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Page 1: Master Thesis


Cancer is the second leading cause of death worldwide. In the United

States in 1999, it is estimated that over 1.2 million persons will be

diagnosed with invasive forms of cancer, and over 1,500 people will die

as a result of cancer each day. Cancer is an ailment that affects more or

less 200 types of cells. The major characteristic is the lack of control of

the cell proliferation, differentiation and health, invading organs and

tissues. There are many difficulties in the treatment but the more

frequently are the drug resistance,less toxicity and low specificity.

Medicinal plants play a key role in human healthcare. About 80% of the

world population relies on the use of traditional medicine, which is

predominantly based on plant materials.Scientific studies indicate that

the promising phytochemicals can be developed from the medicinal

plants for many health problems. Natural products from plants have been

valuable sources for anticancer drug discovery. Often the different

components in a herb have synergistic activities or buffer toxic effects.

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Mixtures of herbs are even more complex and so might have more

therapeutic or preventive activity than single products alone. In fact,

several studies have demonstrated that extracts from several herbal

medicines or mixtures had an anticancer potential in vitro or in

vivo.There is thus increased interest in alternative treatment modalities

that include chemotherapy, hormonal supplements, surgery, radiation

therapy, complementary or alterative medicine, used alone or in

combination. The administration of herbal drugs by patients without a

physician's prior counseling is increasing globally and there is a

possibility of herb–drug interactions too. Besides this Herbal drugs or

extracts themselves contain a combination of active constituents, which

interact within themselves and also between other prescribed

pharmaceutical drugs to either enhance (synergize) or decrease

(antagonize) the therapeutic effect. All phytochemicals tend to increase

the therapeutic effect by blocking one or more targets of the signal

transduction pathway, by increasing the bioavailability of the other drug

or, by stabilizing the other drug in the system. It has been a common

observation that herbal formulations are better than the synthetic drugs

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as they do not possess serious side effects and chronic toxicity. Plant

secondary metabolites also show promise for the cancer

chemoprevention, which has been defined as the use of non-cytotoxic

nutrients or pharmacological agents to enhance physiological

mechanisms that protect the organism against mutant clones of

malignant cells. Phenolic and flavonoids contents provide antioxidant

activities that may underlie the anticancer potential. Several plants have

reputed applications and are deliberately used in treatment of cancer and

inflammatory diseases. Plant derived natural products such as

flavonoids, terpenes, alkaloids and so on have received considerable

attention in recent years due to their diverse pharmacological properties

including cytotoxic and cancer chemopreventive effects. The rich and

diverse plant sources of India are likely to provide effective anticancer

agents. One of the best approaches in search for anticancer agents from

plant resources is the selection of plant based on ethno medical leads and

testing the selected plant’s efficacy and safety in light of modern

science[1] .

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Cytotoxicity assays are widely used in in vitro toxicology studies. The

LDH leakage assay, a protein assay,the neutral red,trypan blue assay

and the MTT assay are the most common employed for the detection of

cytotoxicity or cell viability following exposure to toxic substances[2].

The predictive value of in vitro cytotoxicity tests is based on the idea of

‘basal’ cytotoxicity – that toxic chemicals affect basic functions of cells

which are common to all cells, and that the toxicity can be measured by

assessing cellular damage. The development of in vitro cytotoxicity

assays has been driven by the need to rapidly evaluate the potential

toxicity of large numbers of compounds,to limit animal experimentation

whenever possible,and to carry out tests with small quantities of

compound. There are 3 basic parameters upon which these

measurements are based:-

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1.The LDH leakage assay is based on the measurement of lactate

dehydrogenase activity in the extra cellular medium. Reliability, speed

and simple evaluation are some of the characteristics

of this assay .It has been employed as an indicator of cytotoxicity in

Jurkat cells following exposure to Herbal extracts in different

concentration.The loss of intracellular LDH and its release into the

culture medium is an indicator of irreversible cell death due to cell

membrane damage[2] .

LDH cytotoxicity assay measures cell death in response to chemical

compounds using a coupled two step reaction.In the first step, LDH

catalyzes the reduction of NAD+ to NADH and H+ by oxidation of

lactate to pyruvate.In the second step of the reaction,diaphorase uses the

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newly formed NADH and H to catalyze the reduction of a tetrazolium

salt(INT) to highly- coloured formazan which absorbes Strongly at 490-

520 nm.The amount of formazan produced is proportional to the amount

of LDH released into the culture medium as a result of cytotoxicity.[3]

2.The MTT assay is another cell viability assay often used to determine

cytotoxicity following exposure to toxic substances. It has been used in

jurkat cells after exposure to herbal extracts. MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water

soluble tetrazolium salt,which is converted to an insoluble purple

formazan by cleavage of the tetrazolium ring by succinate

dehydrogenase within the mitochondria. The formazan product

is impermeable to the cell membranes and therefore it accumulates in

healthy cells. This reduction takes place only when mitochondrial

reductase enzymes are active, and therefore conversion can be directly

related to the number of viable (living) cells. When the amount

of purple formazan produced by cells treated with an agent is compared

with the amount of formazan produced by untreated control cells, the

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effectiveness of the agent in causing death of cells can be

deduced,through the production of a dose-response curve. Mitochondrial

dehydrogenases of viable cells cleave the tetrazolium ring, yielding

purple MTT formazan crystals which are insoluble in aqueous solutions.

The crystals can be dissolved in acidified isopropanol. The resulting

purple solution is spectrophotometrically measured. An increase in

cell number resultsin an increase in the amount of MTT formazan

formed and an increase in absorbance[4] .

This is a colorimetric assay that measures the reduction of yellow 3-(4,5-

dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by

mitochondrial succinate dehydrogenase. The MTT enters the cells and

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passes into the mitochondria where it is reduced to an insoluble,

coloured (dark purple) formazan product. The cells are then solubilised

with an organic solvent (eg. isopropanol) and the released, solubilised

formazan reagent is measured spectrophotometrically. Since reduction

of MTT can only occur in metabolically active cells the level of activity

is a measure of the viability of the cells[5].

3.The Trypan Blue assay is used to measure cell viability. It has been

used as an indicator of cytotoxicity in Jurkat cell lines and other cell

lines. Trypan Blue is a vital stain used to selectively color dead tissue or

cells.It is diazo dye. Living cells or tissues with intact cell

membrane are not coloured. Since cells are very selective in the

compounds that pass through the membrane,in a viable cells trypan blue

is not absorbed;however it traverses the membrane in the dead

cells.Hence dead cells are shown as a distictiveblue colour under the

microscope.Since living cells are excluded from staining,this staining

method is also described as a Dye Exclusion Method[6].

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Dye exclusion is a simple and rapid technique measuring cell viability

but it is subject to the problem that viability is being determined

indirectly from cell membrane integrity.Thus, it is possible that a cell’s

viability may have been compromised (as measured by capacity to grow

or function) even though its membrane integrity is (at least transiently)

maintained. Conversely, cell membrane integrity may be abnormal yet

the cell may be able to repair itself and become fully viable[7] .

Toxicological Parameters

1. IC50

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The half maximal inhibitory concentration (IC50) is a measure of

the effectiveness of a compound in inhibiting biological or

biochemical function. This quantitative measure indicates how

much of a particular drug or other substance (inhibitor) is needed

to inhibit a given biological process (or component of a process,

i.e. an enzyme, cell, cell receptor or microorganism) by half. IC50

represents the concentration of a drug that is required for 50%

inhibition in vitro[8]

The IC50 is a measure of how effective a drug is. It indicates how

much of a particular drug or other substance is needed to inhibit a

given biological process,in this case growth of Jurkat Cells[9].The

IC50 of a drug can be determined by constructing a dose-response

curve and examining the effect of different concentrations of

antagonist on reversing agonist activity. IC50 values can be

calculated for a given antagonist by determining the concentration

needed to inhibit half of the maximum biological response of the


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2. LD50

LD stands for "Lethal Dose". LD50 is the amount of a material,

given all at once, which causes the death of 50% (one half) of a

group of test animals or cells. The LD50 is one way to measure the

short-term poisoning potential (acute toxicity) of a material. In

toxicology, the median lethal dose, LD50 of a toxin, radiation, or

pathogen is the dose required to kill half the members of a tested

population after a specified test duration. LD50 figures are

frequently used as a general indicator of a substance's acute

toxicity. This type of test is also referred to as a "quantal" test

because it is measures an effect that "occurs" or "does not


3. EC50

The term half maximal effective concentration (EC50) refers to

the concentration of a drug, antibody or toxicant which induces a

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response halfway between the baseline and maximum after some

specified exposure time. It is commonly used as a measure of

drug's potency.

The EC50 of a graded dose response curve therefore represents the

concentration of a compound where 50% of its maximal effect is

observed. The EC50 of a quantal dose response curve represents the

concentration of a compound where 50% of the population exhibit

a response, after a specified exposure duration. Concentration

measures typically follow a Sigmoidal curve, increasing rapidly

over a relatively small change in concentration. The point at which

the effectiveness slows with increasing concentration is the IC50[11]


4. Percentage cytotoxicity

It is another measure of drug potency. It measures the extent to

which cell died after exposure to drug for specific duration of time

in a given conditions.It is the ratio of absorbance of extract treated

cell divided to the absorbance of untreated or control cells

substracted from 100.

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% cytotoxicity = 100-[(A540 in extract treated cells/A540 in untreated cell)×100]

5. Lymphocyte proliferation Index

It is relative measure of lymphocyte proliferation in untreated cell

compared to extract treated cell.It is simply the ratio of absorbance of

extract treated cell to the absorbance of untreated cells at 540nm.

LPI = A540 in extract treated cell/A540 in untreated cells × 100

This parameter is calculated using MTT assay.

Plant Material

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1. Piper longum

Piper longum is of South Asian origin and is found almost all over India. Its a

slender aromatic climber With perennial woody roots.Piper longum has diverse

pharmacological uses including nerve depressant & antagonistic effect on

electro-shock and chemo-shock seizures as well as muscular in-coordination and

anticarcinogenic effects.Pepper long is the dried fruit of Piper longum which is a

slender, aromatic plant with creeping jointed stems and perennial woody .Pepper

contains volatile oil, the crystalline alkaloids: piperine, piperidine and piperettine,

and a resin[12].

Uses of Piper longum :

Piper longum is widely used in Ayurvedic and Unani systems of medicine

particularly for diseases of respiratory tract most of them s cough, bronchitis, includes

cough,bronchitis, asthma etc and has also anticarcinogenic effects

Long pepper is locally applied to counter-irritant and act as analgesic for

muscular pains and inflammation.

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Long pepper acts as a general tonic and hematinic and widely used in

Ayurveda as good rejuvenator (Rasayana).

Piper longum is known to enhance the bio-availability of food and drugs. In

fact Piper Longum is taken along with Quinine.

Piper longum is widely used as a carminative.

The Piper longum fruit contain 1% volatile oil,resin, a waxy alkaloid. It is used

for several medicinal properties. It has much pharmacological action such as

antifungal, anti-inflammatory, antioxidant and anti cancer effect and it is known to

have insecticidal activity against mosquitoes and flies[13].The plant grows all over

India, in evergreen forests and is cultivated in Assam, Tamil Nadu and Andhra

Pradesh. Piper longum contains a component called Piperine. Piperine is an

alkaloid found naturally in plants belonging to the pyridine group of Piperaceae

family, such as Piper nigrum and Piper longum. It is widely used in various herbal

cough syrups and it is also used in anti inflammatory, anti malarial, anti leukemia

treatment.Piperine is widely used in various herbal cough syrups for its potent

anti-tussive and bronchodilator properties.Recent medical studies have shown that

it is helpful in increasing the absorption of certain vitamins, selenium, beta-cartene,

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also increase the body’s natural thermogenic activity[14].

Piperine is known to exhibit a variety of biological activities which include

anti-pyretic anti-metastatic antithyroid and antidepressant. Piperine exhibits a toxic

effect against hepatocytes and cultured hippocampal neurons.Simultaneous

supplementation with black pepper or piperine in rats fed high fat diet lowered

thiobarbituric acid reactive substances (TBARS) and conjugated dienes levels and

maintained superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase

(GPX),glutathione-S- transferase (GST) and glutathione (GSH) levels close to

control rats. It has also been observed that supplementation of piperine caused

inhibition of Phase I and II enzymes, elevation of glutathione metabolizing

enzymes, reduction in DNA damage and DNA protein cross-links in benzo (a)

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Figure (1): Piperine

pyrene induced lung carcinogenesis in mice[15]. Piperine prevent the

formation of oral carcinoma, probably due to its antilipidperoxidative

and antioxidant potential as well as its modulating effect on the

carcinogen detoxification process.Many scientist observed that piperine

displayed an anti-proliferation effect at 24 hours and statistically

significant inhibition at 48 and 72 hours at 100 - 200 μM concentration

against cultured human colon cancer cells (DLD-1). Also has been

investigated the influence of piperine on chromosomes in rat bone

marrow. piperine administered to Wister male rats at dose of 100, 400

and 800 mg/kg, b wt, (p.o.) for 24 hrs then challenged with

cyclophosphamide at a dose of 50 mg/kg, b wt, (i.p.). They

demonstrated that piperine at a dose of 100 mg/kg, gave a statistically

significant reduction in chromosomal aberration[16].

Piperaldehyde is one of the important constituent of piper longum Linn.

It was isolated from the fruits of the piper longum by extracting with

methanol as solvent. Studies shows that the pet alcoholic extract and

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piperaldehyde shows significant DPPH scavenging activity. The extract

and piperaldehyde were also found to exert protective effective in the

myocardial narcotic rats.They have protected myocardium from the

harmful effects of lipid per oxidation and even maintained the gluthione

levels to normal. Hence it can be concluded that the alcoholic extract as

well as piperaldehyde are useful in exerting protective activity in case of

myocardial ischemia in treated animals. Piperaldehyde showed dose

dependent inhibitory activities on platelet aggregation induced by

collagen, AA, and PAF, except for that induced by thrombin.

Piperaldehyde had the most potent antiplatelet effect. Piperaldehyde

inhibited platelet aggregation induced by collagen[17].

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Figure (2): Piperaldehyde

Another compound isolated from metahanolic extract of Piper longum ,

piperettine, has properties similar to that of piperine.

Figure : Piperettine

2. Piper Nigrum

Black pepper (Piper nigrum) is a flowering vine in the family

Piperaceae, cultivated for its fruit, which is usually dried and used as a

spice and seasoning. The fruit, known as a peppercorn when dried, is

approximately 5 millimetres (0.20 in) in diameter, dark red when fully

mature, and, like all drupes, contains a single seed. Peppercorns, and the

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powdered pepper derived from grinding them, may be described simply

as pepper, or more precisely as black pepper, white pepper, or green

pepper. Green peppercorns are simply the immature black


Black peppers are native to India and are extensively cultivated there

and elsewhere in tropical regions. Currently Vietnam is by far the

world's largest producer and exporter of pepper, producing 34% of the

world's Piper nigrum crop as of 2008[19].

Dried ground pepper has been used since antiquity for both its flavor and

as a medicine. Black pepper is the world's most traded spice. It is one of

the most common spices added to European cuisine and its descendants.

The spiciness of black pepper is due to the chemical piperine. It may be

found on nearly every dinner table in the industrialized world, often

alongside table salt.Piper Nigrum or Black pepper oil can be used to help

in the treatment of pain relief, rheumatism, chills, flu, colds, increase

circulation, exhaustion, muscular aches, physical and emotional

coldness, nerve tonic and fevers. It furthermore increases the flow of

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saliva, stimulates appetite, encourages peristalsis, tones the colon

muscles and is a general digestive tonic. Sometimes it is used in place of

cubebs for gonorrhoea. As a gargle it is valued for relaxed uvula,

paralysis of the tongue. On account of its stimulant action it aids

digestion and is especially useful in atonic dyspepsia and turbid

condition of the stomach. It will correct flatulence and nausea. It has

also been used in vertigo, paralytic and arthritic disorders. It has also

been advised in diarrhoea, cholera, scarlatina and in solution for a wash

for tinea capititis. Externally it is used for its rubefacient properties and

as a local application for relaxed sore throat and some skin diseases. Its

oleoresin has bacteriostatic and fungistatic properties. In Ayurvedic

medicine, this herb is used for treatment of various disorders such as

asthma, colon toxins, obesity, chronic indigestion, sinus congestion,

fever, cold extremities, intermittent fever, cholera, colic pain, diarrhea,

gastric ailments, worms and piles. Various health benefits of black

pepper are described here in this article[19].

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Health Benefits of Piper nigrum

Some of the medicinal properties of Piper nigrum are as follows:

Appetizer – Piper nigrum is an effective home remedy for various

digestive disorders. The herb increases production of gastric juices and

saliva. To increase appetite, mix powdered black pepper with malted

jaggery. To get relief from indigestion and stomach heaviness, you can

take piper powder mixed in the buttermilk.

Dentrifice – Mix piper powder and salt to prevent foul breath, dental

caries, toothache, bleeding and painful gums. Mix pepper powder with

clove oil and apply it on the caries to prevent toothache.

Fever – Piper is effective for cure of fever and severe cold. To get relief

from cold in the head, 20 grams of powdered pepper boiled in milk and

added a pinch of turmeric can be taken once a day for three days.

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Rheumatism – Small amount of pepper powder fried in a little amount

of sesame oil. This mixture can be applied as an analgesic for treatment

of rheumatic and myalgia pains.

Potency – 6 peppers are eaten with 4 almonds with milk to treat

impotency and it is also a never tonic[20].

Many of the secondary metabolites isolated from the piper nigrum has

been found to show anticancer properties. Pellitorine , an compound

isolated from the roots of Piper nigrum, showed strong cytotoxic

activities against many cancer cell line. Two other alkaloids were also

found from Piper nigrum. They are (E)-1-[3’,4’ (methylenedioxy)

cinnamoyl]piperidine and 2,4-tetradecadienoic acid isobutyl amide.

These compounds,having anticarcinogenic properties, were isolated

Figure (2): Pellitorine

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using chromatographic methods and their structures were elucidated

using MS, IR and NMR techniques[21].These compounds are found to

have anti proliferative activity and showed cytotoxicity activity against

cancer cell line.Piper nigrum contains small quantities of

chemopreventive and the antimutagenic activity of black pepper could

be related to the large number of these potent chemopreventive

compounds such as β-carotene,piperine, tannic acid and capsaicin[22].

Black pepper has been reported to be rich in glutathione peroxidase,

glucose-6-phosphate dehydrogenase, and vitamin E. Both water extract

and ethanol extract of black pepper exhibited a strong antioxidant

activity. Piperine, an active alkaloid, is known to possess several

pharmacological actions such as antimetastatic , antimutagenic and

antioxidant. Besides this Piperine modulates benzo[a]pyrene metabolism

by direct interaction with the cytochrome P4501A1 enzyme and it

counteracts aflatoxin B1 toxicity by suppressing cytochrome P-450-

mediated bioactivation of the mycotoxin. The recent studies suggested

that piperine might protect human from various types of cancer . It

reduces experimentally induced colon, liver, and pulmonary cancers .

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some studies has reported reported the protective effect of piperine

against benzo[a]pyrene -induced experimental lung carcinogenesis in

Swiss albino mice by altering the tricarboxylic acid (TCA) cycle

enzymes, reducing phase I enzymes and by enhancing the activity of

glutathione-metabolizing enzymes. In addition, piperine plays an

important role against benzo[a]pyrene induced lung carcinogenesis in

Swiss albino mice by protecting the glycoprotein levels in serum

and tissue, as it is one of the indicators of tumorigenesis.Piperine

demonstrates not only chemopreventive effect , but also genoprotective

effects against benzo[a]pyrene induced mutagenesis in Swiss albino

mice. It can induce apoptosis in benzo[a]pyrene –induced lung

carcinogenesis in Swiss albino mice. Furthermore, black pepper

supplementation can protect the colon by decreasing the activity of

bacterial enzymes β-glucuronidase and mucinase, in the presence of the

procarcinogen 1,2-dimethyl hydrazine (DMH) in rats . Piperine,one of

the constituent of piper nigrum, has been demonstrated in in vitro studies

to protect against oxidative damage by inhibiting or quenching free

radicals and reactive oxygen species. Black pepper or piperine treatment

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has also been evidenced to lower lipid peroxidation in vivo and

beneficially influence cellular thiol status, antioxidant molecules and

antioxidant enzymes in a number of experimental situations of oxidative

stress.However, Piperine is known to induce antiproliferative pro-

apoptotic effects in a diverse range of tumor cells by arresting G2/M,

down-regulating NF-kappa B, Akt, cyclin D, c-myc and initiating PARP

cleavage/DNA fragmentation.This herb root is also known to contain

sapogenins, phytosterols, beta-sitosterol and campesterol, which may

contribute in part to the tumoricidal properties[23].

Figure (3): Phytosterols

3. Zingiber officinale

Ginger is the rhizome of the plant Zingiber officinale, consumed whole

as a delicacy, medicine, or spice. It lends its name to its genus and

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family (Zingiberaceae). Ginger cultivation began in South Asia and has

since spread to East Africa and the Caribbean. It is sometimes called

root ginger to distinguish it from other things that share the name

ginger. The characteristic odor and flavor of ginger is caused by a

mixture of zingerone, shogaols and gingerols, volatile oils that compose

one to three percent of the weight of fresh ginger. In laboratory animals,

the gingerrols increase the motility of the gastrointestinal tract and have

analgesic, sedative, antipyretic and antibacterial properties. Ginger oil

has been shown to prevent skin cancer in mice and a study at the

University of Michigan demonstrated that gingerols can kill ovarian

cancer cells[24].

[6]-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone)

is the major pungent principle of ginger. The chemopreventive potentials

of [6]-gingerol present a promising future alternative to expensive and

toxic therapeutic agents.Ginger contains up to three percent of a fragrant

essential oil whose main constituents are sesquiterpenoids, with (-)-

zingiberene as the main component. Smaller amounts of other

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sesquiterpenoids (β-sesquiphellandrene, bisabolene and farnesene) and a

small monoterpenoid fraction (β-phelladrene, cineol, and citral) have

also been identified but its principal constituent are zingiberene, and

zingiberol[25].Ginger is valued in medicine as a carminative and stimulant

to the gastro-intestinal tract.It is much in vogue as a household remedy

for indigestion dyspepsia,flatulence and colic. Ginger is reported to

contain an antihistaminic factor.It is included among antidepressants and

it forms an ingredient of some anti-narcotic preparations. Ginger is

primarily used to treat nausea, but it is also used as an anti-

inflammatory, a pain remedy, a warming remedy and a cholesterol-

lowering herb. Randomized controlled trials support its use in

preventing nausea[26].

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Figure (4): Gingerols

Gingerols isolated from Zingiber officinale inhibit growth & spread of

various cancers including that of the ovary, cervix, colon, rectum, liver,

urinary bladder, oral cavity, neuroblastoma and leukaemia by inducing

apoptosis. The most active individual component, 6-shogaol, isolated

from Zingiber officinale, inhibit growth & spread of many cancers

particularly the ovarian cancer by blocking formation of new blood

vessels and by inducing apoptosis & autophagy. It is effective even in

chemotherapy resistant ovarian cancer. Zingiber officinale also

possesses antioxidant, antimutagenic and anti-inflammatory properties

and reduces side effects of chemotherapy & radiotherapy[27]. The

pungent vanilloids, gingerol and paradol found in ginger, are very highly

effective in killing cancer cells. They achieve this both by direct

cytotoxic activity against the tumour and indirectly by inducing

apoptosis in the cancer cells[28]. Ginger was also shown to have

anticancer effect by inducing apoptosis in rat liver cancer cells via

upregulation of the expression of pro-apoptotic protein, caspase-8 and

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downregulation of the expression of antiapoptotic protein Bcl-2. b-

Elemene, an anticancer drug extracted from the ginger plant, triggered

apoptosis in non-small-cell lung cancer cells through a mitochondrial

release of the cytochrome c-mediated apoptotic pathway.[6]-gingerol a

major phenolic compound derived from ginger, inhibited TRAIL

induced NF-kB activation by impairing the nuclear translocation of NF-

kB, suppresses cIAP expression and increased TRAIL-induced caspase-

3/7 activation in gastric cancer cells. On the other hand, [6]-shogaol

alone reduced viability by damaging microtubules, arrested cell cycle in

G2/M phase and reduced viability in a caspase-3/7-independent manner

in gastric cancer cells.The other compound, zerumbone antagonises the

processes of both tumour initiation and promotion. It does this by

inducing antioxidant enzymes and by weakening the pro-inflammatory

signalling pathways associated with communication between cancer

cells. Zerumbone found in subtropical ginger Zingiber zerumbet exhibits

antiproliferative and antiinflammatory activities. Zerumbone mediates

its activity through the modulation of NF- B activation, zerumbone

suppressed NF- B activation induced by tumor necrosis factor (TNF),

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okadaic acid, cigarette smoke condensate, phorbol myristate acetate, and

H2O2 and that the suppression was not cell type specific[29].

Figure (5): zerumbone

Cancer is often associated with inflammatory processes and ginger’s

potent anti-inflammatory activity reduces the risk of inflammation-

induced malignancy. Ginger is an effective COX-2 inhibitor, curtailing

the activity of potentially damaging COX-2 enzymes, the

overproduction of which may cause harm to several tissue

types[30].Ginger is listed among the herbs possessing the highest

antitumor activities. The observed evidence from in vitro experiments

warrant further investigations into the potential anti-tumor activity of

ginger. This antitumor activity was shown to be related to its vanilloids,

[6]-gingerol and [6]-paradol. Both compounds were shown to block the

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epidermal growth factor responsible for cell transformation, thus

inhibiting cellular proliferation.Ginger may also produce its antitumor

effect by inducing “programmed cell death,” also known as apoptosis, in

cancer cells[31]. In one study, the cytotoxic effect of [6]-gingerol and

Figure (6): Paradols

[6]-paradol was associated with the induction of apoptosis in human

promyelocytic leukemia cells (HL-60). A third mechanism of ginger’s

antitumor protection is its modification to the carcinogen-metabolizing

enzymes in the liver. Both glutathione S-transferase and aryl

hydrocarbon hydroxylase activity in the liver were elevated following

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administration of ginger oil to mice for 14 consecutive days. [6]-paradol

induced apoptosis in cancer cells , but concentrations of 50 μM or

greater resulted in apparent necrotic cell death . Therefore, [6]-paradol

appears to exert its primary inhibitory effect on cell transformation

through the induction of apoptosis. The application of [6]-gingerol to the

shaven backs of mice prior to applying cancer-promoting agents

significantly inhibited skin cancer formation. gingerol has been shown to

inhibit cell adhesion, invasion, and motility in ER-negative (estrogen

independent) human breast cancer cells in the laboratory. [6]-gingerol

and[ 6]-paradol block EGF-induced cell transformation and although

closely related structurally, act through different mechanisms.[ 6]-

Gingerol inhibit EGF-induced AP-1 transactivation by blocking EGF-

induced AP-1 DNA binding activity in a concentration-dependent

manner, and in contrast, [6]-paradol had no effect on AP-1 activation.

Therefore, in exerting their antitumorigenic effects,[ 6]-gingerol appears

to act through inhibition of AP-1 activation, whereas [6]-paradol appears

to act through induction of apoptosis[32].

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4.Glycyrrhiza glabra

Glycyrrhiza glabra, also known as licorice and sweetwood, is native to

the Mediterranean and certain areas of Asia. Historically, the dried

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rhizome and root of this plant were employed medicinally by the

Egyptian, Chinese,Greek, Indian, and Roman civilizations as an

expectorant and carminative. In modern medicine, licorice extracts are

often used as a flavoring agent to mask bitter taste in preparations, and

as an expectorant in cough and cold preparations. Licorice extracts have

been used for more than 60 years in Japan to treat chronic hepatitis, and

also have therapeutic benefit against other viruses, including human

immunodeficiency virus (HIV), cytomegalovirus (CMV), and Herpes

simplex. Deglycyrrhizinated licorice (DGL) preparations are useful in

treating various types of ulcers, while topical licorice preparations have

been used to sooth and heal skin eruptions, such as psoriasis and herpetic

lesions[33]. A number of components have been isolated from licorice,

including a water-soluble, biologically active complex that accounts for

40-50 percent of total dry material weight. This complex is composed of

triterpene saponins, flavonoids, polysaccharides, pectins, simple sugars,

amino acids, mineral salts, and various other substances[34].Glycyrrhizin,

a triterpenoid compound, accounts for the sweet taste of licorice root.

This compound represents a mixture of potassium-calcium-magnesium

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salts of glycyrrhizic acid that varies within a 2-25 percent range. Among

the natural saponins, glycyrrhizic acid is a molecule composed of a

hydrophilic part, two molecules of glucuronic acid, and a hydrophobic

fragment, glycyrrhetic acid. The yellow color of licorice is due to the

flavonoid content of the plant, which includes liquiritin, isoliquiritin (a

chalcone), and other compounds[35]. The isoflavones glabridin and

hispaglabridins A and B have significant antioxidant activity,and both

glabridin and glabrene possess estrogen-like activity.The beneficial

effects of licorice can be attributed to a number of

mechanisms.Glycyrrhizin inhibit hepatic metabolism of aldosterone and

suppress 5-ßreductase, properties responsible for the well-documented

pseudoaldosterone syndrome. The similarity in structure of glycyrrhetic

acid to the structure of hormones secreted by the adrenal cortex accounts

for the mineralocorticoid and glucocorticoid activity of glycyrrhizic


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Figure :(7) glycyrrhizic acid Figure :(8) Glycyrrhetinic acid

Both compounds possesses a great variety of pharmacological and

biological properties such as anti-inflammatory, anti-ulcerogenic, anti-

allergic, anti-oxidant, anti-hepatotoxic, anti-tumor, and antiviral


Certain licorice constituents possess significant antioxidant and

hepatoprotective properties. Glycyrrhizin and glabridin inhibit the

generation of reactive oxygen species (ROS) by neutrophils at the site of

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inflammation. In vitro studies have demonstrated licorice isoflavones,

hispaglabridin A and B,inhibit Fe3+-induced mitochondrial lipid

peroxidation in rat liver cells. Other research indicates glycyrrhizin

lowers lipid peroxide values in animal models of liver injury caused by

ischemia reperfusion. Licorice constituents also exhibit hepatoprotective

activity by lowering serum liver enzyme levels and improving tissue

pathology in hepatitis patients[38].

Figure (9): Glycyrrhizin

Glycyrrhizin and other licorice components appear to possess

anticarcinogenic properties as well. Although the exact mechanisms are

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still under investigation,research has demonstrated they inhibit abnormal

cell proliferation, as well as tumor formation and growth in breast, liver,

and skin, cancer. Glycyrrhizin is a triterpenoid saponin found in

Glychyrrhiza glabra (licorice). Chemically, glycyrrhizin is a sulphated

polysaccharide named (3-beta,20-beta)-20-carboxy-11-oxo-30-norolean-

12-en-3-yl 2-O-beta-D-glucopyranuronosyl-alpha-D

glucopyranosiduronic acid[39]. It is considered to the active constituent of

the drug and the standardization of licorice is based on glycyrrhizin

content. The standardized extracts of licorice sold in the market contain

20% of glychrrhizin. Glycyrrhizin is converted into glychyrrhetic acid

by an enzyme, glycaronidase.Constituents of licorice includes

triterpenoids, such as glycyrrhizin and its aglycone glycyrrhizic acid,

various polyphenols, and polysaccharides. A number of pharmaceutical

effects of licorice are known or suspected (anti-inflammatory, antivirus,

antiulcer, anticarcinogenesis, and others). Licorice and its derivatives

protect against carcinogen-induced DNA damage and may be

suppressive agents as well. Glycyrrhizic acid is an inhibitor of

lipoxygenase and cyclooxygenase, inhibits protein kinase C, and

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downregulates the epidermal growth factor receptor. Licorice

polyphenols induce apoptosis in cancer cells. These and other activities

of licorice are reviewed, and a rationale is suggested for combinations of

agents in preventive clinical trials. G.glabra extract has been used in

herbal formulations for treatment of cancers.G.glabra appear to induce

the bcl-2 phosphorylation and G2/M cycle arrest in tumor cell lines.70%

methanol soluble fraction of G.glabra extract was found to induce

apoptosis in human monoblastic leukaemic U937 cells.The compound

was identified to be licocoumarone also responsible for antioxidant and

antimicrobial activity.Activator protein-1 (AP-1) is a nuclear

transcription factor.Blocking of tumour promoter induced AP-1 activity

could be used to arrest the induced cellular transformation.It was found

that Glycyrrhizin induced AP-1 activity in untreated cells whereas

inhibited TPA induced AP-1 activity in TPA treated cells[40].

A variety of phytochemicals present in root extract of glycyrrhiza

exhibit a potential antioxidant activity.Licochalcones B and D exhibit a

potential activity by inhibiting the microsomal lipid

peroxidation.Retrochalcones exhibit mitochondria lipid peroxidation and

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prevent red blood corpuscles from oxidative hemolysis.Isoflavones like

glabridin & hispaglabridin A present in glycyrrhiza were found to have a

very potential antioxidant activity against NADH dependent

peroxidation injury[41].

Figure: Licochalcones

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4. Phyllanthus emblica

Amla (phyllanthus emblica) is a medium-sized deciduous tree. It is also

named Emblica officinalis. It belongs to the plant family

Euphorbiaceae.The fruits of P.emblica L. known as amla, are consumed

as fruit, or in the form of food products. The species is native to India

and also grows in tropical and subtropical regions. It primarily contains

tannins, alkaloids, and phenolic compounds, but flavonoids derived from

amla shows maximum beneficial in medicinal aspect. Ayurveda and

Siddha systems of medicine have recognized the importance of this

plant. It is one of the strongest rejuvenatives among Indian medicinal

plants due to its antimicrobial , antifungal , radioprotective ,

chondroprotective , antimutagenic, and anticancer properties , but its

most extraordinary features are its anti-inflammatory and antioxidative

properties . A clinical study has also found that amla showed significant

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antioxidant properties . The fruits of Emblica officinalis are rich in

tannins. The fruits have 28% of the total tannins distributed in the whole

plant. The fruit contains two hydrolysable tannins Emblicanin A and B,

which have antioxidant properties, one on hydrolysis gives gallic acid,

ellagic acid and glucose wherein the other gives ellagic acid and


Phyllanthus emblica is a medicinal fruit used in many Asian traditional

medicine systems for the treatment of various diseases including cancer.

Eighteen main compounds, including four norsesquiterpenoids and 14

phenolic compounds isolated from Phyllanthus emblica, together with a

main constituent, proanthocyanidin polymers identified from the roots,

were found to be highly effective for their antiproliferative activities

against many cancer cell line.Norsesquiterpenoid glycosides,

phyllaemblicins B and C , exhibited significant inhibitory effects on

many cancer cell proliferation[43].

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Figure (10): Norsesquiterpenoid glycosides

All phenolic compounds (5—18 )showed inhibitory activity against the

growth of the many tumor cell lines besides their antioxidant properties.

Research showed that the antitumor activity of polyphenols might be

linked to their anti-inflammatory properties.Since P. emblica has been

used widely for its anti-inflammatory and antipyretic effects by local

people in its growing areas. Phylanthus emblica significantly inhibited

hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in

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experimental animals.20 In addition to its hepatoprotective activities,

Phylanthus emblica appears to be functional in acute necrotizing

pancreatitis, reducing inflammation and the damage to acinar cells[44].

The studies have showed that Emblica officinalis has significant ulcer

protective and healing effects and this might be due to effects both on

offensive and defensive mucosal factors.27 The breast cancer is one of

the most common cancers in women.Lipid-metabolizing enzymes, lipids

and lipoproteins have been associated with the risk of breast cancer.The

elevated levels of free cholesterol, total cholesterol,triglycerides,

phospholipids and free fatty acids and decreased levels of ester

cholesterol in plasma, kidney and liver found in cancer suffering animals

were reverted back to near normal levels on treatment with methanolic

extracts of Emblica officinalis.Besides it antiproliferative properties

Phylanthus emblica has been found to enhance natural killer cell activity

and antibody dependent cytotoxicity in tumor bearing mice, enhancing

lifespan to 35% beyond the control animals. Phylanthus emblica has

been shown to significantly reduce the cytotoxic effects of sodium

arsenite when administered orally in experimental animals. Amla

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resulted in an enhanced cell survival, decreased free radical production

and higher antioxidant levels similar to that of control cells. Phylanthus

Emblica enhance cell survival, increases phagocytosis and gamma-

interferon (Γ-IFN) production. Phylanthus Emblica has beneficial effects

such as memory improving property, cholesterol lowering property and

anticholinesterase activity.Phyllembin, isolated from the ethanolic

extract of the fruit pulp has been found to potentiate the action of

adrenaline in vitro and in vivo. It showed a mild depressant action on

Central Nervous System and also had a spasmolytic activity. Phyllembin

antagonize the spasmogenic effect of acetylcholine, bradykinin and

serotonin on the ileum. It also antagonize serotonin and acetylcholine-

induced contractions of oestrogenised uterus[45]. It increased the

amplitude of cardiac contraction and heart rate transiently.Alcoholic

extract of a plant (1g/kg) has shown to increase the cardiac glycogen

and a decrease in serum GOT, GPT and LDH rats, suggesting a

cardioprotective action[46].

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5. Trikatu

Trikatu is a traditional Ayurvedic herbal formulation consisting of three

herbs, Black pepper(Piper nigrum), Long pepper (P. longum) and

Ginger (Zingiber officinale). Trikatu means the ‘three pungents’ .It

consists three crude drug black pepper (Piper nigrum Linn.), dried fruits

of long pepper (Piper longum Linn.) and dried rhizomes of ginger

(Zingiber Officinalis Rosc) in the ratio of (1:1:1; w:w). It is an essential

ingredient of numerous formulations and prescriptions of Ayurvedic

medicine, used for a wide range of diseases.It is traditionally used for

lung and nasal problems with the hot spices drying excessive mucus

production. Trikatu is also used as a food supplement to help maintain

healthy respiratory, digestive and circulatory systems. It is used in

Ayurveda as part of a weight loss regime to increase metabolism, digest

fats and balance appetite.Trikatu works οn уοur skin аnd іt very

helpfully tο prevent аnу allergies. It hаѕ anti-inflammatory аnd anti-

helminthes properties. It helps іn removing thе worms out οf thе body

аnd thus improving thе digestive functions. Trikatu helps tο maintain thе

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healthy digestive system аnd removes thе toxins frοm thе body. It helps

іn rejuvenating уοur body cells аnd аlѕο helps іn preventing аnу

infection οf inflammation οf thе digestive organs. Trikatu hаѕ natural

anti-histamine аnd thus іt helps tο prevent аnу infection аnd

inflammation οf thе gastric mucosa. It prevents уοur body frοm аnу

allergic reaction. Regular intake οf thіѕ product helps іn increasing уουr

appetite. Thе digested food іѕ used bу уουr body cells tο give уου

energy tο perform various activities. Trikatu enhances thе metabolic

activity аnd rapid absorption οf thе nutrients. Trikatu іѕ аlѕο a very gοοd

product tο treat thе bаd odor frοm mouth. Trikatu аlѕο helps tο maintain

thе normal flow οf blood іn уοur body cells.Trikatu mainly has

antioxidant properties. An antioxidant is a molecule capable of slowing

or preventing the oxidation of other molecules.Trikatu contains

combined properties of ginger,piper longum and piper nigrum as has

been discussed.

Composition and constituents

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As mentioned above, Trikatu consists of equal parts Black pepper (Piper

nigrum dried fruit), Long pepper (P.longum dried fruit) and Ginger

(Zingiber officinale dried rhizome). Both Black and Long pepper contain

as their major active constituent the alkaloid piperine, which is chiefly

responsible for the pungency of these peppers.

Figure : Piperine

Black pepper contains 5-9% of the alkaloids piperine and piperettine and

1-2.5% of volatile oil, the major constituents of which are alpha- and

beta-pinene, limonene and phellandrene. In one study, the essential oil of

Black pepper was found to comprise 33.7% beta-caryophyllene.

Long pepper was found to contain about 1.25% piperine as well as about

1% volatile oil, the major constituents of which were beta-caryophyllene

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(17%), pentadecane (17.8%) and beta-bisabolene (11.16%). Long pepper

also contains an amide, which has demonstrated coronary vasorelaxant

activity.The major pungent compound in dried Ginger rhizome is [6]-

shogaol, the dehydration product of [6]-gingerol, which is the primary

pungent compound in fresh Ginger. [6]-Shogaol is more pungent than

[6]-gingerol. Ginger also contains a volatile oil, which shows

considerable variation depending on geographical origin. Ginger from

India typically yields a volatile oil containing high levels of zingiberene

and ar-curcumene[46].

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