a search for hepatoprotective agents of natural...

A search for hepatoprotective agents of natural origin

Department of Chemical Technology, Dr.Babasaheb Ambedkar Marathwada University, Aurangabad

1

NATURE always stands as a golden mark to exemplify the

outstanding phenomenon of symbiosis. The plants are indispensable to man

for his life. Nature has provided a complete storehouse of remedies to cure

all ailments of mankind.

In the past, almost all the medicines used were from the plants, the

plant being man's only chemist for ages. Today, vast store of knowledge

concerning therapeutic properties of different plants has accumulated.

The history of herbal medicines is as old as human civilization. The

documents, many of which are of great antiquity, revealed that plants were

used medicinally in China, India, Egypt and Greece long before the beginning

of Christian era.

Ayurveda is believed to be prevalent since last 5000 years in India. It

is one of the most noted systems of medicine in the world. Ayurveda is based

on the hypothesis that everything in the universe is composed of five basic

elements viz. space, air, energy, liquid and solid. They exist in the human

body in combined forms like Vata (space and air), Pitta (energy and liquid)

and Kapha (liquid and solid). Vata, Pitta and Kapha together called Tridosha

(three pillars of life). It is believed that they are in harmony with each other

but in every human being one of them is dominating which in turn is called

as the prakruti of that person1.

Considerable scopes of ethanobotanical studies are found in different

parts of India; mainly use natural plant product directly as drugs to get rid

from various diseases. Much work in the field of medicinal plants has

accumulated in India during 20th century. Therapeutic use of plants for

treatment of human illness dates back many millennia. Evidenced of their



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effectiveness in the diagnosis, cure and prevention of disease state exist in

every culture throughout the world2.

Plants constitute one of the major raw materials for drugs for treating

various ailments of human being, although there has been significant

development in the field of synthetic drug chemistry and antibiotics. In all

over the world now considerable changes have taken place during last two

decades. Due to the awareness of toxicity associated with the long use of

synthetic drugs and antibiotic, the western society prefers the drug from

natural sources than the synthetics.

Moreover modern medicine does not have a suitable answer for many

conditions such as liver disorder and for chronic conditions such as asthma,

arthritis etc. and this leads to increase interest in herbal drugs3.

Today "traditional medicine" characterized by the use of herbs and

other natural products, still remains regular component of health care in

countries such as China, Japan, India, South America and Egypt. Even

today more than 40% of drugs in allopathy have their origin from plants.

Keeping this significant contribution, there is a need that we should

understand the original system of medicine and stop calling them as

"Alternative Systems"4.

Hepatic endothelial and kupffer Cell specific delivery of drugs

Fibrosis or scarring of the liver occurs after damage to liver tissue.

Most chronic liver diseases eventually result in excess scarring leading to

liver cirrhosis. This fatal disease, to date can only be effectively treated with

a liver transplantation. Since this is a costly procedure, hampered by the

lack of donor organs among other technical factors, much effects has been



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put into developing new drugs. The drugs available are not sufficiently

effective and/or cause too many adverse side-effects. Therefore, drug

targeting is an option in trying to maximize efficacy and minimize adverse

drug reactions.

Since drug targeting implies the manipulation of drug distribution in

the whole body, emphasis should be put on in vivo studies. In contrast to in

vitro studies, studies in the intact organism will provide more definite insight

into the cell specificity of carrier systems, the potential toxicity,

immunogenicity and the ability of the carrier system to pass anatomical

barriers enroute to the target cells. Moreover, it is of the utmost importance

that these parameters are also studied in the diseased state, since the

targeting potential of carriers can change dramatically under pathological

conditions. In vitro studies with various liver preparations can be used to

study endocytosis, carrier degradation and intracellular release of the

targeted drug in more detail. In addition, the concept of drug targeting

should also be tested in human tissue.

The Liver

At the crossroads between the digestive tract and the rest of the body

resides the largest solid organ of the body, the liver. Because of its

interposition, the liver has a dual blood supply.

Nutrient-rich blood arrives through the portal vein and oxygen-rich

blood through the hepatic artery. Together these channels import a large

variety of endobiotics and xenobioties, ranging from nutrients to toxic

substances derived from the digestive system. The main function of the

liver, therefore, is to maintain the body’s metabolic homeostasis. This



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includes the efficient uptake of amino acids, carbohydrates, lipids, vitamins

and their subsequent storage, metabolic conversion and release into blood

and bile, synthesis of serum proteins, hepatic biotransformation of

circulating compounds. It is a process which converts hydrophobic

substances into water-soluble derivatives that can be secreted into bile or

urine as well as phagocytosis of foreign macromolecules and particles such

as bacteria.

Classically the liver has been divided into hexagonal lobules centered

around the terminal hepatic venules. Blood enters the liver through the

portal tracts that are situated at the corners of the hexagon. The portal tracts

are triads of a portal vein, an hepatic artery and a common hepatic bile duct.

The vast expanse of hepatic tissue, mostly consisting of parenchymal cells

(PC) or hepatocytes, is serviced via terminal branches of the portal vein and

hepatic artery, which enters the tissue at intervals. The hepatocytes are

organized into cords of cells radially disposed about the central hepatic

venule. Between these cords are vascular sinusoids that transport the blood

to the central hepatic venules. The blood is collected through the hepatic

venules into the hepatic vein which exits the liver into the inferior vena cava

(Figure 1.1)



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Figure 1.1 Representation of architecture of liver

Blood enters the liver through the portal vein (PV) and hepatic

arteries (HA), flows through the sinusoids and leaves the liver again via the

central vein (CV), Kupffer cells (KC), sinusoidal endothelial cells (SEC),

hepatic stellate cells (HSC) and bile duct (BD).

The Sinusoids are lined by the discontinuous and fenestrated

SEC that demarcate the extrasinusoidal space of Disse. The abundant

microvilli of the hepatocytes protrude into this space, which also contains the

fat-containing lipocyte or HSC. At a strategic position along the luminal side

of the endothelial cells are the resident tissue macrophages, which are KC.

Also located on the endothelial lining is the Pit cell that corresponds to the

large granular lymphocytes with natural killer activity. Between the abutting

hepatocytes are bile canaliculi, channels in between the plasma membranes

of facing hepatocytes that are delineated from the vascular space by tight

junctions. These intercellular spaces constitute the outermost reaches of the



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biliary tree. The canaliculi emanate from the centriobular regions,

progressively drain into the canals of Hering at the fringes of the portal

tracts, and biliary fluid finally collects in the interlobular bile ducts.

The Parenchymal Cell (PC)

The liver consists mainly of parenchymal cells (PC) or hepatocytes.

Most drug-targeting preparations designed for liver targeting of therapeutic

compounds are directed towards this cell type, generally aiming at the

asialoglycoprotein receptor using galactose residues coupled to a core

molecule for binding 5-7.

Hepatocytes make up 60-70 % of the total number of liver cells. They

have a well-organized intracellular structure with huge numbers of cell

organelles to maintain the high metabolic profile. At the apical side or

canalicular membrane the cell is specialized for the secretion of bile

components. There are several ATP dependent transport carriers located on

this side of the membrane, which transport bile salts, lipids and xenobiotics

into the canaliculus. On the sinusoidal side, the cells specialize in uptake

and secretion of a wide variety of components. To increase the surface of the

membrane for this exchange with the bloodstream, the sinusoidal domain of

the membrane is equipped with irregular microvilli. The microvilli are

embedded into the fluid and matrix components of the space of Disse and are

in close contact with the sinusoidal blood because of the discontinuous and

fenestrated SECs. To facilitate its metabolic functions numerous membrane

transport mechanisms and receptors are situated in the membrane.



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The Sinusoidal Endothelial Cell (SEC)

The endothelial lining of the sinusoids in the liver differs from the

other capillaries in the body and is adapted to form a selective barrier

between blood and hepatocytes. The basement membrane is composed of

non-fibril-forming collagens including types IV, VI and XIV glycoproteins and

proteoglycans. The lining is discontinuous and the SECs are perforated by

numerous fenestrae that lack diaphragms. This allows direct contact of the

hepatocytes with most plasma proteins in the space of Disse, but prevents

direct contact with blood cells, large chylomicrons, bacteria and viruses.

SECs play an important role in the pathogenesis of several acute and chronic

inflammatory liver diseases. Consequently they are attractive target cell for

anti-inflammatory therapies.

The SECs account for 20% of all liver cells and are the first cells,

together with the KCs to encounter potentially harmful materials present in

the portal blood. They are therefore equipped with scavenger capabilities

and certain defense mechanisms to prevent damage to other cell types. The

SECs have an active scavenging system for the majority of physiological and

foreign soluble (waste) macromolecules8,9. Clearance mechanisms include

receptor mediated endocytosis, transcytosis and phagocytosis. To regain

local homeostasis after ingestion of injurious substances and after other

determental events, the SECs can also produce cytokines, eicosanoids and

adhesion molecules for the mobilization of their hepatic cell types and cells of

the immune system.



8

Receptor-mediated Endocytosis

Targeting to SECs should be directed at specific receptors present on

this cell type-A wide range of proteins and other molecules can be taken up

by SECs through receptor-mediated endocytosis. For example, SECs play an

important role in the uptake of degradation products of the extracellular

matrix. For this purpose they have hyaluronan10, procollagen and

fibronectin receptors11. The first two receptors are uniquely located on SECs.

Elevated levels of serum hyaluronan and fibronectin that are often found in

liver disease12 are usually the result of dysfunction of the clearance capacity

of SECs combined with an increased production by HSCs13.

Scavenger receptors on the SECs are instrumental in another

important endocytic mechanism. They recognize and endocytose modified

proteins that have a high net negative charge13. SECs predominantly express

two type of scavenger receptors viz. the class AI and the class AII scavenger

receptor14. Physiological substrates for these receptors were found to be the

N-terminal propeptides of types I and III procollagen and the lipid A moiety of

endotoxin15,16. The SECs are further equipped with a receptor that

specifically interacts with mannose and N-acetylglucosamine terminated

glycoproteins. Unlike the scavenger receptor, binding of ligands to this so-

called mannose receptor is Ca2+- dependent, but is also followed by rapid

endocytosis and degradation in lysosomes17. The receptor is thought to be

involved in the uptake of microorganisms like yeasts, bacteria and parasites,

but has also been shown to be involved in uptake of tissue-type plasminogen

activator18,19. In addition, the receptor is involved in antigen uptake for



9

subsequent antigen presentation. This indicated that SECs may also be

involved in cell-mediated immune responses in the liver20.

Phagocytosis and Transcytosis

SECs are normally able to internalize only small particles (up to 0.23

µ) in conditions of impaired KC function. However, they have also been

found to phagocytose larger particles21. They are also responsible for the

receptor mediated transcytosis of several compounds such as insulin22 and

transferrin23.

Regulation of the Inflammatory Process by SECs

Exposure of the SECs to pathogens or cytokines produced by other

cells during stress induces activation of the SECs and subsequent

production of cytokines, eicosanoids, and/or adhesion molecules. For

instance, after activation with LPS, a main component of the walls of gram

negative bacteria and a major inducer of inflammation and non-specific

immune functions24, SECs produce a number of pro and anti-inflammatory

cytokines. Pro-inflammatory cytokines shown to be produced were a) tumor

necrosis factor alpha (TNFα)25, b) interleukin-1 alpha/beta(IL-1α/β)26, c) the

major inducer of acute phase proteins interleukin-6 (IL-6)27 and d) the

neutrophil chemo-attractant interleukin-828. Anti-inflammatory cytokines

shown to be produced were interleukin-1026 (IL-10) and hepatocyte growth

factor (HGF) 29.

Eicosanoids are the oxidative metabolites derived from the cell

membrane component arachidonic acid. Arachidonic acid is released from

the cell membrane by phospholipase A2 and enzymatically converted to either

prostaglandins (PGs) by cyclo-oxygenase or leukotrienes (LTs) by



10

lipoxygenase. Eicosanoids is the collective name of prostaglandins and

leukotrienes. SECs and KCs are the major sources of eicosanoids, whereas

the PCs are considered to be the most important target cells for them. The

main eicosanoid produced by SECs was found to be PGE230, although PGD2

has also been reported to be a major product31. The type of PG released may

be a result of the difference in the induction stimulus used. Eicosanoid

production is induced by many circulating substances; LPS interferon

gamma (IFNγ), TNFα and platelet activating factor (PAF). PGE2 is postulated

to be involved in liver regeneration32 and inhibition of hormone-stimulated

glycogenolysis30, PGD2 was found to induce glycogenolysis33.

SECs, like the vascular endothelium, play an active part in the control

of leucocyte recruitment in cases of acute and chronic inflammatory

conditions. Leucocyte recruitment from the blood compartment is a crucial

determinant for the induction of immunity and inflammation. SECs control

this process by producing cytokines that activate leucocytes and by

expressing adhesion molecules. Under inflammatory conditions upregulation

of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion

molecule 1 (VCAM-1)34, 35 as well as expression of E-selectin and P-selectin36

were found. Together with the expression of CD4 on SECs it has been

postulated that these adhesion molecules might also be involved in the

adhesion of KC cells to the sinusoidal wall24.

The Kupffer Cell (KC)

Kupffer cells are the largest reservoir of fixes tissue macrophages and

are quantitatively the most important cell type for the removal of circulating

micro organisms, LPS, tumor cells, immune complexes, other circulating



11

tissue and microbial debris37. They account for about 15% of the liver cell

population in number and they are preferentially located in the periportal

areas38.

Receptor-mediated Endocytosis

Similar to the targeting of compounds to SECs drug targeting

preparations designed to modify KC functions have to be directed at KC-

specific receptors. KCs are able to remove numerous soluble and particulate

substances from the circulation and they possess many receptor systems

that mediate this clearance, some of which have also been described for

SECs Like SECs, they possess fibronectin receptors, mannose receptors, Fe

receptors, CD14 receptors and the scavenger receptors class AI and AII39. In

addition to these receptors, KCs also possess the novel member of the class A

scavenger receptor family, the macrophage receptor with collagenous

structure (MARCO) 40. Besides these types of scavenger receptors, they also

have macrosialin scavenger receptors for the uptake of oxidized LDL and

scavenger receptors class BI for the removal of high-density lipoproteins

(HDL)41. For the uptake of unmodified LDL, KCs also have special LDL

receptors42.

Mannose receptors on KCs essentially recognize the same

molecules as the mannose receptors present on SECs, but they exhibit

different kinetics43. Besides the mannose receptors, KCs have two other

carbohydrate-specific receptors. One is the galactose particle receptor,

recognizing galactose terminated oligosaccharides on particles and mediating

endocytosis of desialylated erhyhrocytes44. The other is the fucose receptor



12

which interacts not only with fucose terminated glycoproteins, but also with

galactose exposing neoglycoproteins45.

KCs also possess receptors for the complement components Clq and

C3ba46,47. The complement system is one of the main defense mechanisms of

the body against invading pathogens. It is composed of a group of serum

proteins that are part of a multienzymatic cascade. Activation of complement

generates membranolytic components and protein fragments that enhance

phagocytosis and mediate immune responses. KCs have the optimal capacity

to remove complexes coated with complement from the circulation.

Phagocytosis

Not all KCs are phagocytic to the same extent; periportal KCs

generally have a higher level of phagocytic activity than those in other

regions of the liver48. Prior to phagocytosis, particulate material like viruses,

bacteria and erythrocytes may be opsonized and bound by specific receptors,

but this is not essential for phagocytosis49.

Hepatic Inflammation and Fibrosis

Virtually any insult to the liver can cause hepatocyte destruction and

parenchymal inflammation. If the insult is minor and occurs only once, local

restoration mechanisms will suffice to repair the damage. If however, the

insult is major or persistent, an inflammatory response will be generated.

This inflammation is the result of cytokine-mediated activation of sinusoidal

cells, their subsequent release of pro-inflammatory cytokines and their

expression of adhesion molecules for the recruitment of circulating

leucocytes. Once the damage is under control and the inciting insult has

been eliminated, the inflammatory process will end and local mechanisms



13

will proceed until the damage is repaired. Usually little scar tissue will be

detectable, because of extracellular matrix remodeling. During conditions of

chronic liver injury, however, the repair process does lead to scar tissue

formation, which is deposited with in the liver until impairment of liver

function occurs. This process is called liver fibrogenesis and the end stage,

or irreversible stage, is referred to as liver cirrhosis (Figure 1.2)

Figure 1.2 Diagram outlining the pathogenesis of liver fibrosis.

Injury to PC results in the activation of KC and SEC and the recruitment of

IC. These cells release cytomines, growth factors and reactive oxygen species

that induce activation and proliferation of HSC. HSCs gradually transform

into MF, the major producers of ECM proteins.

After damage or infection, monocytes and KCs in the area

detect the damaged cells or infectious agent and respond with release of

primary mediators such as TNFα, IL-1 and some IL-6. These cytokines

activate the surrounding cells that respond with a secondary amplified

release of cytokines. This second wave includes large amounts of IL-6 which



14

induce the synthesis of acute phase proteins in hepatocytes and

chemoattractants such as IL-8 and MCP-1. These events will then lead to the

typical inflammatory reactions. Both IL-1 and TNFα activate the central

regulatory protein of many reactions involved in immunity and inflammation,

nuclear factor kappa B (NFkB). These cytokines cause dissociation of NFkB

from its inhibitor IkB, which makes translocation of NFkB to the nucleus

possible. In the nucleus active NFkB induces the transcription of the

‘Second wave’ cytokines.

The release of TNFα and IL-1 also upregulates adhesion molecules like

ICAM-1 and VCAM-1 on SECs that are subsequently responsible for the

adhesion and recruitment of circulating neutrophils. KCs and PCs release

IL-8, which is a potent neutrophil chemoattractant. The attracted neutrophils

and KCs are stimulated to release large amounts of reactive oxygen species

(ROS: hydrogen peroxide, superoxide anion and nitric oxide (NO) radicals).

The production of NO is also mediated through the NFkB pathway. The

enzyme responsible for the increased synthesis of NO, inducible NO

synthetase (i-NOS), is increasingly ex-pressed through NFkB mediated

stimulation of the i-NOS promoter region.

TGFβ and TNFα produced by KCs and PDGF produced by SECs

subsequently play an important role in the activation HSCs. TGFβ appears

to be the most important cytokine in stimulating the production of scar

tissue components like collagens by HSCs. The mechanism of activation is

probably via the IGF-II/M6P receptor, which is also increasingly expressed

on activated HSCs. As yet unknown factors produced by KCs stimulate

expression of PDGF receptors on the surface of HSCs. In the presence of



15

PDGF the HSC will now proliferate as well. On chronic stimulation HSC

stimulation, but substances directly released by PCs are also found to be

mitogenic50.

Since not every insult necessarily results in liver fibrosis, counter-

regulatory mechanisms must also exist. During inflammation, elimination of

ROS by SECs and KCs is enhanced via increased expression of radical

scavengers like superoxide dismutases and glutathione peroxidase. The

radical nitric oxide itself also has an anti-inflammatory role. It has been

described to prevent leucocyte adhesion to the endothelium51 and to block an

activation pathway of thrombocytes by stimulating guanylyl cyclase52.

Furthermore, both PGE2 and IL-10 can downregulate cytokine produced by

macrophages53,54 and can also inhibit the antigen-presenting properties of

SECs and KCs20,55. HGF produced by KCs, SECs and quiescent HSCs is a

potent mitogen for PCs and stimulates liver regeneration. It is probably aided

by PGE2 which also stimulates DNA synthesis in PCs56. Finally, scar tissue

formation is not only regulated by production of extracellular matrix

components, but also by the degradation of matrix components. Activated

and quiescent HSCs, KCs and SECs produce matrix metalloproteinase that

are responsible for matrix degradation57.

Liver Cirrhosis: Causes and Therapy

Cirrhosis is among the top 10 causes of death in the Western World.

This is largely the result of alcohol abuse, viral hepatitis and biliary

diseases58. The causes for cirrhosis can be roughly divided into six

categories;

1. Chronic exposure to toxins such as alcohol, drugs or chemicals



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2. Viral hepatitis resulting from infection with the Hepatitis B,C

or D viruses

3. Metabolic disorders such as Wilson’s disease (copper storage

disease) and haemochromatosis (iron overload disease)

4. Autoimmune disease such as primary biliary cirrhosis (PBC),

primary selerosing cholangitis (PCS) and autoimmune hepatitis

5. Venous outflow obstruction

6. Cirrhosis of unknown causes

Obviously the best treatment for cirrhosis is removal of the injurious

event. In the case of viral hepatitis, viral load can at least be temporarily

reduced with anti-viral agents such as lamivudine, ribavirin and/or IFNα59.

Unfortunately, complete removal of the injurious event is frequently not

possible. Moreover, by the time cirrhosis is diagnosed the fibrotic process has

usually progressed beyond the point of no return and removal of the

injurious event will have little effect. Successful pharmacological treatment to

reverse the fibrotic process is not yet available. Several drugs have been

tested in clinical trials, by the most effective treatment remains a liver

transplantation.

The bile acid ursodeoxycholic acid has shown some promise in

slowing down the fibrotic process in cholestatic patients, especially those

suffering from PBC and PSC60,61. Its mechanism of action, however, is still a

matter of debate.

Penicillamine, an inhibitor of collagen cross linking, was evaluated in

PBC, but failed to demonstrate any efficacy62. More promising results were

found for colchicine, which inhibits collagen synthesis and secretion and



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enhances collagenase activity. Long-term use of colchicines prolonged

survival in patients with mild to moderate cirrhosis, regardless of the

cause60,63. Other types of collagen synthesis inhibitors like the prolyl

hydroxylase inhibitors have been studied in experimental animal models64,

but have not yet found their way into the clinic.

Several types of immunosuppression have also been tried.

Azathioprine alone was found to have no effect on PBC65, but additional

beneficial effects were found in combination with ursodeoxycholic acid and

corticosteroids61.Cyclosporine showed some success, especially in

corticosteroid-resistant autoimmune hepatitis66, but its use is generally

considerably limited by severe side-effects. Corticosteroids were effective in

the management of several types of autoimmune chronic active hepatitis67,68

and in the management of acute alcoholic hepatitis69. Their use, however,

has to be brief in order to minimize side-effects. In the treatment of PBCs,

corticosteroids alone were found to be toxic and had only limited efficacy60.

A promising new development in drug therapy is the endothelin-

antagonists70,71.Though not yet clinically tested, these compounds show

potential in the management of portal hypertension, a hallmark of cirrhosis.

Again, uptake of these antagonists by other parts of the body hampers their

applicability72, which might be circumvented by drug targeting.

Drug Targeting to the Liver

With no effective drugs available and the unacceptable side-effect

profile of those drugs which have been studied so far, liver cirrhosis might

benefit from the targeting of drugs to cells within the liver. There are several

ways to intervene in the fibrotic process. One way is the targeting of drugs to



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SECs and KCs to modulate their release of pro-inflammatory mediators. This

may arrest the inflammatory process leading to cirrhosis. Another way is the

delivery of drugs to HSCs to inhibit collagen production or to enhance their

extracellular matrix degrading capabilities. Drug targeting to the liver focuses

on targeting to KCs and SECs to influence the inflammatory process that is

the basis of most forms of liver cirrhosis. As mentioned before these cells

have a number of specific entry mechanisms that could be used for cell-

specific delivery of drugs. By either enclosing drugs in particles or by

coupling drugs to macromolecular carriers with high affinity for certain

uptake mechanisms, drugs can be concentrated in the target cells without

causing side-effects else where in the body. The choice for a type of carrier is

determined by a number of considerations, depending on the specificity of

the carrier, the potency of the drug and the entry mechanism during

pathological conditions. The possible carriers directed to KCs and SECs show

a considerable overlap, because these cells share many receptor mediated

endocytotis uptake mechanisms, such as uptake mediated by scavenger

receptors of mannose receptors. Most of the carriers directed to SECs and

KCs are designed for these receptors.



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References

1. Kokate C.K.; Purohit, A.P.; Gokhale, S.B.; “ Pharmacognacy” Nirali prakashan, 1998, pp. 1-4.

2. Kapur, S.K.; Indian Drugs; 50, 1991,210.

3. Naik, S.R.; Eastern Pharmacist, 29:36, 1986, 346. 4. Narauan, D.B.; Eastern pharmacist, 21, 1998,487. 5. Flume, L.; Di Stefano, G.; Busi C.; Mattioli, A.; Bonino, F.; Torrani-

cerenxi; M.; Bertini M.; Gervasi G,B.; J.Viral. hepat. 4, 1997, 363-370.

6. Smith, R.M.; Wu G.Y.; Liver Dis., 19, 1999, 83-92. 7. Meijer, D.K.F.; Molema, G.; Liver Dis., 15, 1995, 202-256. 8. Martinez, I.; Sveinbjornsson, B.; Vidal Vanaclocha, F.; Asumendi, A.;

Smedsr onari, J.; Gorry, F.; Boillot, O. Biochem. Biophys. Res. Commun., 27, 1995.

9. Melkko, J.; Hellevik, T.; Risteli, L.; Risteli, J.; Smedsrod, B.; J. Exp.

Med. 179, 1994, 405-412. 10. Mcgary, C.T.; Yannariello-Brown, J.; Kim, D.W.; Stinson, T.C.; Weigel,

P.H.; Hepatology 18,1993, 1465-1476. 11. Smedsrod, B.; De Bleser, P.J.; Braet, F.; Lovisetti, P.p; Vanderkerken,

K.; Wisse, E.; Geerts, A.; Gut, 35, 1994,1509-1516. 12. Ichida, T.; Sugitani, S.; Satoh, T.; Matsuda, Y.; Sugiyama, M.; Yonekura,

K.; Ishikawa, T.; Asakura, H.; Liver, 16, 1996, 365-371. 13. Smedsrod, B.; Pertoft, H.; Gustafson, S.; Laurent, T.C.; Biochem. J.,

266, 1990, 313-327. 14. Van Oosten, M.; van de Bilt E.; Van Berkel T.J.C.; Kuiper, J.; Infect.

Immun. 66, 1998, 5107-5112. 15. Smedsrod, B.; Melkko, J.; Araki, N.; Sano, H.; Horiuchi, S.; Biochem.

J., 322, 1997, 567-573. 16. Hampton, R.Y.; Golenbock, D.T.; Penman, M.; Krieger, M.; Raetz, C.R.;

Nature, 352, 1991, 342-344. 17. Magnusson, S.; Berg, T.; Biochem. J., 257, 1989, 651-656.



20

18. Ezekowitz, R.A.B.; Williams, D.J.; Koziel, H.; Armstrong, M.Y.K.; Warner, A.; Richards, F.F.; Rose, R.M. Nature, 351,1991,155-158.

19. Noorman, F.; Rijken, D.C.; Fibrin. Proteol. , 11, 1997, 173-186. 20. Knolle, P.A.; Uhrig, A.; Hegabnarth, S.; Loser, E.; Schmitt, E.; Gerken,

G.; Lohse, A.W.; Clin. Exp. Immunol., 114, 1998, 427-433. 21. Kanai, M.; Murata, Y.; Mabuchi, Y.; Kawahashi, N.; Tanaka, M.; Ogawa,

T.; Doi M.; Soji, T.; Herbert, D.C.; Anat. Rec., 244,1996, 175-181. 22. Bergman, R.N.; Recent. Prog. Horm. Res., 52, 1997, 359-385. 23. Omoto, E.; Minguell, J.J.; Tavassoli, M.; Pathobiology, 60, 1992, 284-

288. 24. Scoazes, J.Y.; Feldman, G.; Hepatology, 14, 1991, 789-797.

25. Nagano, 1.; Kita, 1.; Tanaka, N.; Int. J. Exp.Pathol., 73,1992, 675-683.

26. Rieder, H.; Meyer zum Btischenfelde K.H.; Ramadori, G.;J. Hepatol.15, 1992,237-250.

27. Knolle, P.A.; Loser, E.; Protzer, U.; Duchmann, R.; Schmitt, E.;

Zumbuschenfelde, K.H.M.; Rosejohn, S.; Gerken, G.; Clin. Exp. Immunol., 107, 1997, 555-561.

28. Ohkubo, K.; Masumoto, 1.; Horiike, N.; Onji, M.; J. Gastroemerol.

Hepatol. 13, 1998, 696-702. 29. Noji, S.; Tashiro, K.; Koyama, E.; Nohno, 1.;, Ohyama, K.; Taniguchi, S.;

Nakamura, 1.; Biochem. Biophys. Res., 173, 1990, 42-47.

30. Hashimoto, N.; Watanabe, 1.; Shiratori, Y.; Ikeda, Y.; Kato, H.; Han, K.; Yamada, H.; Toda, G.; Kurokawa, K.; Hepatology, 21, 1995,1713-1718.

31. Kuiper, J.; Zijlstra, F.; Kamps, J.A.; Van, Berkel, T.J.; Biochem.

Biophys. Acta, 959, 1988, 143-152. 32. Tsujii, H.; Okamoto, Y.; Kikuchi, E.; Matsumoto, M.; Nakano, H.;

astroenterology, 105, 1993, 495-499. 33. Kuiper, J.; De Rijke Y.B.; Zijlstra, F.J.; Van Waas, M.P.; Van Berkel,

T.J.; Biochem. Biophys. Res., 157, 1988, 1288-1295. 34. Van Oosten, M.; van de, Bilt E.; de Vries H.E.; Van Berkel T.J.C.; J,

Hepatology, 22, 1995, 1538-1546. 35. Steinhoff, G.; Behrend, M.; Schrader, B.; Duijvestijn, A.M.; Wonigeit, K.;

Am. J. Pathol., 142, 1993, 481-488.



21

36. Lopez, S.; Prats, N.; Marco, A.J.; Am. J. Pathol., 155, 1999, 1391-1397. 37. West, M.A.; Heaney, M.L.; In: Hepatocyte and Kupffer Cell

Interactions (Eds. Billiar TR, Curran RD), CRC Press, London, Tokyo 1992 pp. 209-241.

38. Bioulac Sage, P.; Kuiper, J.; Van Berkel T.J.; Balabaud, C.;

Hepatogastroenterology, 43,1996, 4-14. 39. Toth, C.A.; Thomas, P.; Hepatology, 16, 1992, 255-266. 40. van der Laan, L.J.; Dopp, E.A.; Haworth, R.; Pikkarainen, 1.; Kangas,

M.; Elomaa, 0.; Dijkstra, C.D.; Gordon, S.; Tryggvason, K.; Kraa,l G.; J. Immunology., 162, 1999, 939-947.

41. Fluiter, K.; vanderWesthuijzen, D.R. Vanberkel, T.J.C.; J. Biol. Chem., 273, 1998, 8434-8438.

42. Kamps, J.A.; Kruijt, J.K.; Kuiper, J.; Van Berkel, T.J.; Biochem. J., 276, 1991, 135-140.

43. Sano, A.; Taylor, M.E.; Leaning, M.S.; Summerfield, J.A.; J. Hepatol.,

10, 1990, 211-216.

44. Shimada, K.; Kamps, J.A.A.M.; Regts, J.; Ikeda, K.; Shiozawa, T.; Hirota, S.; Scherphof, G.L.; BBA-Biomembranes, 1326, 1997,329-341.

45. Biessen, E.A.; Bakkeren, H.F.; Beuting, D.M.; Kuiper, J.; Van Berkel

T.J.; Biochem. J., 299, 1994, 291-296. 46. Armbrust, T.; Nordmann, B.; Kreissig, M.; Ramadori, G.; Hepatology,

26, 1997, 98-106. 47. Maruiwa, M.; Mizoguchi, A.; Russell, G.J.; Narula, N.; Stronska, M.;

Mizoguchi, E.; Rabb, H.; Arnaout, M.A.; Blum, A.K. J.Immunol., 150, 1993, 4019-4030.

48. Bouwens, L.; Baekeland, M.; De Zanger, R.B.; Wisse, E.; Hepatology, 6, 1986, 718-722.

49. Coleman, D.L. Eur. J. Clin. Microbiol., 5, 1986,1-5. 50. Gressner A.M.; J. Hepatol., 22, 1995, 28-36. 51. Kanwar, S.; Kubes, P.; New. Horiz., 3, 1995, 93-104. 52. Radziszewski, W.; Chopra, M.; Zembowicz, A.; Gryglewski, R.; Ignarro, L

diol., 51, 1995, 211-220.



22

53. Goss, J.A.; Mangino, M.J.; Callery, M.P.; Flye, M.W.; Am. J. Physiol., 264, 1993, 601-608.

54. Goss, J.A.; Mangino, M.J.; Flye, M.W.; J. Surg. Res., 52, 1992, 422-428.

55. Fennekohl, A.; Schieferdecker, H.L.; Jungermann, K.; Puschel, G.P.; J.Hepatol., 30, 1999, 38-47.

56. Callery, M.P.; Mangino, M.J.; Flye, M.W.; Hepatology, 14, 1991, 368-372.

57. Alcolado, R.; Arthur, M.J.P.; lredale, J.P.; Clin. Sci., 92, 1997, 103-112s.

58. Scholmerich, J.; Holstege, A.; Drus., 40 (Suppl. 3) ,1990, 3-22. 59. Hoofnagle, J.H.; Digestion, 59, 1998, 563-578. 60. Kaplan, M.M.; Semiln.Liver. Dis., 17, 1997, 129-136.

61. Wolfhagen, F.H.; van Hoogstraten, H.J.; van Buuren, H.R.; Berge-Henegouwen, G.P.; Ten Kate, F.J.; Hop WC, van der Hoek, E.W.;

Kerbert M.J.; van Lijf, H.H.; den Ouden, J.W.; Smit A.M.; de Vries, R.A.; van Zanten R.A.; Schalm S.W.; J. Hepatol., 29, 1998, 736-742.

62. Messner, M.; Brissot, P.; Drugs, 40 (Suppl. 3), 1990, 45-57.

63. Kershenobich, D.; Vargas, F.; Garcia-Tsao, G.; Perez, T.R.; Gent, M.; Rojkind, N.; N.Engl. J. Med., 318, 1988, 1709-1713.

64. Bickel, M.; Baringhaus, K.H.; Gerl, M.; Gunzler, V.; Kanta, J.; Schmidts, L.; Stapf, M.; Tschank, G.; Weid-mann, K.; Werner, U.; Baringhaus, K.H.; Gunzler, V.; Hepatology, 28, 1998, 404-411.

65. Crowe, J.; Christensen, E.; Smith, M.; Cochrane, M.; Ranek, L.; Watkinsol Tygstrup, N.; Williams, R. Gastroenterology, 78, 1980, 1005-1010.

66. Fernandes, N.F.; Redeker, A.G.; Vierling, J.M.; Villamil, F.G.; Fong,

T.L.; Am. J. Gastroenterol., 94, 1999, 241-248.

67. Chazouilleres, O.; Wendum, D.; Serfaty, L.; Montembault, S.; Rosmorduc, O.; Poupon, R.; Hepatology, 28, 1998, 296-301.

68. Czaja, A.J.; Ann. Intern. Med., 125, 1996, 588-598.



23

69. Batey, R.G.; Alcohol Suppl., 2, 1994, 327-333.

70. Kawada, N.; Seki, S.; Kuroki, T.; Kaneda, K.; Biochem. Biophys. Res. Commun., 266, 1999, 296-300.

71. Baveja, R.; Kresge, N.; Bian X.; Yokoyama, Y.; Sonin N.V.; Clemens, M.G.; Zang, J.X.; Hepatology, 30, 1999, 232A.

72. Poo, J.L.; Jimenez, W.; Maria, M.R.; Bosch-Marce. M.; Bordas, N.; Morales-Ruiz, M.; Perez, M.; Deulofeu, R.; Sole, M.; Arroyo, V.; Rodes, J.; Gastroenterology, 116, 1999, 161-167.

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