Extraction of anti-cancerous compounds from Annonamuricata&Simaroubaglauca

“EXTRACTION OF ANTI-CANCEROUS
COMPOUNDS FROM
Annona muricata, Simaroba glauca
AND IT’S OPTIMIZATION FOR
ANTI-CANCEROUS ACTIVITY”
Anju M T, Department of biotechnology, university of Calicut, METS
School of engineering, Mala, Thrissur.
DR. V. M. Nishad, Asst Professor, Department of Biotechnology, METS
School of engineering Mala, Thrissur.

ABSTRACT
Cancer is a general term used to refer a condition where the body’s cells begin to grow
and reproduce in an uncontrollable way. These cells can then invade and destroy healthy
tissues including organs. There are hundreds of different kinds of cancer. The most
common cancers are breast, lung, prostate, and skin cancer. Certain natural occurring
compounds such as annonacin and acetogenin from the plant species of Annona
muricata and Simaroba glauca are found to have direct effect on these tumour cells
.These can be extracted from their respective sources using different methods of
extraction. The properties of these extracts have to be confirmed by DPPH method and
their composition is required to be analyzed using HPLC method. The analyzed data
obtained from the extracts should be made free of alkaloids like Coreximine and
Reticuline, which may cause some movement disorders and their composition especially
the concentration of annonacin and acetogenin should be compared with that of current
commercially used chemotherapeutic drug to optimize the required concentration of
desired compounds. Then extracted compounds has to be tested on tissues by cell line
culture methods and if found effective there in experimented on animals.
Traditional way of cancer treatment using Annona muricata and Simaroba glauca is
going on with reference to some of authorized as well as unauthorized agencies. There
has been several numbers of patients who has been identified as recovered from different
types of cancer by traditional cancer treatment centres. So that a detailed survey is
required to examine Annona muricata and Simaroba glauca and its different package of
utilization practices as chemotherapeutic alternative medicine. Different case studies are
required on patients by classifying them into three groups (Patients with chemotherapy
drugs, patients with both chemotherapy drugs and traditional herbal medicine package,
patients with Annona muricata and Simaroba glauca package alone)to observe the effect
of traditional practice in curing cancer by examining medical reports . Correlation of
these results obtained for chemo therapy drugs, Annona muricata and Simaroba glauca
package in patients with the curative effect of the extracts obtained during the present
study can be made possible to conclude the possibility for developing a new natural
chemotherapy drug.
KEY WORDS: Annona muricata, Simaroba glauca, Anti-oxidant activity, Quassinoids,

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 Extraction of anti-cancerous compounds from Annonamuricata&Simaroubaglauca

Chemo-preventive, Annonacin, Actogenin, Natural chemotherapy drug, Drug designing.

1. INTRODUCTION
I. CANCER

Cancer is one of the major causes of death worldwide. Anti-cancerous activity is

the effects of natural, synthetic, biological chemical agents to reverse suppresses or
prevent carcinogenic progression. Several synthetic agents are used to cure the disease,
but they have their toxicity. Hence the research is going on to investigate plant derived
chemotherapeutic agents.

Anti-neoplasm (anti-cancerous) activity is defined as effects of natural, synthetic

or biological chemical agents used to reverse, suppress or prevent carcinogenic
progression (Madhuri and Pandey, 2009). Worldwide cancer is the second largest cause
of death after cardiovascular disease and kills about 3500 million people annually
(Turgay et al., 2005). Several synthetic or natural chemo preventive agents are used
worldwide to cure the disease. Chemically synthesized agents have their toxicity and
DNA damage induction potential which prevents their uses (Sasaki et al., 2002).
Because of these synthetic chemo-preventive agents, the research is going to investigate
the plant derived chemotherapeutic agents. Hence plant derived compounds plays an
important role as clinically useful anti- cancer agents without toxicity.

Bio-prospecting for plants with anti-cancer activity has been a major focus in the
search for plant based cures (Raskin et al., 2002). Taxol and Camptothecin were among
the most important anti-cancer compounds derived from plants available today (Suhas et
al., 2007).India has a rich history of using plants for health care in general (Misra et al.,
2008) and treatment of cancer in particular without causing toxicity (Madhuri and
Pandey, 2009). Cancer has become an important Public Health Problem with over
800,000 new cases occurring every year and is one of the ten leading causes of death in
India. It has been reported that there are nearly 2.5 million cases in the country with
nearly 400,000 deaths occurring due to cancer. Cancer incidence in India is estimated to
be around 70-90 per 100,000 populations (Devi et al., 2009).

CANCER TREATMENTS

Many agents are needed to fight cancer, primarily because it arises after several
normal mechanisms break down, and because cancer preys on the body in numerous
ways simultaneously, and because no single agent, whether chemotherapeutic or natural,
has yet been found that has enough anti-neoplastic strategic effects to reverse all of
those abnormalities in all patients, in effect, to be “the cure” for cancer (Colleen et al.,
2013). Surgery, chemotherapy, immunotherapy, hormone therapy, radiation therapy, and
combined-modality therapies often are effective methods for treating patients with
cancer. The specific treatment used depends on the type, stage, and location of the
cancer and the patient's general health.

Surgery which can be practiced in case of metastasis

Radiation Therapy which affect both normal and cancerous tissue equally

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Chemotherapy with some side effects

Immunotherapy: the manipulation of immune response, immunotherapy is also tried
in association with chemotherapy.

II. CHEMOTHERAPY DRUGS

Chemotherapy is the use of any drug (such as aspirin or penicillin) to treat any
disease. But to most people, chemotherapy refers to drugs used for cancer treatment. It’s
often shortened to “chemo.”Chemotherapy is used to treat many cancers. More than 100
chemotherapy drugs are used today – either alone or in combination with other drugs or
treatments. These drugs vary widely in their chemical composition, how they are taken,
their usefulness in treating specific forms of cancer, and their side effects.
Chemotherapy is a drug treatment that uses powerful chemicals to kill fast-growing cells
in your body.

Chemotherapy is most often used to treat cancer, since cancer cells grow and
multiply much more quickly than most cells in the body. Many different chemotherapy
drugs are available. Chemotherapy drugs can be used alone or in combination to treat a
wide variety of cancers. Though chemotherapy is an effective way to treat many types
of cancer, chemotherapy treatment also carries a risk of side effects. Some
chemotherapy side effects are mild and treatable, while others can cause serious
complications. Chemotherapy is used to kill cancer cells in people with cancer.

III. ALTERNATIVE MEDICINE FOR CANCER TREATMENT

In an effort to develop effective alternative strategies that increase the

therapeutic efficacy and minimize the systemic toxicity of chemotherapeutic agents,
more efforts are being directed towards the investigation of dietary supplements and
other Phyto-therapeutic agents for their synergistic efficacy in combination with
anticancer drugs. Cancer involves disordered cell replication that is death and
disorganization of organ structure. Nutritional experts are searching for suitable
naturally occurring dietary factors which are or may be anti-carcinogenic.
There is a demand for natural source in food, cosmetics & therapeutic industry.
Due to their low cost, high stability, compatibility and environment friendly, we found
that plants are the good source of natural antimicrobial agents due to the presence of
phytochemicals. Plant extracts appears to be one of the better alternatives as they are
known to have minimal environmental impact and danger to consumers in contrast to
synthetic drugs. The use of and search for drugs and dietary supplements derived from
plants have accelerated in recent years. Ethno- pharmacologists, botanists,
microbiologists, and natural products chemists’ are combining the Earth for
phytochemicals and "leads" which could be developed for treatment of infectious
diseases. Many pathogenic microbes have capability to develop resistance against
synthetic formulation.

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IV. THE MEDICINAL PLANTS WITH CHEMOPREVENTIVE EFFECT

Plant based formulation are least toxic and better for environment balance so it
can be replace by synthetic formulation. Antimicrobial activity of plants is mainly due to
the presence of secondary metabolites. Plants are rich in wide variety of secondary
metabolites such as tannins, terpenoids, alkaloids, and flavonoids, which have been
found in vitro to have antimicrobial properties.
The NCI (National Cancer Institute) has examined more than 30,000 plants with
anticancer activity (Ipek et al., 2005). However, the use of plants as a means treatment is
still very limited. From 250,000 to 500,000 species plants, a small percentage has been
examined for its medicinal properties. The World Health Organization estimates that
80% of the inhabitants of the earth choose traditional medicine for primary health needs,
much of which relies on the use of essential oils from plants.
Medicinal plants have been used as remedies for human diseases for centuries.
The reason for using them as medicine lies in the fact that they contain chemical
components of therapeutic value (Nostro et al., 2000). The medicinal value of plants lies
in some chemical substances (usually secondary metabolites), that produce a definite
physiological action on the human body. The most important of these bioactive
compounds of plants are alkaloids, flavonoids; tannins and phenolic (Edeoga et al.,
2005). Phyto chemicals are non-nutritive plant chemicals that work with nutrients and
dietary fibre to protect one against diseases. There are more than a thousand known
phytochemicals. They are not essential nutrients and are not required by the human body
for sustaining life.
Research suggests that phytochemicals, found in fruits, vegetables and nuts, may
help slow the aging process and reduce the risk of many diseases, including cancer,
heart disease, stroke, high blood pressure, cataracts, osteoporosis, and urinary tract
infections. Foods containing phytochemicals are already part of our daily diet. In fact,
most foods contain these chemicals except for some refined foods such as sugar or
alcohol. Some foods, such as whole grains, vegetables, beans, fruits, and herbs, contain
many phytochemicals.
The easiest way to get more phytochemicals is to eat more fruit and vegetables.
These biologically active micronutrients exhibit their activity as antioxidants,
detoxifying agents or simply by physic-chemical means in the case of dietary fibre.
They can have complementary and overlapping mechanisms of action in the body,
including antioxidant effects, modulation of detoxification enzymes, stimulation of the
immune system, modulation of hormone metabolism, and antibacterial and antiviral
effect (ArtiGoel, 2013).
Although very few people nowadays would abandon the benefits of modern
pharmaceutical science or the convenience of technology, a growing interest in
medicinal plants and their uses occurs which began in early 1980s. It has been reported
that almost 80% of the global population in developing countries use plant materials for
health care (Farnsworth et al., 1985). Plants are a good source of chemical compounds
many drugs originate from a natural compound isolated from a medicinal plant.
Nevertheless, this extended medicinal use reported does not incorporate or result in
respective drug discovery. According to estimates only 20% of all plant species have
been chemically or biologically evaluated (Cordell, 2003) and therefore the evaluation
of a natural product seems to be a great prospect in the seek out for new drugs.

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V. SOME FRUITS AND VEGETABLES USED AS ANTI-CANCEROUS

AGENTS
Green leafy vegetables exhibits higher antioxidant activity followed by nutritive
whole grains, however cooking reduces this activity. Anti-mutagenic effect of ethanolic
extracts of fruits and vegetables like Liquorice, Kinu, Carrot, Broccoli, Pineapple,
Onion, Garlic, green pepper figs ,grape fruit ,grape then tea, aloe, milk thistle(Sheela ,
Salar, et al., 2010). Herbal medicine uses plants, or mixtures of plant extracts, to treat
illness and promote health. It aims to restore your body's ability to protect, regulate and
heal itself. It is a whole body approach, so looks at your physical, mental and emotional
well-being. It is sometimes called phyto-medicine or phyto-therapy or botanical
medicine.
To date, many of herbal medicines (HM) and its active ingredients have been
identified as potential modifiers of cancer. Herbal medicines are, however, yielding
important breakthroughs in cancer prevention and treatment. HM is presently used first
line in numerous cultures across the world. Despite a rapidly growing body of
experimental evidence supporting the cancer preventive properties of herbal medicines,
minimal data exists regarding actual dietary intake levels of herbal medicines to use as
preventive agents. Consequently, researchers are limited in their ability to compare the
effective exposures of culinary herbal medicines and their bioactive components from
experimental studies, which are primarily animal studies, using approximate human
intake levels. Furthermore, information on the uptake, distribution, and excretion of
most non-nutritive dietary components is sparse and little data related to human, despite
these limitations, the exact mechanism still a challenge. Research indicates several
possible mechanisms of action for herbal medicines, or their bioactive components, may
act alone or in concert to reduce cancer risk through their anti-oxidant, and anti-
tumorigenic properties, as well as their direct suppressive effect on carcinogen
bioactivities. Hence, there is an increasingly convincing rationale for employing HMs
against cancer. However, before acceptance of HMs into wide scale prevention use can
be achieved, one critical issues need to be addressed; this include a clearer
understanding of their precise mechanism of action.
Medicinal plants have been used for healing and preventative health for
thousands of years all around the world. For example, the therapeutic efficacy of the
following herbs is well known. The use of HMs in cancer prevention has not been
thoroughly evaluated despite growing evidence that the public is utilizing a wide range
of HMs. HMs may be used supportively, prophylactically, symptomatically, or
correctively. The pharmacodynamics parameters were not stated clearly.
Pharmacodynamics may be defined as the study of the actions and effects of HMs on
organ, tissue, cellular, and subcellular levels. Therefore, pharmacodynamics provides us
with information about how HMs brings about their beneficial effects and how they
cause their side effects. Pharmacodynamics considers the sites, modes, and mechanisms
of actions of HMs.

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VI. Mechanism of Action of Anticancer Activity for Herbal Medicine

(a) General preventive mechanism of action

a) Antioxidant
Oxidation in biological condition is a chemical reaction that passes electrons or
hydrogen from a substance to an oxidizing agent. Oxidation reactions lead to produce
free radicals (FR). When the chain reaction happens in a biological cell, it can cause
damage or death to the cell. Antioxidants stop these chain reactions by removing free
radical intermediates, and inhibit other oxidation reactions. They do this by being self
oxidized , so antioxidants are often reducing agents such as or polyphenols. Free radicals
(FR) are identified to play a dual role in biological systems, since they can be either
harm or beneficial to living systems. Beneficial effects of FR involve physiological roles
in cellular responses to anoxia, as for example in the function of a number of cellular
signalling systems and in fight against infectious agents. One further beneficial example
of FR at small concentrations is the induction of a mutagenic response.
Specific Treated Mechanism of Actions
a) Apoptosis
Apoptosis is a hereditarily controlled method of cell death participated in the
regulation of tissue homeostasis. The pathways of apoptosis are two major both of
which are found in the cytoplasm , which include extrinsic (Fas and other TNFR
superfamily members and ligands) and the intrinsic (mitochondria-associated)
pathways . The extrinsic pathway is triggered by death receptor engagement, which
starts a signalling cascade mediated by caspase-8 activation. Caspase-8 both feeds
directly into caspase-3 activation and stimulates the release of cytochrome c by the
mitochondria. Caspase-3 activation starts to the degradation of cellular proteins
primarily to maintain cell survival and integrity.
The intrinsic pathway occurs when various apoptotic stimuli trigger the release
of cytochrome c from the mitochondria [independently of caspase-8 activation).
Cytochrome c interacts with Apaf-1 and caspase-9 to promote the activation of
caspase-3. Latest studies point to the ER as a third subcellular compartment
implicated in apoptotic execution. Changes in Ca2+ homeostasis and accumulation of
mis folded proteins in the ER cause ER stress. Prolonged ER stress can result in the
activation of BAD and/or caspase-12, and help in apoptosis
b) Cyclooxygenase 2 inhibition
Chronic inflammation and cancer involves cytokines and mediators of
inflammatory pathways, which act during the different steps of tumorigenesis, have
been admitted. Inflammatory mediators, generated by cyclooxygenase (COX), are
implicated in some cancer pathogenesis such as the colorectal cancer (CRC). Inhibiting
COX may therefore have anti-carcinogenic effects. Expression of 5-lipoxygenase (5-
LOX) is up-regulated in colorectal adenoma and cancer.The inducible COX-2 isoform,
which is up-regulated during cancer. COX-2 was described to modulate cell
proliferation and apoptosis in different tumours, in solid tumours [colorectal, breast,
and prostate cancers) and in haematological malignancies.

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c) Angiogenesis inhibition
Angiogenesis inhibitors for the treatment of cancer have now been approved by
the FDA in US and other developed countries. Angiogenesis, the formation of new
blood vessels from existing vasculature, plays an essential role in tumour growth,
invasion and metastasis. Vascular endothelial growth factor (VEGF) is one of the key
factors responsible for its regulation. High expression of VEGF has been observed in
many cancers, and is associated with worse survival. Non-nutritive plant substances that
possess health-protective effects, natural dietary agents including fruits, vegetables, and
spices have drawn a great deal of attention from both the scientific community and the
general public due to their various health promoting effects including suppression of
cancers . Each type of herbal remedy may have its own side effects. Some are safe to use
and don’t have any noticeable side effects. But some plants are poisonous to humans and
can have serious and severe side effects. There is currently no strong evidence from
studies in people that herbal remedies can treat, prevent or cure cancer. There is no
substantial evidence that herbal remedies can prevent cancer. Some plants or plant
extracts have been found in laboratory tests to have anti-cancer effects and have been
turned into cancer drugs (such as Taxol from the yew tree). But there is no scientific
evidence from patient trials that herbal medicine can cure cancer. Many people assume
that because a product is marketed as natural or herbal, this means it’s safe to use. Some
herbal medicines are safe but others can have serious and dangerous side effects .Some
herbal medicines may interact with treatments from your doctor, including cancer drugs
or radiotherapy. Some herbal treatments may affect the way drugs are broken down by
your body, or the way drugs are carried around your body.
Herbal medicine is still the main source for agent that can be used for cancer
treatment and prevention. Herbal medicine is considered the second method to fight
cancer utilized by cancer patient in developed countries . It considered as a first line in
developing countries because the availability and the affordable cost. For long time
many plants were well known for anticancer property and such as Nigella sativa (black
seed), Cinnamomum cassia (Cinnamon), Panax ginseng (Ginseng), Camellia sinensis
(Green tea), Allium sativum (garlic), ZingiberofficinalRosc. (Ginger). Several
experimental and case report studies have been done and most of conducted studies
showed supportive outcomes. Moreover, several clinical trials have been conducted for
some herbal products. Beside old anticancer plants, exploring for new plants have
anticancer has been going on.
Many screening for anticancer activity plant studies have been recorded. Some
of these plants have shown promising anticancer activity. However all these affords
done, but still the research required to be more focus to be able to produce product from
herbal medicine used as preventive medication. Some of these products their dosage and
frequency of taken have been standardised but not all as one the limitation for using
food supplement as cancer prevention.
Most of Cancer diseases (90%) are due to external factors only 10% is due to
genetic factors. Preventive medicine will be very effective to fight cancer and reduce
prognoses. The herbal products are the most suitable since have traditionally and
experimental based evidences from other alternative medicines. The HM only required
conducting study to use as preventive medicines. The technology today can help us to
conduct because there are animal can carry human genes. Long epidemiological studies

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can be carried out. Many cancer patients use herbal medicine as alternative medicines
to, or following the failure of standard cancer therapy (Eisenberg et al.,
1998).Experimental studies indicate that phytochemicals with anti-oxidative and anti-
inflammatory properties can inhibit tumour initiation, promotion and progression
(Sharma et al., 2009). Annona muricata and Simaroba glauca are the herbal plants,
which are believed to have anti-cancerous effect and traditionally used for cancer
treatment.
Annona muricata
Annona muricata commonly known as graviola or soursop, belongs to the family of
Annonaceae (Feo, 1992). It promises to be a cure for cancer targeting only malignant
cells without having any side effects. Annona muricata is a slender, evergreen tree, 5-10
m in height and 15 cm in diameter; trunk straight; bark smooth, dull grey or grey-brown,
rough and fissured with age; inner bark pinkish and tasteless; branches at first ascending
with the crown forming an inverted cone, later spreading; crown at maturity spherical
due to lack of apical dominance; twigs brown or grey, bearing minute raised dots
(lenticels); root system extensive and superficial, spreading beyond the diameter of the
crown although shallow rooted.

GRAVIOLA HERBAL PROPERTIES AND ACTIONS

MAIN ACTIONS OTHER ACTIONS STANDARD DOSAGE
 Kills cancer  relieves Leaves
cells depression Infusion- 1 cup 3 times daily
 Slows tumour  reduces
growth spasms Tincture- 2-4 ml 3times daily
 Kills bacteria  kills viruses
 Kills parasites  reduces fever Capsules- 2g 3 times daily
 Reduces blood  expels worms
pressure  stimulates
 Lowers heart digestion
rate  stops
 Dilates blood convulsions
vessels

Many active compounds and chemicals have been found in graviola, as

scientists have been studying its properties since the 1940s. Most of the research on
graviola focuses on a novel set of chemicals called Annonaceous acetogenins. Graviola
produces these natural compounds in its leaf and stem, bark, and fruit seeds. Three
separate research groups have confirmed that these chemicals have significant
antitumorous properties and selective toxicity against various types of cancer cells
(without harming healthy cells). These groups have published eight clinical studies on
their findings. Many of the acetogenins have demonstrated selective toxicity to tumour
cells at very low dosages—as little as 1 part per million.
Four studies were published in 1998 which further specify the chemicals and
acetogenins in graviola that are demonstrating the strongest anti cancerous, anti
tumorous, and antiviral properties. Annonaceous acetogenins are only found in the

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Annonaceae family (to which graviola belongs). These chemicals in general have been
documented with antitumorous, antiparasitic, insecticidal, and antimicrobial activities.
Mode of action studies in three separate laboratories have recently determined that these
acetogenins are superb inhibitors of enzyme processes that are only found in the
membranes of cancerous tumour cells. This is why they are toxic to cancer cells but
have no toxicity to healthy cells. In 1997, Purdue University published information with
promising news that several of the Annonaceous acetogenins “not only are effective in
killing tumours that have proven resistant to anticancer agents, but also seem to have a
special affinity for such resistant cells(Wu et al.,1995). Previous reports over the years
have demonstrated that the leaf, bark, root, stem, and fruit seed extracts of Annona
muricata are anti-bacterial (Misas et al., 1979; Sundarrao et al., 1993), antifungal
(Heinrich et al., 1992) and anti-malarial (Arkcoll, 1990; Antoun et al., 1993). Its leaves
extract were also found to possess antioxidant (Baskar et al., 2007) and molluscicidal
properties (Santos, et al., 2001; Luna et al., 2006). Recently, it has also been reported to
exhibit anti-inflammatory and analgesic effects (De Sousa et al., 2010; Roslida et al.,
201 Among the chemical constituents found in the leaf of Annona muricata are
alkaloids (Le Bouef et al., 1981: 1982), essential oils (Pelissler et al., 1994; Kossouh et
al., 2007) and acetogenins (Wu et al., 1995; Zeng et al., 1996; Kim et al., 1998; Chang
et al., 2003). Annonaceous acetogenins, from Annona muricata were found to be a
promising new anti-tumour and anticancer agent in numerous in vitro studies. These
acetogenins demonstrated to be selectively toxic against various types of the cancerous
cells without harming healthy cells (Rieser et al., 1993; Wu et al., 1995a; Zeng et al.,
1996).Acetogenins inhibit complex 1 NADH/ Ubiquinoneoxidoreductase in Electron
transport system inhibiting oxidative phosphorylation in cancer cells and induce
apoptosis.
SimarobaGlauca
Simaroba glauca locally known as Lakshmi tharu, comes under the family of
simarobaceae, Simarouba is a medium-sized tree which has 20 m high, with a trunk 50
to 80 cm in diameter. It produces bright green leaves 20 to 50 cm in length, small white
flowers, and small red fruits. The leaves and bark of Simaroba glauca have a long
history of use as a natural medicine in the tropics. Simaroba glauca was first imported
into France from Guyana in 1713 as a remedy for dysentery.Simarouba also has a long
history in herbal medicine in many other countries It is taken internally for diarrhoea,
dysentery, malaria, and colitis; it is used externally for wounds and sores. In Belize the
tree is called Negritoor dysentery bark. There the bark and, occasionally, the roots are
boiled in water to yield a powerful astringent and tonic used to wash skin sores and to
treat dysentery, diarrhoea, stomach and bowel disorders, haemorrhages, and internal
bleeding. In Brazil it is employed much the same way against fever, malaria, diarrhoea,
dysentery, intestinal parasites, indigestion, and anaemia. In high dosages it is The bark
and leaf extract of Simaroba is well known for its different types of pharmacological
properties such as haemostatic, anthelminthic, antiparasitic, antidysentric, antipyretic
and anticancerous (Angela et al., 2002).The exact mechanism of action of the single
agents remains unclear, some agents have been shown to affect protein synthesis in
general, or specifically membrane polarization and the apoptotic machinery (Iasmineet
al., 2014). Main Phytochemicals present in the plant is: Ailanthinone, benzoquinone,
canthin, dehydroglaucarubinone, glaucarubine, glaucarubolone, glaucarubinone,

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holacanthone, melianone, simaroubidin, simarolide, simarubin, simarubolide, sitosterol,

and tirucalla (Oncol Res, 1998).
Humans have used plants as foods and natural medicines since ancient times.
Crude drugs, typically safer than synthetic drugs, have been used as both spices and
supplements. Natural medicines like Annona muricata and Simaroba glauca have been
used as anti-cancer agents by inhibiting the promotion process, and it is important that
these are consumed in small quantities for extended periods of time. The study of cancer
prevention using these plants is generating vast amounts of information regarding their
benefits. This paper provides, an outline of studies focusing on plant extracts of
Annonamuricata and Simarobaglauca. Several active components have been isolated,
and their chemical structures have been and continue to be determined In addition,
structure-activity relationships, elucidation of physiological activities at the molecular
level, and development of strategies that allow for the production of sufficient supplies
of these agents are issues for further investigation using computer aided drug designing
program. The continued search for natural medicines is necessary for finding additional
sources of active components that are suitable for clinical application. For this purpose,
we will harness the strength of researchers from various fields with the goal for
chemotherapy prevention.

3. MATERIALS AND METHODS

3.1 Sample collection
The leaves of Annona muricata and Simaroba glauca were collected from nearby
sources. These leaves were washed in tap water and dried under shade for 30 days.
3.2 Extraction from Annona muricata
3.2.1 Water extraction (traditional method)
An amount of 15 leaves of Annona muricata were boiled in 600ml water
for 15 mins and made up to 200 ml(as per traditional method followed by pulse
palliative care, figure 3.1 ).The extract was concentrated using Rota vapor eeping the
temperature at c and dried the concentrated extract by keeping it laminar air flow

Figure 3.1: Brochure of Procedure Followed In Pulse Palliative Care Thrissur

3.2.2 Water Extraction

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An amount of 15 leaves of Annona muricata were dried and powdered

using mixer grinder and boiled in 1000 ml till the volume became half (500 ml) at
controlled temperature ( not above 65ºc). The extracts were concentrated in rotavapor
by eeping the temperature at c. the concentrated samples were kept in laminar air
flow for further drying.
3.2.3 Sohxlet extraction
An amount of 40gm leaves of Annona muricata were weighed after
drying. These were made in to fine powder by grinding it using mixer grinder. Ground
sample were packed in whatmann no 1 filter paper and subjected to solvent extraction
by ethanol in sohxlet apparatus (Balabharatiet.al, 2013). Solvent used was 300 ml
ethanol. After completion of 6 cycles (6 hours), the extract was collected. The extract
were concentrated using Rota vapor by keeping the temperature at c and the
concentrated samples were kept in laminar air flow for further drying.
3.3 Extraction from Simaroba glauca
3.3.1 Water extraction (traditional method)
An amount of 18 shade dried leaves of Simaroba glauca were boiled in
200 ml water for 15 minutes and made up to 100 ml. ( as per traditional method
followed by pulse palliative care Thrissur figure 3.1).The extract was concentrated using
Rota vapor and dried by keeping it at laminar air flow.
3.2.2 Water Extraction
An amount of 15 leavesof Simaroba glauca were dried and powdered and
boiled in 1000 ml till the volume became half (500 ml) at controlled temperature ( not
above 65ºc). The extracts were concentrated in rotavapor and kept in laminar air flow
for further drying.
3.3.3 Sohxlet extraction
An amount of 40gm leaves were weighed after drying and made in to
fine powder by grinding it using mixer grinder. The ground sample were packed in
whatmann no 1 filter paper and subjected to solvent extraction by ethanol (300 ml) in
sohxlet apparatus. .After completion of 7 cycles (6 hours) the extracts were collected.
These were concentrated using Rota-vapor eeping the temperature at c and further
dried by keeping it in laminar air flow chamber.

Figure3.2: Extraction Using Soxhlet Apparatus

3.4. Water extract with fungal growth

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The concentrated aqueous extracts of both Annona muricata and Simaroba

glauca when kept in the laminar air flow for drying, fungal growth was observed on it
after two days. The funguses were sub cultured in sabrose dextrose agar. Fungal staining
using bromophenol blue was done to identify the fungus. The stained slide was observed
under electronic microscope.
3.5 Determination of Anti-Oxidant Activity

The determination of antioxidant activity through DPPH scavenging system was

carried out for all the samples extracted using different methods. DPPH assay is a
common antioxidant assay. The hydrogen atoms, or electron donation ability, of the
corresponding extract were measured from the bleaching of purple color of DPPH
solution. Each 1 μL of various concentrations of the extracts/ascorbic acid was added
to 1 μL of ethanol (99%) solution of DPPH. After a 30 minutes incubation period at
room temperature, the absorbance was read compared to a blank at the wavelength of
517 nm. Ascorbic acid was taken as the standard reference. The antioxidant activity was
expressed as

Anti-oxidant activity = (control value + substrate blank value) - sample

(Control value + substrate blank value)

Table 3.1: procedure for preparation of samples for DPPH assay

Reagent B Sample1 Sample2 Sample3 Sample4 Sample5

Sample - 100 200 300 400 450
DPPH - 500 500 500 500 500
Ethanol 1000 400 300 200 100 50

Table 3.2: procedure for preparation of substrate blank and control

Reagent Substrate Substrate Substrate Substrate Substrate Control

blank 1 blank 2 blank 3 blank 4 blank 5
DPPH 900 800 700 600 500 400
Ethanol 100 200 300 400 500 600

 All samples were done in triplicates.

 All values are in µl

3.6 LCMS Analysis

The solvent extracted samples of Annona muricata and Simaroba glauca (each
10mg/ml concentration) were subjected to LC-MS analysis for confirming the presence
of annonaceous acetogenin and quassinoids which are believed to be present in the
plants Annona muricata and Simaroba glauca respectively. This test was done at CARE-
KERALAM Ltd, KINFRA PARK. The eluent used was ethanol.

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 Extraction of anti-cancerous compounds from Annonamuricata&Simaroubaglauca

Figure: 3.2 samples sent for LC-MS assay

3.7 Computer Aided Drug Designing

Computer aided drug designing using acetogenin, annonacin and quassinoids
which are extracted from Annona muricata and Simaroba glauca respectively. This is
the inventive process of finding new medications based on the knowledge of
a biological target. The drug is most commonly an organic small molecule that activates
or inhibits the function of a biomolecule such as a protein, which in turn results in
a therapeutic benefit to the patient. In the most basic sense, drug design involves the
design of molecules that are complementary in shape and charge to the bio-molecular
target with which they interact and therefore will bind to it. Drug design frequently but
not necessarily relies on computer modelling techniques. This type of modelling is often
referred to as computer-aided drug design. This designing includes three distinct
aspects:
(i.) Utilizing QSFR (quantitative stability to flexibility relationship) in
computational molecular design of protein sequences or variations of molecular
structures, to have specific stability and flexibility relationships deemed
important for biochemical function for specified thermodynamic and solvent
conditions
(ii.) Utilizing QSFR to optimize thermodynamic and/or solvent conditions
necessary for a specified molecular system to exhibit protein function
characteristics, such as improved catalytic efficiency.
(iii.) Utilizing QSFR to computationally screen or design small molecules and/or
design its putative receptor so as to exhibit a desired effect on protein function.
 Using CHEMSKETCH software to draw the structure of molecules of
annonacin, acetogenin and quassinoids.
 Then with the help of OPENBABEL software ,we are converting the format of
the mol file to pdb format
 Preparation of our target proteins like BCL2, cyclin D1, EGRF, RAS, RAF,
MEK, ERK1, ERK2.
 EGRF structure is taken from Swiss prot (database).
 Energy minimisation was done with the help of spdBv (Swisspdb viewer).
 Molecular docking is done with the help of AUTODOCK4.2. Here is the
creation of gpf (grid parameter file) and .dpf (dock parameter file).

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 Run the docking algorithm using CYGWIN software .Here we will create glg
file and dlg file using different commands.
 After successful completion got docked protein.
 Using software UCSF CHIMERA we are visualization and analysing was done.
 These molecules were now ready for drug likeness score, ADMET studies. For
this we used the website http://molsoft.com/mprop/ .
 For toxicity and bioactivity we used the website
http:/www.organicchemistry.org/prog/peo/
 For ADMET studies we use https://ilab.acdlabs.com/iLab2/.
3.8 MTT Assay
Cytotoxicity of Simaroba glauca and Annona muricata and Metformin at different
concentration was tested in pancreatic cancer cells, using the MTT assay as described
(Wilson. 2000). The cells seeded in 96-well plate with 5 X 103cells / 200µl and incubated
for 24 hrs at 37ºC. Cells grown to 70-85% confluence were treated with different
concentrations of the samples (0.5,1.0, 1.5and 2.0 mg/ ml) dissolved in 5% DMSO,
Metformin (5 µM) and DMSO 0.1 % (v/v) we added and incubated the cells for desired time
periods.
The control and treated cells were incubated for 48hrs in 37ºC, 5% C02. After
incubation, media was removed and replaced with MTT and incubated for 4 hr. After
incubation, DMSO was added to dissolve insoluble crystals and Absorbance was read at
570nm using a 96 well micro plate reader

Percentage viability = ×

3.9 Case Study Analysis

The list of patients following the traditional package was collected from Pulse
Palliative Care, Anchery, Thrissur. They were selected based on their sex, age group
(between 30-50), stage of cancer and duration of taking medicine (from January 2015
onwards). The details were collected from patients along with their medical reports of
each 15 days interval. The reports were analysed and observations were tabulated.

4. RESULTS AND DISCUSSIONS

4.1 DPPH Results of Water Extracts (traditional method)

The water extract (traditional method) of both Annona muricata and Simaroba
glauca were subjected to DPPH assay and the results showed negative values. The
results of assay clearly confirming that the samples obtained by water extraction may
not have any antioxidant properties.
Table 4.1: DPPH Results of water extracts (traditional methods)
Concentration(mg\ml) Annona muricata Simaroba glauca

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5 -0.362 -0.477
4 -0.477 -0.715
3 -0.547 -0.724
2 -0.575 -0.976

4.2 DPPH Results of Water Extracts

The water extract of both Annona muricata and Simaroba glauca were subjected
to DPPH assay with varying sample concentrations. The results obtained for all
concentration from very small (1 mg/ml) to maximum possible concentration (10
mg/l) showed nil or very little anti-oxidant activity (Table 4.2) for both samples.
Table 4.2: DPPH Results of water extract
Concentration(mg\ml) Annona muricata(%) Simaroba
glauca(%)
1 0 0
2 0 0
3 0 0
4 0 7
5 10 11
6 13 16
7 17 18
8 19 20
9 20 22
10 20 23
Note: The values obtained for all the samples were triplicated and averaged

Figure 4.1 concentration v/s antioxidant activity of water extract

4.2 DPPH Results of Soxhlet Extraction

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The soxhlet extract of both Annona muricata and Simaroba glauca were
subjected to DPPH assay with varying sample concentrations. At lower
concentrations, the anti-oxidant activities of both samples were almost similar. At
higher concentrations, Simaroba glauca showed higher anti-oxidant activity.

Figure 4.2: Dried Ethanolic Extract Samples of Annona muricata And Simaroba
glauca.
Table 4.3: DPPH Results of Soxhlet extracts
Concentration(mg\ Annona Simaroba
ml) muricata(%) glauca(%)
1 15 17
2 18 22
3 26 30
4 45 38
5 52 45
6 60 54
7 68 56
8 75 61
9 79 66
10 82 71
Note: The values obtained for all the samples were triplicated and averaged

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Figure 4.3 concentration v/s antioxidant activity of ethanolic extract

4.3 Water Extract With Fungal Growth
The dried water extract powder kept for incubation in petri dishes showed fungal
growth on its surface just after 3 days of shelf life (figure.4.3). The extract of both
Annona muricata and Simaroba glauca with fungal growth were subjected to DPPH
assay with varying sample concentrations. The values obtained for the assay showed
a higher level of anti-oxidant activity from lower concentrations onwards (Table 4.3).
Hence it reveals that the fungal growth may increases the anti-oxidant property of
both the plant extracts. The dominant fungal growth with anti-oxidant properties was
identified as Aspergillus niger through lacto phenol cotton blue staining (fig 4.4).

Figure 4.3 Fungal Growths Observed On Water Extract

Table 4.4 DPPH Results of water extracted samples with Fungal growth
Concentration Simaroba Annona
(mg/ml) glauca(%) muricata(%)

10 90 95

9 89 91

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8 84 82

7 71 75

6 63 69

5 52 56

4 44 45
3 39 37
2 30 24

1 21 16
Note: The values obtained for all the samples were triplicated and averaged

Figure 4.4 concentration v/s antioxidant activity of water extract with fungal growth

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Figure 4.5 fungus observed under microscope (Aspergillus niger)

4.4 Comparative analysis of antioxidant activity between different methods of
extraction observed for the samples of Annona muricata and Simaroba gluaca.
4.4.1 Simaroba Glauca
The data obtained in the DPPH assay (the anti-oxidant activity) of the extracted
samples by different methods of extraction for Simaroba glauca were statistically
analysed using Duncun’s multiple range test (Table 4.5).
Table 4.5 Data analysis of anti-oxidant activity of Simaroba glauca
METHODS 10mg/ml 9mg/ml 8mg/ml
Ethanolic 3 3 3
*82.06±0.404 C 78.7333±0.378 b 74.9667±0.251 a
extraction

Aqueous extract 2 2 2
17.7000±0.360 c 17.1667±0.208 b 16.7000±0.265 a

Aqueous extract 89.5000±0.458 1 85.8667±0.776 b

1
82.7333±0.750 a
1
c
with fungal growth

*Mean ± SD
Values in a row followed by same subscript alphabets are not significantly different,
according to Duncan’s multiple range test
Values in a column followed by same super script numerals are not significantly
different, according to Duncan’s multiple range test
4.4.2 Annona muricata
The data obtained in the DPPH assay (the anti-oxidant activity) of the extracted
samples by different methods of extraction for Annona muricata were statistically
analysed using Duncun’s multiple range test (Table 4. ).

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Table 4.6 Data analysis of anti-oxidant activity of Annona muricata

METHODS 10mg/ml 9mg/ml 8mg/ml

Ethanolic extraction 3 3 3
*70.20±0.700 c 66.1000±0.360 b 60.300±0.251 a

Aqueous extract 2 2 2
19.77±0.3.511c 19.0667±0.208 b 18.660±0.305 a

Aqueous extract with 94.300±0.435 1 91.44±1.276 b

1
84.233±1.09 a
1
c
fungal growth

*Mean ± SD
Values in a row followed by same subscript alphabets are not significantly different,
according to Duncan’s multiple range tests.
Values in a column followed by same super script numerals are not significantly
different, according to Duncan’s multiple range tests.
4.5 LC-MS Assay Results
The compound of interest in Annona muricata and Simaroba glauca extracts were
established as acetogenin and quassinoids respectively from the previously published
papers. The anti-cancerous properties of both the compounds were found to be
effective in chemotherapeutic applications.
The Annona muricata and Simaroba gluaca extracts were subjected to Liquid
Chromatography – Mass Spectrometry analysis and the results obtained were
compared with standards in references. The m/z values of graphs obtained were
compared with standard LC-MS m/z values of acetogenin and quassinoids (Pierre
Champy et.,al 2009).

TABLE 4.5.1 Comparison of m/z value of sample and standards of acetogenin

Compound Sample m/z Standard Reference paper
value m/z value

C35H62O8 641.4 641.4 (Pierre et.,al 2009).

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C35H64O8 651.4 651.4 (Pierre et.,al 2009).

C37H66O4 613.4 613.4 (Pierre et.,al 2009).

C35H64O8 651.5 651.5 (Pierre et.,al 2009).

C35H64O7 635.4 635.4 (Pierre et.,al 2009).

C35H64O7 619.4 619.4 (Pierre et.,al 2009).

C35H64O9 651.4 651.4 (Pierre et.,al 2009).

From the table it is clear that the m/z value of 7 compounds present in Annona
muricata extracts is matching with the value of acetogenin compounds of plant
extracts in the previously published papers. Hence, it is confirmed that the acetogenin
compounds are present in the extract of Annona muricata. Similarly, the m/z value
of one compound present in Simaroba glauca extracts is matching with the value of
quassinoids compound of plant extracts in the previously published papers. So, the
result confirmed the presence of quassinoids in the extract of Simaroba glauca.

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4.6 Computer aided drug designing using annonaceous acetogenin and

quassinoids which are extracted from Annona muricata and Simaroba
glauca respectively

Fig 4.6.1chemsketch drawing of quassinoids

Fig 4.6.2:chemsketch drawing of annonaceous acetogenin

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Fig 4.6.3: chemsketch drawing of acetogenin

Fig 4.6.4: Converting mol file to pdb format using OPEN BABEL software

Fig 4.6.5Auto docking of minimized energy molecule done in docked tools

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 Extraction of anti-cancerous compounds from Annonamuricata&Simaroubaglauca

Fig 4.6.6: Visualization of docked protein of Annonaceous acetogenin in UCSF

CHIMERA SOFTWARE

Fig 4.6.7: Visualization of docked protein of quassinoids in UCSF CHIMERA

SOFTWARE

Fig 4.6.8: Molecular properties & drug likeness score of quassinoids using molsoft
website

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 Extraction of anti-cancerous compounds from Annonamuricata&Simaroubaglauca

Fig 4.6.9: comparative graph got from analysis for quassinoids

Fig4.6.10: Molecular properties & drug likeness score of annonaceous acetogenin

using molsoft website

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Fig 4.6.11: comparative graph got from analysis for annonaceous acetogenin

Fig 4.6.12: properties & drug likeness score of acetogenin using molsoft website

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Fig 4.6.13: comparative graph got from analysis for acetogenin

4.7 MTT Assay Results

The MTT assay results showed that Simaroba glauca (sample B) is having
higher necrosis activity than Annona muricata (sample A) in highly concentrated
samples (figure 4.7.1) From this assay it indicated that Simaroba glauca extract is
having higher anti cancerous activity than the extracts of Annona muricata .The LC-MS
assays showed the presence of higher number of compounds in the extract of Annona
muricata than Simaroba glauca. These conflicts in result making an assumption that
concentration of compound is more significant than quantity of compounds present.
That means, the concentration of quassinoids in the extract of Simaroba glauca may
sufficient enough to perform anti-cancerous property compared with the combination of
7 acetogenin compounds of Annona muricata extract. But, further clarification with
reference to concentration of each compounds are required to confirm the anti-cancerous
property of the compounds.
Table 4.7.1 cell survival rate of samples
CONCENTRATION (mg/ml) Annona muricata (%) Simaroba glauca (%)
Sample A Sample B
0 100 100
0.5 94.8 88.35
1 87.88 66.03
2 93.2 58.9

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Figure 4.7.1 concentration of samples v/s cell survival rate of cancer cell lines

4.8 Case Study Analysis

The correlation of case studies produced the following observations (Table 4.7).
The patients do not believe that the traditional package alone can cure their disease even
though they felt some betterment while undertaking these medicines. The patients also
believe that the diet and habit changes which were taken along with the intake of
traditional medicines may also have some role in curing the disease.

Table 4.7 case study analysis observations

PATIENT SEX AGE TYPE TREATMENT PERIOD OF PERCENTAGE OF
NO: OF TAKEN WITH TRADITIONAL SATISFACTION
CANCER NUMBER OF TREATMENT
DOSAGE

1 Female 47 Breast Surgery,chemo- 5 months 5

10

2 Female 49 Breast Surgery , 3 months 5

chemo-8
Radiation -12

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3 Female 45 Breast Surgery, 10 months 7

chemo-10
Radiation 10

4 Male 44 Lungs Surgery chemo- 12 months 9

8

5 Female 37 Tongue Surgery 12 months 9

Chemo-8

6 Female 32 Uterus, Surgery, 12 months 9

Breast Chemo -10

7 Male 38 Bone Chemo-10 5 months 0

marrow Radiation 5

8 Female 43 Breast Surgery, 2 months 3

chemo- 5

9 Male 34 Head Radiation 27 8 months 7

Excellent-10, Very good-9, Good-7, Average-5, Poor-3, Very poor-1, No use-0

5. CONCLUSION

Humans have used plants as foods and natural medicines since ancient times.
Crude drugs, typically safer than synthetic drugs, have been used as both spices and
supplements. Natural medicines like the extracts of Annona muricata and Simaroba
glauca have been used as anti-cancer agents for the last several years. Current way of
practice in using the Annona muricata and Simaroba glauca as alternative medicine to
replace chemotherapy is not scientifically proven as effective with reference to the
following findings.
 The water extracts (traditional method) of both Annona muricata and Simaroba
glauca has not showed any anti-oxidant property. Hence this method of
extraction found as ineffective for treatment practices.
 Soxhlet extraction using solvents (especially with ethanol) can improve the
release of anti-oxidant compounds from plant leaves.

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 The water extracted powder of both Annona muricata and Simaroba glauca
subjecting microbial (Fungal growth) decomposition may improve its anti-
oxidant properties.
 A higher number of Annonacious acetogenin (7 compounds) and higher
concentration of quassinoids were observed in the extracts of Annona muricata
and Simaroba glauca by LC- MS analysis.
 The chemsketch analysis confirms that the compound present in the extract of
Annona muricata, annonacious acetogenin needs to be modified in to its
acetogenin structure for a higher anti-cancerous property (drug likeliness).
 Case studies showed non-conformance due to the lack proper information with
the patience.
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35Department of BTE