Introduction
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.
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
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] .
Assay
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:-
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
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
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
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].
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
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
agonist[8].
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
occur"[10].
3. EC50
The term half maximal effective concentration (EC50) refers to
the concentration of a drug, antibody or toxicant which induces a
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.
% 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
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.
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,
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)
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
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].
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
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
peppercorns[18].
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
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].
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.
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
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 .
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
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
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
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].
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
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),
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
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
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].
4.Glycyrrhiza glabra
Glycyrrhiza glabra, also known as licorice and sweetwood, is native to
the Mediterranean and certain areas of Asia. Historically, the dried
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
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
acid[36].
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
activities[37].
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
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
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
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
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
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
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
glucose[42].
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].
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
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
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].
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
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
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
(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].
REFERENCES:
[1]ijprd.com/IN%20VITRO%20CYTOTOXICITY%20OF%20CASSIA%20ITALICA%20MILLER.pdf
[2] linkinghub.elsevier.com/retrieve/pii/S037842740900085X
[3] http://www.nshtvn.org/ebook/molbio/Current%20Protocols/CPI/ima03b.pdf