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2. REVIEW OF LITERATURE 2.1 Inflammation and Inflammatory Diseases

Inflammation is considered as the most potent defense in the immune system

(Mogensen et al., 2009). It is a part of complex biological response by the vascular

tissues to harmful stimuli, such as pathogens, tissue injury or irritants (Eming et al.,

2007). A set of events that follows the wound or invasion of a pathogen, which may

result in a specific immune response for the clearance of the invasion or the invader

by the innate immune system is called the inflammatory response (Kindt and Kuby,

2007). It can be recognized based on symptoms like swelling, pain, heat and redness

in the affected tissue. It may occur around a skin infection like a boil or within a

tendon (tendinitis), a joint (arthritis) or a vital organ. Inflammation is mediated by

immune cells by releasing specific mediators which control local circulation and

cell activities. It can also occur when the host fights infection. It is a protective

attempt by the organism to remove the injurious stimuli and to initiate the healing

process.

Inflammation can be as either acute or chronic. The inflammatory response in the

former one is short-lived but in the latter the response stays relatively much longer.

Acute inflammation usually is highly helpful in isolating the damaged tissue and

healing the affected region. It is the initial response of the body to the harmful

stimuli and is achieved by the increased movement of plasma and leucocytes from

the blood into the injured tissues. This is followed by a cascade of biochemical

events that proceeds with the inflammatory response. On the other hand during

chronic inflammation, there will be prolonged secretion of various inflammatory

factors. Although chronic inflammation seems to be advantageous, the prolonged

effect has its own consequences of leading to various kinds of disorders such as hay

fever, atherosclerosis, rheumatoid arthritis and cancer. Balkwill and Mantovani

(2001) rightly mentioned that cancer is a fire lighted by the genetic mutations but

the inflammatory response may be the fuel for the flames of the cancer.

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2.2 Inflammation and Cancer

In 1863, Virchow (Balkwill et al., 2001) in his hypothesis stated that some of the

irritants have potential for inducing cancer through inflammation. Later this was

found to be due to the irritants along with the tissue injury, which are for the wound

healing resulting in enhanced cell proliferation. As the wound gets healed these

inflammatory factors recedes from the site. But due to the chronic inflammation and

the presence of inflammatory factors along with various agents including DNA

damaging agents, there is a chance that some cells undergo mutations and continue

to proliferate in the nutrient rich microenvironment resulting in cancer (Coussens

and Werb, 2002). There are many factors which triggers the cancer via

inflammation which include autoimmune disorder (colon cancer–inflammatory

bowel disease), microbial factors (gastric cancer-Helicobacter pylori infections) and

miscellaneous factors (prostate cancer- prostitis) (Table: 1).

Peyton Reus reported that many factors including viral infections result in the sub

threshold neoplastic states (Rous, 1910). This part is referred to as “initiation” step

of cancer. During cancer the first step is always followed by secondary signals,

including irritants and chemicals like phorbol esters and chemicals produced at the

site of wound healing, organ resection etc. This step is referred to as “promotion”

step. This step is where the cells which have the mutations, in the presence of

various inflammatory factors continue to proliferate and at later stage results in a

tumor (Cossens and Werb, 2002).

The host leucocytes including macrophages, dendritic cells and lymphocytes are

present in the inflammatory microenvironment both in the supporting stroma and

the tumor (Lu et al., 2006). Tissue mast cells have also shown to play a major role

in inflammation. All these factors prepare a provisional extracellular matrix where

the endothelial and fibroblast cells grow and produce an environment where the

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“promoted” cells grow. These conditions prevail during the wound healing of

injured tissues also. During tissue injury, platelet aggregation results in release of

thrombin which initiates the blood clotting preventing the loss of nutrients. Apart

from this, the platelet aggregation also induces various inflammatory processes by

secreting various proteins and α-granules to the affected site thus initiating

inflammation. During chronic inflammation, the process continuously goes on

resulting in possible mutations and suitable microenvironment for the cancer cells to

grow, thus resulting in tumor (Cossens and werb, 2002). Based on this Dovorak

(1986) called tumors as wounds that do not heal.

There are many inflammatory disorders. Some of them are not harmful to the body.

But some inflammatory bowel disorders like ulcerative colitis and Crohn’s disease

have strongest association with the tumor development in colon. Apart from these

schistosomiasis also plays a major role in colon carcinoma whereas the chronic

infection by H. pyroli is the leading cause for the development of stomach cancer.

The Gram-negative bacterium was proved to be the causative agent for gastric

cancer, where the mechanism is believed to be the DNA damage arising as a result

of chronic inflammation. Hepatatis C infection in liver also has strong influence

over the development of hepatocarcinoma. Here in this thesis, the effect of various

plant extracts on the possible inflammatory damage sites like liver (hepato

protection), stomach (ulcer) and external wounds (wound healing) with respect to

immune modulation was checked. Apart from this various extracts has been checked

for the potential anti-oxidative properties too.

Table 1: List of cancer and the related chronic immunological conditions

(Balkwill and Manowani, 2012)

Malignancy Inflammatory stimulus/condition

Bladder Schistosomiasis Cervical Papillomavirus

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Ovarian Pelvic inflammatory disease/talc/tissue remodeling

Gastric H. pylori induced gastritis MALT lymphoma H. pylori Oesophageal Barrett’s metaplasia Colorectal Inflammatory bowel disease Hepatocellular Hepatitis virus (B and C) Bronchial Silica, asbestos, cigarette smoke Mesothelioma Asbestos Kaposi’s sarcoma Human herpesvirus type 8

2.3 Importance of plant extracts for treatment

Extracts from plants contains compounds which are used for curing various

disorders. Ayurveda is the Indian traditional medicine which utilizes the plant and

plant derived compounds for the treatment of various disorders including cancer.

Based on Ayurveda, cancer can be developed from both inflammation and non-

inflammatory disorders. But for development of tumors, inflammation plays a major

role (Garodia et al., 2007). But the use of plant extracts for the treatment is now

limited to use in a particular region. First one is the variations and the number of

herbs used for the preparation of the extracts. This might in turn result in its effect in

the levels of the alkaloids present in the extracts. Among all plants, active principle

has been defined only for certain plants and for only some the chemical structures

are known. Therefore it cannot be completely ascertained that the cure/side effect is

because of a particular compound or multiple compounds. The second reason

attributed is the lack of complete clinical studies. Therefore safety of the plant

extracts was the concern of the scientists (liver herbal products). Therefore more

studies have to be performed in order to find new plant sources and new compounds

for the treatment of various disorders.

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2.4 Inflammation and Liver

Liver is an important organ in the human body. It controls the major part of the

human internal environment through various biochemical pathways. Some of the

major functions of the liver include protein and lipid metabolism, detoxification etc.

A summary of the functions of the liver is shown in the Table. 2. But the liver is

always subjected to huge amount of stress because of the pesticide contamination in

the food, alcoholism etc. which leads to increased oxidative stress in the liver.

Although liver has the capacity to regenerate itself, but if the contamination goes

beyond threshold limit, there will be change in the metabolism in the liver. Due to

this oxidative stress various potential disorders may raise in the liver including

inflammation.

Table 2: Summary of major functions of liver (table adopted from Treadway,

1998)

Carbohydrate Metabolism

Produces and stores glycogen (glycogenesis), produces glucose from liver glycogen and other molecules (gluconeogenesis) and releases it into the blood

Lipid Metabolism Oxidizes fatty acids to acetyl-CoA for energy production, synthesizes cholesterol, phospholipids, and bile salts, and excretes cholesterol in bile

Protein Metabolism Deamination of amino acids and produces urea, albumin, plasma transport proteins, and clotting factors Forms the intermediate product in the synthesis of active vitamin D hormone Stores iron as ferritin, and stores large amounts of vitamins A, D, and B12, and smaller amounts of other

Formation and Storage of Vitamins and Minerals

B-complex vitamins and vitamin K. Conjugates and excretes steroid hormones.

Detoxification of Blood

Biotransforms endogenous and exogenous compounds via Phase I and Phase II pathways of detoxif ication (glucuronidation, etc.)

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Hepatic fibrosis is the response to the wounds formed in the liver due to chronic

hepatic injury (Curcumin inflammation). Hepatic injuries may arise due to hepatitis,

fatty liver, cirrhosis, biliary cirrhosis and alcoholic liver disease (Treadway, 1998).

This condition is characterized by the abnormal formation of extracellular matrix

(ECM) in the wounded site. Research showed that the hepatic stellate cells and

kupffer cells secrete various factors including PDGF-β during this condition. The

key event in HSC activation followed by hepatic fibrosis is the inflammation caused

due to oxidative stress. Carbon tetrachloride (CCl4) induced hepatic fibrosis has

been used as an experimental model system. The reponse to chronic administration

of CCl4 by the liver tissue in rats is similar to human cirrhosis (Tamayo, 1983).

CCl4 induces lipid peroxidation and production of free radicals in the liver (Basu,

2003) which leads to necrosis in the hepatocytic cells, inflammation and finally

mimics the conditions of hepatic fibrinogenesis (Curcumin inflammation).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 1: Image showing the major factors that lead to the inflammation of liver

which finally leads to cancer (Image adopted from

http://www.in.gov/isdh/17438.htm).

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Figure 2: Possible pathways which might trigger ROS and DNA damage which

later leads to hepatocarcinoma (Image modified and adopted from Sun and Karin,

2012).

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2.5 Ulcer- inflammation

Now it has been proven through various studies that there is a strong relationship

between H. pylori infection in stomach and adenocarcinoma. There is a hypothesis

porposed that H. pylori infection causes gastric inflammation which may lead to

atrophic gastritis and this finally may lead to gastric cancer. Moreover the

organisms infection is also related to the gastric and peptic ulcerations, as described

by many studies which have shown the role of H. pylori in idiopathic peptic ulcer.

But most of the infections which may lead to chronic gastric inflammation remain

clinically silent (Blazer et al., 1995).

Figure 3: Image showing the involvement of H. pylori in the inflammation induced

at gastric epithelial cell (Adapted from Smith et al., 2006).

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Figure 4: Role of H. pylori in the induction of various factors leading to gastric

cancer (Image adopted from Kwiecien et al., 2002).

IL-1β is the prime most cytokine activated by the H. pylori infection. The infection

not only induces inflammation but also the neoplastic stage of the intestine. IL-1β is

a proinflammatory cytokine and inhibits the secretion of acid in the stomach.

Therefore when the bacterial virulence factors and the inflammatory factors

combine, the effect will be multi-factorial and may result in cancer (Smith et al.,

2006). Besides, during inflammation Tumor necrosis factor-α is also secreted which

along with IL-1β act as proinflammatory factors leading to various disorders.

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Figure 5: Possible involvement of various factors that induce COX-2 as a factor

that induces epithelial neoplasms which may end in cancer (Image adopted from

Kwiecien et al., 2002).

These inflammatory factors invite various cells to the affected site including

neutrophils. Neutrophils secrete various reactive oxygen species including super

oxide (O2-). This superoxide radical reacts with various lipids present in the cell

forming lipid peroxides. Therefore chronic inflammation enhances the production of

ROS and further may result in cancer (Kwiechen et al., 2002). Apart from

cytokines, Cyclooxygenase -2 (COX-2) was also found to play a major role in the

healing of ulcer. Various Non-steroidal anti-inflammatory agents (NSAIDs) have

been used for the treatment of gastric and peptic ulcers. When inhibitors for COX-1

were administered, the ulcer healed very slowly than the non-specific inhibitors for

the COX-2. Therefore, COX-2 was considered to be one of the candidates which are

targeted for treating gastric and peptic ulcer inflammation (Halter et al., 2001).

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2.6 Inflammation – ROS – antioxidative system

Reactive Oxygen Species (ROS) is a name given to particular set of ions and

radicals which include super oxide (O2), peroxyl (RO2), hydroxyl (OH), alkoxyl

(RO) group. Apart from these there are certain other members like HOCl, Ozone

(O3), singlet oxygen (O2) and H2O2 which can be easily converted into radicals also

fall in to this category. Mutations induced by ROS can lead to cancer and can occur

in the following three ways.

1. Alterations in base pairs – ROS induced alternations in DNA may result in

mutations in proto-oncogenes and tumor suppressor genes which may lead to

cancer.

2. Affect the cytoplasmic signal pathways – enhanced H2O2 production may lead to

loss of the inhibitory segment in NF-κB which continuously result in

transcriptional activation.

3. Modulation of the stress related genes – H2O2 can activate c-jun and MAP kinase

(Wiseman and Halliwell, 1996).

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Figure 6: Possible mutation sites for the ROS to exert its effects in leading to

cancer (Image modified and adopted from Nelson and Montgomery, 2003).

Oxidative stress is a major factor that triggers hepatic fibrosis, which in turn has

been shown to enhance the possibility of cancer (Curcumin inflammation liver).

Similarly H. pylori and other agents are shown to increase the ROS in the stomach

which in turn may result in cancer (Smith et al., 2006). Therefore, in both the cases

of liver and stomach, ROS plays a major role in inflammation and cancer. Therefore

inhibition of enhanced ROS production may be a possible strategy for the treatment

of the disorders in liver and stomach.

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Figure 7: Both extrinsic (eg. radiations) and intrinsic pathways can induce ROS

production where the antioxidant system can block the production of ROS (Image

modified and adapted from Perera and Bardeesy, 2011).

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2.7 Cancer – Immunomodulation

Many chemotherapy agents induce their effect through anti-proliferative and

cytotoxic effects. But the same agents had the capacity for immunosuppression as

the drug reduces the proliferation of immune cells which multiplies at a fast rate.

Some agents which exhibit the property of immunomodulation augmented the

treatment of cancer through the modulation of the immune system (Ehrke and Jane,

2003). Cyclophosphamide is a very good example for the immunomodulatory drug.

The drug has different role at higher concentrations but when the concentration is

lowered, administered alone or with some other agents exhibited anti-cancer and

immunomodulatory properties and cured cancer in mouse models. Apart from

cyclophosphamide, several other agents are shown to modulate the immune system,

nitrosoreus compounds such as adriamycin, arabinosylcitrosine etc. have been

shown to have the same potential (Ehrke et al., 1996).

 

Figure 8: Use of immune modulators in the treatment of prostate cancer, where the

T cells are activated by either threshold reduction or by enhancing the life cycle of T

effector cells (Image adopted from Kwek et al., 2012).

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Immunomodulation is used as a method in the treatment various cancers including

prostate cancer where, by inhibiting the activity of an immune checkpoint protein,

Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4), a crucial impedance can

be removed. This is followed by activation of T cells by lowering the threshold or

by eliminating the inhibitory signals which attenuate the effector T cells (Kwek et

al., 2012).

2.8 Plants as anti-microbials

Apart from H. pylori in causing inflammatory effect, humans in day to day life have

been infected by various microorganisms, which range from infections which are

easily controlled by the host to serious infections resulting in severe morbidity and

mortality. This prompted many scientists to look for new anti-microbial agents

from various sources including plants. Since time immemorial, human have used

plants and their extracts to treat various infectious diseases. Some plants like

cranberry (Vaccinium macrocarpon) was used to treat urinary infections, garlic

(Allium sativum), lemon (Melissa officinalis) etc have been used as antimicrobial

agents. The compounds can be used either directly as phytomedicine for a particular

disease or used as a base compound from which new compounds can be derived

(Iwu et al., 1999).

The search for newer drugs from plant products continues every day since the

scientists predicted that the average effective of every antibiotic is limited, which

kindles the way to produce new antibiotics for the use of mankind.