Eastern herbal remedies. One of the
GASTROENTEROLOGY Vol. 117, No. 5
herbal preparations that has received most attention is silymarin, a standardized extract from milk thistle, the principal active component of which is the flavonoid silybinin, representing 60%–70% of silymarin. Among other actions, silybinin has been shown to be a scavenger of reactive oxygen species (Biochem Pharmacol 1994;48:753– 759); its antioxidant activity prevents or limits the extent of lipid peroxidation as well as preserves the lipid profile of hepatocyte membranes (Gastroenterology 1995;109:1941–1949). Silybinin has been shown to inhibit the proinflammatory leukotriene B4 in Kupffer cells (Hepatology 1996;23:749–754) and the oxidation of low-density lipoproteins. The compound also possesses antifibrotic properties, because it prevents the proliferation of stellate cells in vitro and retards collagen accumulation in chronic bile duct–ligated rats (Hepatology 1987;26:643–649). There have been numerous clinical trials involving silymarin. However, after some positive clinical studies (Scand J Gastroenterol 1982;17:517–522, J Hepatol 1989;9:105–113), the latest results in a multicenter, double-blind, randomized, controlled trial of silymarin vs. placebo in patients with alcoholic cirrhosis showed no effect on survival or the clinical course of the patients ( J Hepatol 1998;28:615–621). In the present study on TJ-9, the investigators found that the active components were baicalin and balicalein, both flavonoids with chemical structures very similar to that of silybinin. Each molecule contains a 2-phenyl-1-benzopyrane-4-one (flavone) structure (Hepatology 1999;29:282–284). Thus, Shimizu et al. confirm previous reports that TJ-9 prevents fibrosis by the inhibition of stellate cells previously shown in a different animal model, the choline-deficient rat ( J Hepatol 1998;28:298–306), as well as in isolated stellate cells ( J Hepatol 1998;29:642–649). They also provide very strong evidence for at least one mechanism for fibrosis by the prevention of the effects of oxidative stress, which stimulates stellate cell transformation, via the release of tumor necrosis factor ␣ ( J Hepatol 1998;29:836–847) in the same way as the related molecule, silybinin. Technically this study is thorough, well conceived, and well executed. But there are still some points to consider. In the in vitro studies, the investigators used stellate cells, hepatocytes, and mitochondria isolated from healthy livers that were oxidatively stressed by various stimuli. We feel it would have been more relevant and informative to perform these studies on cells isolated from sick or diseased livers. Second, small sample sizes of 3 or 4 were used for some assays, giving a potential type 1 error. Because oxidative stress has been hypothesized as a trigger for fibrogenesis, we are surprised that the authors did not examine the effects of TJ-9 on Kupffer cells, a primary source of reactive oxygen species in the liver. The effect of TJ-9 and its the bioactive fractions on superoxide anion generation, using xanthine-xanthine oxidase assay, was measured by continuously monitoring the changes in absorbance of cytochrome C over 30 minutes at 37°C. There is a potential problem in presenting and interpreting the data because the reaction is enzymatic and temperature dependent. After 30 minutes, all the cytochrome C will be consumed, irrespective of the presence of TJ-9. Hence, it is preferable to perform the experiment at lower temperatures and compare the data at different time points up to 30 minutes to show a different pattern of cytochrome C consumption in the 2 groups. The time points at which the investigators did observe differences between the 2 groups were not reported. Again, the authors performed and reported an effect of TJ-9 on the basal rate of superoxide release from healthy stellate cells and hepatocytes. Because we do not know the basal rates in cells from diseased livers, we have to be cautious in any extrapolation of the possible beneficial effects of TJ-9 and bioactive ingredients in patients.
November 1999
SELECTED SUMMARIES 1251
What can we learn from this information? Certainly, the studies so far point to the possibility that this group of flavonoids have antifibrotic properties both in vitro and in animal models of liver fibrosis, and that one mechanism at least is via their antioxidant effect. However, with the explosion of the use of herbal remedies by patients with liver disease, it is of the utmost importance that the medical establishment continues in a cautious and scientific way to study these properties in a clinical setting. It must be emphasized that at the same time the medical profession continues to take the lead in this area, as urged by a recent editorial (N Engl J Med 1998;339:839–841). We must, however, remain cautious, because natural product does not necessarily mean harmless product, and we must therefore be aware of possible harmful side effects of herbal remedies (Gastroenterology 1997;113:1408–1412). Nevertheless, in this instance, the long history of use of TJ-9 in Japan is reassuring. Because there have been disappointments in the past, rigorously randomized controlled clinical studies must be carried out to verify this encouraging in vitro and animal data before extrapolation to patients. Finally, even if the results of the clinical studies with TJ-9 are disappointing, the work of Shimizu et al. and previous studies on this group of flavonoids indicate that this group of compounds should be a fruitful area to explore for new antifibrotic agents. LAURIE BLENDIS, M.D. ARIEH BOMZON, Ph.D. FLORENCE WONG, M.D.
Editor’s Note: The following is a Reply to a Selected Summary that appeared in the July 1999 issue of GASTROENTEROLOGY (117;270– 272), H. pylori and Nonulcer Dyspepsia: Not Guilty as Charged (by M. L. Schubert). Reply. Dr. Schubert noted the unusually low placebo response in the study by McColl et al. (N Engl J Med 1998;339:1869–1874) as a possible explanation for their observation of a statistically significant, albeit weak, beneficial effect of Helicobacter treatment on dyspeptic symptoms. In contrast, we (N Engl J Med 1998;339:1875–1881) observed a similar degree of symptomatic improvement after Helico-
bacter treatment but no significant difference from placebo. We would like to propose some explanations for our differing conclusions. McColl et al. may have inadvertently included many ulcer patients who are known to benefit from Helicobacter treatment (Gut 1997;41: 43–48, Aliment Pharmacol Ther 1993;7:429–442). They conducted their study in Scotland, an area known for its high ulcer prevalence, (Aliment Pharmacol Ther 1993;7:429–442), and they performed diagnostic endoscopy with a 2-week delay after initial assessment; many ulcers initially present would be expected to heal during this period (Scand J Gastroenterol 1986;121:46–52). There were other major differences between our studies. First, routine repeated endoscopies were not part of the McColl protocol. Only 3 of their 32 antibiotic-treated patients and 6 of their 39 control patients with severe recurrent dyspepsia underwent reendoscopy in the second part of the study (ⱖ10 points on the Glasgow Dyspepsia Severity Score, corresponding to severely symptomatic ulcer patients [Eur J Gastroenterol Hepatol 1996:8:967–971]). Four of the 6 control patients had an ulcer at reendoscopy, indicating that many more patients in this study had dyspepsia due to ulcer disease. In our study, routine endoscopy was performed at 3 and 12 months. The annual ulcer incidence was 1% in the antibiotic-treated group and 4% in the control group. Blinding in the McColl study may have been compromised by 2 weeks of high-dose treatment with metronidazole, which is known to produce more frequent side effects than 1-week treatment (Gastroenterology 1997;113:S131–S148). Bias may also have arisen from the unduly long recall period of 6 months for dyspepsia-related events at the final interview (Health Serv Res 1997;32:367–384, J Clin Epidemiol 1998;51:237–244). The cutoff value between success and failure could be one single day with dyspeptic symptoms over a 6-month period, which the patient was supposed to remember at the end of the study. Finally, Helicobacter treatment did not improve the quality of life, the mean Glasgow Dyspepsia Severity Index, or the risk of suffering from severe dyspepsia in the McColl study. Therefore, we are not convinced that McColl et al. have shown a favorable effect of Helicobacter treatment; their data rather confirm our observation of a lack of effectiveness. ANDRE´ L. BLUM, M.D.