Anti Cancer Activity on Graviola, an Exciting Medicinal Plant Extract vs Various Cancer Cell Lines and a Detailed Computational Study on its Potent Anti-Cancerous Leads 02 April 2019

Anti cancer activity on graviola, an exciting medicinal plant extract vs various cancer cell lines and a detailed computational study on its potent anti-cancerous leads

Nature is the world’s best chemist: Many naturally occurring compounds have very complicated structures that present great challenges to chemists wishing to determine their structures or replicate them. The plant derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. Their high ligand binding affinity to the target introduce the prospect of their use in chemo preventive applications; in addition they are freely available natural compounds that can be safely used to prevent various ailments. Plants became the basis of traditional medicine system throughout the world for thousands of years and continue to provide mankind with new remedies. Here, we present a research study on a medicinal plant, Graviola, a native of North America but rarely grown in India. It has a wide potent anticancerous agents coined as Acetogenins which play a key role towards many varieties of cancer, Acetogenins are potent inhibitors of NADH oxidase of the plasma membranes of cancer cells.

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Patient with Metastatic Breast Cancer Achieves Stable Disease for 5 Years on Graviola and Xeloda after Progressing on Multiple Lines of Therapy 02 April 2019

Patient with metastatic breast cancer achieves stable disease for 5 years on graviola and xeloda after progressing on multiple lines of therapy

Breast cancer (BC) is the most common malignancy in women and is second to lung cancer in terms of cancer mortality. Treatment of BC remains a challenge as current therapies are limited by toxicity and drug resistance. Graviola (Annona muricata) is a tree that grows in the tropics of North and South America. Traditionally, the leaves and stems from the graviola tree have been used for a wide range of human diseases including cancer. In vitro and in vivo studies demonstrate anticancer activity in BC however clinical studies are lacking. We present the first case demonstrating clinical benefit without side effects using graviola in a patient with BC whose disease was refractory to multiple lines of chemotherapy including anthracyclines and taxanes.

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Curcumin inhibits endometriosis endometrial cells by reducing estradiol production 26 April 2018

Curcumin inhibits endometriosis endometrial cells by reducing estradiol production

Endometriosis is a complex estrogen-dependent disease that is defined as the presence of endometrial gland and stroma outside the uterine cavity. Although the exact mechanism for the development of endometriosis remains unclear, there is a large body of research data and circumstantial evidence that suggests a crucial role of estrogen in the establishment and maintenance of this disease. This study is an attempt to assess the effect of curcumin on inhibiting endometriosis endometrial cells and to investigate whether such an effect is mediated by reducing estradiol production.

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Effects of Components of Artemisia annua on Coccidia Infections in Chickens 20 April 2018

Effects of components of artemisia annua on coccidia infections in chickens

Four experiments were run to test the anticoccidial activity of dried Artemisia annua leaves and several of their chemical constituents for possible use as prophylactic feed additives. When fed over a period of 3 wk at a level of 5%, a dried leaf supplement of A. annua provided significant protection against lesions due to Eimeria tenella but not Eimeria acervulina or Eimeria maxima. When fed over a period of 5 wk at a level of 1% to chicks undergoing immunization with a live vaccine, it provided significant protection in partially immunized chicks against E. acervulina and E. tenella lesions from a dual species challenge infection.

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Pharmacokinetic study of artemisinin after oral intake of a traditional preparation of artemisia annua l. (annual wormwood)

Artemisia annua L. (annual wormwood) contains the antimalarial artemisinin. Aqueous preparations of the dried herb are included in the pharmacopoeia of the People’s Republic of China for treatment of fever and malaria. Artemisinin was absorbed faster from herbal tea preparations than from oral solid dosage forms, but bioavailability was similar.

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Flavonoids from Artemisia annua L. as Antioxidants and Their Potential Synergism with Artemisinin against Malaria and Cancer 12 April 2018

Flavonoids from artemisia annua l. as antioxidants and their potential synergism with artemisinin against malaria and cancer

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin.Since artemisinin was discovered as the active component of A. annua in early 1970s, hundreds of papers have focused on the anti-parasitic effects of artemisinin and its semi-synthetic analogs dihydroartemisinin, artemether, arteether, and artesunate. Artemisinin per se has not been used in mainstream clinical practice due to its poor bioavailability when compared to its analogs. In the past decade, the work with artemisinin-based compounds has expanded to their anti-cancer properties. Although artemisinin is a major bioactive component present in the traditional Chinese herbal preparations (tea), leaf flavonoids, also present in the tea, have shown a variety of biological activities and may synergize the effects of artemisinin against malaria and cancer.

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Antiproliferative effect of annona muricata linn. leaves on ehrlich ascites carcinoma

Cancer is a dreadful disease caused by abnormal and uncontrolled cell division. Lung cancer remains a major cause of cancer-related deaths in men and the second most common cancer in women, responsible for 1.59 million deaths worldwide annually. Treatment of cancer usually involves surgery, chemotherapy, and radiation therapy, or a combination of these. Despite these therapeutic options, cancer remains associated with high mortality. Traditional medicine which involves the use of herbs has been used to treat various types of cancer and this has been found to be effective with minimal or no side effects. Annona muricata L. also called “Soursop” or “Sharp Sharp” is traditionally used in treatment of cancer. There are many claims that Annona muricata L. can kill cancer, since its leaves, seeds and fruits contain active compound called annonaceous acetogenins

The present investigation was to screen Antiproliferative effect of Annona muricata Linn. on Ehrlich ascites carcinoma. As per OECD guideline 425, acute toxicity study of methanolic extract of Annona muricata Linn was performed and two doses 200 mg/kg and 400 mg/kg were selected for the study. Injected EAC cell for mice. Four groups (n=6) were selected. Group I served as normal Control, Group II served as EAC control, Group III served as EAC + 200mg/kg, Group IV served as EAC + 200mg/kg. On 90 days fasting blood sample were collected and analyzed for haematological parameters in EAC bearing mice. In Ehrlich ascites carcinoma model, the tumor bearing mice treated with methanol extract of Annona muricata leaves 200 mg/kg and 400 mg/kg once daily orally started 24 h after inoculation significantly reduced the viable cell count. The dose level of 400 mg/kg showed a significant increase in non-viable count with a significant decrease in body weight of animals compared to EAC control animals. a) Before treatment b)After treatment

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Antiallergic effect of the atomized extract of rhizome of Curcuma longa, flowers of Cordia lutea and leaves of Annona muricata 11 November 2016

Antiallergic effect of the atomized extract of rhizome of curcuma longa, flowers of cordia lutea and leaves of annona muricata

Allergies are a problem that greatly affects the population, and hence the use of antiallergic medications is fairly widespread. However, these drugs have many adverse effects. The use of medicinal plants could be an option, but they need to be evaluated. This study was designed to evaluate the antiallergic effect of the atomized extract of rhizome of Curcuma longa, flowers of Cordia lutea, and leaves of Annona muricata. Considering the clinical and histopathological signs, we conclude that the administration of the atomized extract of rhizome of C. longa, flowers of C. lutea, and leaves of A. muricata lacks antigenic effect but could have an antiallergenic effect in a model of dermal irritation in rabbits.

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Anti-cancer effect of Annona Muricata Linn Leaves Crude Extract (AMCE) on breast cancer cell line 17 October 2016

Anti-cancer effect of annona muricata linn leaves crude extract (amce) on breast cancer cell line

Annona muricata Linn which comes from Annonaceae family possesses many therapeutic benefits as reported in previous studies and to no surprise, it has been used in many cultures to treat various ailments including headaches, insomnia, and rheumatism to even treating cancer. However, Annona muricata Linn obtained from different cultivation area does not necessarily offer the same therapeutic effects towards breast cancer (in regards to its bioactive compound production). In this study, anti-proliferative and anti-cancer effects of Annona muricata crude extract (AMCE) on breast cancer cell lines were evaluated.

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11 July 2016

Phyto-chemical screening and anti listerial activity of annona muricata (l) leaf extract

Natural remedies from medicinal plants are found to be safe and effective. Many plant species have been used locally as medicine to treat various ailments. Bio-active compounds from plants continue to play a major role in primary health care as therapeutic remedies in many developing countries. According to the WHO survey, 80% population depends upon the traditional medicines for primary health care needs. It suggested in improving the technologies for cultivation of medicinal plants. It has been reported that there has been an alarming increase in number of diseases and disorders caused by synthetic drugs prompting a switch over to traditional herbal medicine [1-5]. Annona muricata L. (Soursop) is a naturally occurring plant, traditionally used to treat various ailments including cancer. It belongs to the family Annonaceae and is widely distributed in India and Central America. Fruits of Annona muricata, also known as Graviola in South America are taken internally for worms, parasites, fever,as an astringent for diarrhoea and dysentery. The plant is also reported to have good antioxidant property.

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11 July 2016

Phyto-chemical and pharmacological properties of annona muricata: a review

In the present review, an attempt has been made to congregate the traditional, phytochemical and pharmacological studies done on an important medicinal plant Annona muricata, (Family annonaceae). Cyclo hexapeptides, acetogenins, annonaceous acetogenins were the major phytochemical compounds studied from this medicinal plant. The fruit is of economic value and hence cultivated and used widely as an edible food. The plant possess the major pharmacological activities includes cytotoxic, antileishmanial, wound healing, anti-microbial activity. It also has the anticarcinogenic and genotoxic effect. Phytochemical analysis of the plant revealed the presence of tannins, steroids and cardiac glycosides which are the major phytochemical compounds. The pulp obtained from the plant shows the thermal diffusivity property. This review encompasses the potential application of the above plant in the pharmaceutical field due to its wide pharmacological activities. As the fruit of this plant is highly nutritious this paves the ways to work in future on its potential to serve as an edible vaccine.

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18 May 2016

Annona muricata leaves induced apoptosis in a549 cells through mitochondrial-mediated pathway and involvement of nf-κb

For the first time the ethyl acetate extract of Annona muricata showed to inhibit the proliferation of A549 cells, leading to cell cycle arrest and programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway.

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04 May 2016

Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence

Approximately 200 studies that examined the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary are reviewed. A statistically significant protective effect of fruit and vegetable consumption was found in 128 of 156 dietary studies in which results were expressed in terms of relative risk. For most cancer sites, persons with low fruit and vegetable intake (at least the lower one-fourth of the population) experience about twice the risk of cancer compared with those with high intake, even after control for potentially confounding factors. For lung cancer, significant protection was found in 24 of 25 studies after control for smoking in most instances. Fruits, in particular, were significantly protective in cancers of the esophagus, oral cavity, and larynx, for which 28 of 29 studies were significant. Strong evidence of a protective effect of fruit and vegetable consumption was seen in cancers of the pancreas and stomach (26 of 30 studies), as well as in colorectal and bladder cancers (23 of 38 studies). For cancers of the cervix, ovary, and endometrium, a significant protective effect was shown in 11 of 13 studies, and for breast cancer a protective effect was found to be strong and consistent in a meta-analysis. It would appear that major public health benefits could be achieved by substantially increasing consumption of these foods.

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04 April 2016

a.muricata leaves extract was able to suppress tumor initiation as well as tumor promotion even at lower dosage.

Annona muricata L (Annonaceae), commonly known as soursop has a long, rich history in herbal medicine with a lengthy recorded indigenous use. It had also been found to be a promising new anti-tumor agent in numerous in vitro studies. The present investigation concerns chemopreventive effects in a two-stage model of skin papillomagenesis. Chemopreventive effects of an ethanolic extract of A. muricata leaves (AMLE) was evaluated in 6-7 week old ICR mice given a single topical application of 7,12-dimethylbenza(α)anthracene (DMBA 100 μg/100 μl acetone) and promotion by repeated application of croton oil (1% in acetone/ twice a week) for 10 weeks. Morphological tumor incidence, burden and volume were measured, with histological evaluation of skin tissue. Topical application of AMLE at 30, 100 and 300 mg/kg significantly reduced DMBA/croton oil induced mice skin papillomagenesis in (i) peri-initiation protocol (AMLE from 7 days prior to 7 days after DMBA), (ii) promotion protocol (AMLE 30 minutes after croton oil), or (iii) both peri-initiation and promotion protocol (AMLE 7 days prior to 7 day after DMBA and AMLE 30 minutes after croton oil throughout the experimental period), in a dose dependent manner (p<0.05) as compared to carcinogen-treated control. Furthermore, the average latent period was significantly increased in the AMLE-treated group. Interestingly, At 100 and 300 mg/ kg, AMLE completely inhibited the tumor development in all stages. Histopathological study revealed that tumor growth from the AMLE-treated groups showed only slight hyperplasia and absence of keratin pearls and rete ridges. The results, thus suggest that the A.muricata leaves extract was able to suppress tumor initiation as well as tumor promotion even at lower dosage.

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Chemopreventive effect of Annona muricata on DMBA-induced cell proliferation in the breast tissues of female albino mice 03 March 2015

Chemopreventive effect of annona muricata on dmba-induced cell proliferation in the breast tissues of female albino mice

Abstract Background

Breast cancer is the most common type of cancer and leading cause of cancer death in women. Breast cancer and cancer related diseases have been treated using surgery, chemotherapy, and radiation therapy, or a combination of these. Despite these therapeutic options, cancer remains associated with high mortality. Traditional medicine which involves the use of herbs has been used to treat various types of cancer and this has been found to be effective with minimal or no side effects. Aim

This research was aimed at evaluating the potential chemopreventive effect of an ethanolic extract of Annona muricata leaves on 7,12-dimethylbenzeneanthracene (DMBA)-induced cell proliferation in the breast tissues of female albino mice. Materials and methods

A. muricata leaves, thirty (30) female albino mice, and 7,12-dimethylbenzeneanthracene (DMBA) were used for this study. Crude extraction protocol was employed in the preparation of an ethanolic extract of A. muricata leaves. Qualitative and quantitative phytochemical screening of an ethanolic extract of A. muricata leaves was carried out using standard protocol. Agarose gel electrophoresis was used to analyze deoxyribonucleic acid (DNA) extracted from the breast tissues of experimental mice while hematoxylin and eosin staining was used for histological assay. Results

Phytochemical screening revealed the presence of terpenoid, steriod, flavonoids, cardiac glycoside, tannin, phenol, alkaloid, and reducing sugar. Phenol was quantitatively determined to be present in the highest amount. DNA smears obtained from agarose gel electrophoresis suggested possible DMBA-induced damage which was significantly prevented owing to the effect of the leaf extract of A. muricata leaves. Histological assay revealed the presence of DMBA induced lobular alveolar hyperplasia, adenomatoid hyperplasia, fibro adipose stroma, and proliferating sebaceous gland in the histological sections of the breast tissues of treated mice, however, these changes were found to vary in occurrence among the different groups of treated animals. Conclusion

This study has shown that the leaf extract of A. muricata could be used as a prophylactic measure against DMBA-induced cell proliferation in the breast tissues of female albino mice. Abbreviations

DMBA, 7,12-dimethylbenzene[a]anthracene;
HE, hematoxylin and eosin;
A. muricata, Annona muricata;
DNA, deoxyribonucleic acid


Annona muricata;
Breast cancer;
Lobular alveolar hyperplasia
  1. Introduction

Cancer is a multifactorial disease arising from the accumulative effects of gene products of mutant proto-oncogenes, tumor suppressor genes and DNA repair genes, leading to the uncontrolled growth and spread of cancer cells [1] and [2]. There are over 100 different types of cancer, and each is classified by the type of cell that is initially affected, and these include cervical cancer, skin cancer, leukemia, lung cancer, prostate cancer, and so on [3].

Breast cancer represents the most common neoplastic disease in females, accounting for up to one third of new diagnoses of women’s cancer in certain regions of the world [4]. In developing countries traditionally known for low incidence of breast cancer, increase in both incidence and mortality has been recently detected [5]. Azubuike and Okwuokei [6] observed that in Nigeria, the peak age of breast cancer is about ten years earlier than the experience of many western women and were attributed to increasing adoption of western life style and diet. Contrary to previous reports indicating breast cancer as the second leading cause of cancer deaths, breast cancer is now the leading cause of cancer deaths in Nigerian women [7], [8] and [9].

Breast cancer and cancer related diseases have been treated using surgery, chemotherapy, and radiation therapy, or a combination of these. But despite these therapeutic options, cancer remains associated with high mortality [10]. This is basically due to difficulties in early diagnosis, exorbitant cost of treatment, with the often late presentation of breast cancer that generally characterizes cancer diagnosis among Nigerian and other African women [10], [11], [12] and [13]. Owing to these several shortcomings, there is a need for better therapeutic options which will increase the chances of survival of breast cancer patients with minimal or no side effects of treatment. Early diagnoses of breast cancer and its prevention have been suggested as a better means of managing breast cancer because about 5% of human breast cancers have been attributed to inheritance of breast cancer susceptibility genes [2] and [14]. Therefore, people with familial history of breast cancer have better options of diagnosing and preventing its occurrence within their life time.

Among the major problems of breast cancer therapy is the fact that a majority of patients suffering from the disease cannot afford the high cost of therapy [15]. It has also been discovered that more than 70% of all cancer deaths occurred in low and middle-income earners [16]. Nigeria as one of the countries found in the tropics is rich with plants that have been found to possess anticancer therapeutic activity [10]. Annona muricata commonly called soursop or “sharp sharp” is a small erect evergreen tropical fruit tree plant belonging to the family Annonaceae, growing 5–6 m in height [17]. It is one of the easily found plants used traditionally in treating cancer. The leaf decoction is usually taken to lessen the symptoms of cancer [18].

In order to develop a better and cheaper means of preventing breast cancer in people liable to suffering the disease in their life time, this work was therefore aimed at evaluating the chemopreventive effect of an ethanolic extract of A. muricata leaves on 7,12-dimethylbenzeneanthracene (DMBA)-induced cell proliferation in the breast tissues of female albino mice.

  1. Materials and methods 2.1. Experimental animals

The 30 adult mice used for this study were obtained from the Nigeria Institute of Medical Research, Lagos, Nigeria. The animals were housed in standard clean mice cages at 25 °C, fed with standard pellet and tap water ad libitum. They were maintained under uniform conditions of natural photo period (12 h light/dark cycle) and humidity (61–95%). 2.2. Plant collection and identification

Leaves of A. muricata were collected in the month of May 2013, from the botanical garden of the University of Lagos, Lagos, Nigeria. Leaves were identified and confirmed taxonomically at the Department of Botany, University of Lagos, Lagos, Nigeria. A voucher specimen number LUH 6070 of the plant was deposited in the Herbarium of the same department. 2.3. Plant preparation and extraction

A. muricata leaves were washed in a running tap and air-dried in the Cell Biology and Genetics laboratory, University of Lagos. The dried leaves were later blended using an electric blender which had been sterilized with 70% ethanol. The leaf extract was obtained using Crude Extraction Protocol. About 350 g of A. muricata powder was soaked in 70% ethanol for 72 h. This was later filtered using a sieve. The filtrate was concentrated using a water bath set at 40 °C. The final mass of concentrated extract obtained was 40 g. The concentrate was used to prepare the different concentrations used for the experiment and phytochemical screening.

  1. Methodology

At the commencement of this work, thirty (30) adult female albino mice weighing between 21 and 28 g, were divided into 5 groups, each group had 6 mice. The experimental groups received different concentrations of an ethanolic extract of A. muricata prepared with respect to the LD50 result as documented by Arthur et al. [17]. Mice were treated with increasing doses of extract.

Group MA: Mice treated with 20 mg/ml/week of DMBA + 200 mg/ml/day of extract.

Group MB: Mice treated with 20 mg/ml/week of DMBA + 100 mg/ml/day of extract.

Group MC: Mice treated with 20 mg/ml/week of DMBA + 50 mg/ml/day of extract.

Group NC (Negative Control): Mice treated with 20 mg/ml/week of DMBA only.

Group PC (Positive Control): Mice treated with distilled water only.

DMBA and extract were given intragastrically by gavage using a cannula fitted to a feeding needle. Treatment of animals lasted for six (6) weeks. The experimental and control animals were carefully checked daily and their weight taken weekly. Each mouse had 6 pairs of mammary glands that were checked by inspection, touching and palpitation [4]. Mice were sacrificed at the end of the sixth week by cervical dislocation. The breast tissues from each animal were sliced off and divided into two portions using a surgical blade. One portion was fixed in formalin saline for histology using hematoxylin and eosin staining while the other portion was fixed in ethanol for agarose gel electrophoresis. The work was carried out in the animal house of the University of Lagos, Lagos, Nigeria in accordance with the Code of Ethics of The World Medical Association (Declaration of Helsinki) for animal experiment with consent from the University of Lagos Ethics Committee guidelines for experiments with whole animals [19]. 3.1. Phytochemical screening

This was carried out using standard procedures in accordance with Vimala et al. [20]. 3.2. DNA extraction

The DNA extraction protocol used for this study was a modified procedure of Nishiguchi et al. [21].

Group MA: Mice treated with 20 mg/ml/week of DMBA + 200 mg/ml/day of extract.

Group MB: Mice treated with 20 mg/ml/week of DMBA + 100 mg/ml/day of extract.

Group MC: Mice treated with 20 mg/ml/week of DMBA + 50 mg/ml/day of extract.

Group NC (Negative Control): Mice treated with 20 mg/ml/week of DMBA only.

Group PC (Positive Control): Mice treated with distilled water only.
  1. Results

Table 1 shows the result obtained from the qualitative phytochemical screening of the ethanolic extract of A. muricata leaf. Phytochemical screening revealed that terpenoid, steroid, flavonoids, cardiac glycoside, tannin, phenol, alkaloid, and reducing sugar were present while phlobatannin and saponin were absent.

Table 1.

Qualitative analysis of an ethanolic extract of Annona muricata leaves.
S. No.  Phytochemical components    Ethanolic extract
1   Saponin −
2   Terpenoid   +
3   Steroid +
4   Flavonoids  +
5   Cardiac glycoside   +
6   Tannin  +
7   Phenol  +
8   Phlobatannin    −
9   Alkaloid    +
10  Reducing sugar  +

Key: (+), Presence of phytochemical; (−), Absence of phytochemical.

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The quantitative phytochemical screening of selected phytochemicals present in the ethanolic extract of Annona muuricata is shown in Table 2. Phenol was discovered to be present in the highest amount while cardiac glycoside was found to be in the lowest amount.

Table 2.

Quantitative analyses of selected phytochemicals present in an ethanolic extract of Annona muricata leaves.
S. No.  Phytochemical   Annona muricata (mg/100 g)
1   Phenol  162.99
2   Cardiac glycosides  12.92
3   Tannin  121.98
4   Alkaloid    13.74
5   Flavonoids  16.26
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The effects of an ethanolic extract of A. muricata leaves on the survival rate of mice administered with DMBA are shown in Fig. 1. No death was recorded in any group during the first 2 weeks. The highest number of deaths was recorded in NC in the fifth week. No death was recorded in MB throughout the 6 weeks of this experiment.

Full-size image (19 K) Figure 1.

Effects of an ethanolic extract of Annona muricata leaves on the survival rate of DMBA-induced cell proliferation in mice.
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Plate 2 is the genomic DNA smear obtained from the electrophoresis of deoxyribonucleotides (DNAs) obtained from the breast tissues of mice from different experimental groups. Genomic DNA smear from PC had normal smear typical of agarose gel electrophoresis. But smears displayed by other groups deviated from normal; with the deviation being highest in NC. Genomic DNA smear obtained from PC and MB are similar and have better smears compared to NC and MC. DNA obtained from MA showed no smear.

Full-size image (32 K) Plate 1.

DMBA induced-papilloma on the hind limbs of mouse treated with DMBA only. Arrows indicate the location of DMBA induced papilloma.
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Full-size image (33 K) Plate 2.

Genomic DNA smear obtained from the electrophoresis DNAs obtained from the breast tissues of DMBA-induced cell proliferation in mice.
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The histological section of the breast tissues of mouse in the positive control is shown in Plate 3i. This revealed the presence of normal epidermal tissue, sweat glands, sebaceous gland, alveolar duct and terminal bronchiole.

Full-size image (58 K) Plate 3.

Histological sections of the breast tissues of mice from the positive (PC) and negative control (NC) showing the alveolar duct and terminal bronchiole in (i) and DMBA-induced lobular alveolar hyperplasia in (ii).
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Plate 3ii shows the histological section of the breast tissues of DMBA-induced cell proliferation in mouse from the negative control group. Normal keratinocytes, sebaceous glands, ecrine glands and subdermal layer with prominent DMBA-induced lobular alveolar hyperplasia were seen to be present.

Plate 4i reveals the histological section of the breast tissue of DMBA-induced cell proliferation of mice that were treated with 200 mg/ml of extract. The presence of DMBA-induced lobular alveolar hyperplasia, fibrocystic change and DMBA-induced fibroadenomatoid hyperplasia were identified in Plate 4i while lobular alveolar hyperplasia and fibro adipose stroma were identified in Plate 4ii.

Full-size image (54 K) Plate 4.

Histological sections of the breast tissues of DMBA-induced cell proliferation in mouse treated with 200 mg/ml showing fibroadenomatoid hyperplasia in (i) and fibro adipose stroma in (ii).
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The histological section of the breast tissue of DMBA-induced cell proliferation in mouse that was treated with 100 mg/ml of extract is shown in Plate 5. The section revealed the presence of DMBA-induced lobular alveolar hyperplasia.

Full-size image (23 K) Plate 5.

Histological section of the breast tissue of DMBA-induced cell proliferation in mouse treated with 100 mg/ml of extract showing the presence of lobular alveolar hyperplasia.
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Plate 6 shows the histological section of the breast tissue of DMBA-induced cell proliferation in mouse that was treated with 50 mg/ml of ethanolic extract of A. muricata. This reveals the presence of proliferating sebaceous glands and lobular alveolar hyperplasia.

Full-size image (26 K) Plate 6.

Histological section of the breast tissue of DMBA induced cell proliferation in mouse treated with 50 mg/ml of extract showing the presence of lobular alveolar hyperplasia and proliferating sebaceous gland.
Figure options
  1. Discussion

Breast cancer is the most common type of cancer and the leading cause of cancer death in Nigerian women [7] and [8]. Owing to this fact, in addition to a long latency period of many years prior to the development of metastatic disease, this form of cancer could be identified as an ideal target for chemoprevention and/or early intervention. A general concept put forward by several researchers is that the anticancer activity of compounds which are typically present in plants at sub-pharmaceutical doses could synergize to delay or disrupt the development of aggressive disease [22] and [23]. The chemopreventive effect of an ethanolic extract of A. muricata leaf extract on 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in mice was evaluated. DMBA is a well-known potent carcinogen which has been used to induce carcinogenesis in the mammary gland or skin of experimental rodents such as rat and mouse [4], [24], [25], [26] and [27].

The visible evidence of 7,12-dimethylbenz[a]anthracene administration (DMBA) induced-papilloma was seen in the hind limbs of mouse treated with DMBA only (Plate 1) about 4 weeks after DMBA administration. This occurred earlier than reported by Barros et al. [4]. This could be owing to the fact that the modified method of Barros et al. [4] used for this study was of higher concentration. The high death rate recorded in the fifth week among the group of mice treated with DMBA only might be attributed to the toxicity of the carcinogen. A recent study that focused on the immunotoxicity of DMBA given to experimental animals to induce mammary gland showed that DMBA elicits immunotoxicity in the spleen, thymus and bone marrow [28].

The deviation from normal displayed by the genomic DNA smears obtained after electrophoresis, especially in NC (Plate 2), might have arisen as a result of DMBA-induced deoxyribonucleic acid (DNA) damage or mutation. This is in concordance with the report of Nebert et al. and Rundle et al. [29] and [30] who described the mechanism of action of DMBA to involve up-regulation of cytochrome P450 enzymes that metabolize DMBA into a mutagenic epoxide intermediate which readily forms DNA-adducts that are associated with DNA mutations and the malignant transformation that leads to carcinogenesis. As cancer cells usually have mutations in genes regulating DNA damage responses or repair pathways, proto-oncogenes and tumor suppressor genes, they can be more susceptible to cell cycle arrest and death from treatment with carcinogenic agents than normal cells [2] and [31].

The similarity between the genomic DNA smears displayed by DNAs obtained from PC and MB might be due to the prevention of excessive DMBA-induced damage to DNA by an ethanolic extract of A. muricata leaves at a concentration of 100 mg/ml ( Plate 2). Many chemotherapeutic agents such as plant-derived compounds have been shown to exert their therapeutic effect by directly interacting with DNA or DNA-binding proteins [31] and [32]. This interaction triggers DNA damage signaling pathways resulting in the inhibition of cell proliferation or the induction of apoptosis, depending on the extent of the damage [33]. However, the similarity between smears displayed by the DNAs obtained from MC and NC ( Plate 2) suggests the plant extract’s inability to prevent DMBA-induced DNA damage at a dose of 50 mg/ml. Therefore, the extract could be said to have acted in such a way as to prevent or reduce excessive DMBA-induced DNA damage. The preventive effect of an ethanolic extract of A. muricata leaves against DMBA-induced DNA damage could be owing to the presence of the various secondary metabolites (tannins, terpenoids, cardiac glycosides (CGs), and flavonoids) discovered from the phytochemical screening of the ethanolic extract of A. muricata ( Table 1).

Previous studies have shown that tannins, terpenoids, cardiac glycosides (CGs), and flavonoids possess anticancer activities [34], [35], [36], [37], [38], [39], [40] and [41]. Epidemiologic evidence suggested that breast cancer patients treated with cardiac glycosides had a significantly lower mortality rate, and their cancer cells had more benign characteristics than those from patients not treated with it [34]. Phenol was determined to be quantitatively present in the highest amount (Table 2), this might have accounted for the preventive effect of the extract on DMBA-induced cell proliferation. This is in agreement with the previous studies showing the anti-proliferative effects of herbal polyphenols, in various human cancer cell lines [42], [43] and [44].

The presence of DMBA-induced lobular alveolar hyperplasia, and DMBA-induced fibroadenomatoid hyperplasia in the histological sections of the breast tissues of mice treated with DMBA (Plates. 3ii, 4, 5, and 6) suggests a neoplastic transformation which is an indication of DMBA-induced cell proliferation [45]. DMBA-induced ductal hyperplasia has been discovered to be a physiologic precursor to the development of ductal carcinoma in situ [46]. Furthermore, DMBA-induced carcinogenesis had been documented to be associated with ductal carcinomas, fibroadenomas, adenomas, and papillomas [4]. However, these changes were found to vary in occurrence among the different groups used for the present study. These variations might be due to the preventive effect of the different concentrations of extract administered to mice. Terminal end buds had been shown to be preferential targets of DMBA effects (DMBA–DNA linking) in the neoplastic transformation of the mammary gland [26]. The presence of proliferating cells in the stroma as well as among the epithelial and myoepithelial cells (Plate 4ii) strongly suggests that the carcinogen acts on different cells in the breast tissue and, although the stroma itself does not undergo neoplastic transformation, it plays an important role in the carcinogenic process [47].

  1. Conclusion

The present study showed that the ethanolic extract of A. muricata leaves can be used as a preventive measure against DMBA-induced breast cell proliferation in the breast tissues of female albino mice. Agarose gel electrophoresis showed that the plant extract prevented DMBA-induced DNA damage to some extent. Histological assay revealed the presence of a neoplastic transformation which suggests the presence of cells undergoing the initial proliferative stage preceding carcinogenesis. The knowledge obtained from this study can be exploited by person(s) suspected to be linked with familial history of breast cancer. Nevertheless, further studies and more research need to be done to optimize the quality of extract, effective dose and its specificity on breast cancer susceptibility genes.

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23 January 2015

Antiproliferative activity and induction of apoptosis by annona muricata (annonaceae) extract on human cancer cells

Open Access Highly Accessed Research article Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells

Constant Anatole Pieme12, Santosh Guru Kumar2, Mireille Sylviane Dongmo3, Bruno Moukette Moukette1, Fabrice Fekam Boyoum4, Jeanne Yonkeu Ngogang1 and Ajit Kumar Saxena2

* Corresponding authors: Constant A Pieme - Ajit K Saxena

Author Affiliations

1 Department of Physiological Sciences and Biochemistry, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, PO Box 1364, Yaoundé, Cameroon

2 Cancer Pharmacology Division, Indian Institute of Integrative Medicine, 180001, Canal Road, 18001 Jammu, India

3 Department of Biochemistry and Molecular Biology, University of Buea, Buea, Cameroon

4 Department of Biochemistry, Faculty of Sciences, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon

For all author emails, please log on.

BMC Complementary and Alternative Medicine 2014, 14:516 doi:10.1186/1472-6882-14-516

The electronic version of this article is the complete one and can be found online at:

Received: 3 June 2014 Accepted: 9 December 2014 Published: 24 December 2014

© 2014 Pieme et al.; licensee BioMed Central.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated. Abstract Background

Annona muricata (A. muricata) is widely distributed in Asia, Africa and South America. Different parts of this plant are used to treat several diseases in Cameroon. The aim of this study is to determine the in vitro anti-proliferative effects and apoptotic events of A. muricata extracts on HL-60 cells as well as to quantify its phenols content. Methods

The cell viability was measured by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay while the changes in morphology of HL-60 cells, membrane mitochondrial potential (MMP) and the cell cycle were used for assessment apoptosis induction. Results

The results show that the concentration of phenols, flavonoids and flavonols in the extracts varied depending on the part of the plant. All the extracts tested inhibited the proliferation of HL-60 cells in a concentration dependent manner with IC50 varied from 6–49 μg/mL. The growth inhibition of the cells by extracts was associated with the disruption of MMP, reactive oxygen species (ROS) generation and the G0/G1 cell arrest. Conclusion

These findings suggest that the extracts from A. muricata have strong antiproliferation potential and can induce apoptosis through loss of MMP and G0/G1 phase cell arrest. Keywords: Apoptosis; Membrane mitochondrial potential; Antiproliferative; Cell cycle; A. muricata Background

Chemopreventive properties have long been attributed to polyphenolic compounds present in the human diet. The interest on these natural substances is increasing because of their higher potential sources of anticancer compounds. Plants and plant-based medicaments are used as the basis of many modern pharmaceuticals industries today for the treatment of our various ailments [1]. According to world health organization (WHO), more than 80% of the total world’s population depends on the traditional medicines to satisfy their primary health care needs. Several phytochemical molecules from natural products capable of exerting a physiologic action on the human body were studied and characterized. These bioactive compounds such as alkaloids, flavonoids, tannins and phenols were considered to be most important. The phytochemical research that has been done based on the ethno pharmacological information forms the effective approach in the discovery of new anti-infective agents from higher plants [2].

Annona muricata L (A. muricata) commonly known as graviola, soursop or corossol, belongs to the Annonaceae family. It is a widespread small tree and has its native in Central America [3]. It is a typical tropical tree with heart shaped edible fruits and widely distributed in most of tropical countries. All parts of A. muricata tree are used in natural medicine in the tropic including the twigs, leaf, root, fruit and seeds. Generally, the fruit and fruit juice are taken to eliminate worms and parasites, cool fever, increase mother’s milk after child birth, and as an astringent for diarrhea and dysentery [4]. The crushed seeds are used against internal and external parasites, head lice and warms. The twigs, leaf are considered sedative and antispasmodic [4]. A decoction of A. muricata leaf is used to kill bed bug and head lice to reduce fever. For the latter it can have the same effect taken orally or added to bathing water [5]. The creamy and delectable flesh of the fruit consist of 80% water, 1% protein, 18% carbohydrates and fair amount of vitamins B, B2 and C, potassium and dietary fiber [6]. The leaf are lanceolate with glossy and dark green in color had been traditionally used to treat headaches, hypertension, cough, asthma and used as antispasmodic, sedative and nervine for heart condition [7,8]. Previous reports have demonstrated that the leaf, twigs, root, stem, and fruit seed extracts of A. muricata have several biological activities such as anti-bacterial [9], antifungal [10] and anti-malarial [11]. Its leaf extract were also found to possess antioxidant [12] and molluscicidal properties [13]. Recently, it has also been reported to exhibit anti-inflammatory and analgesic effects [14], cytotoxicity and apoptosis inducing activities on T47D breast cancer [15], antiviral activity [16] and antidiabetic activity. Phytochemical investigation of the leaf of A. muricata showed the presence of alkaloids [17], essential oils [18] and acetogenins [19]. These acetogenins demonstrated to be selectively toxic against various types of the cancerous cells without harming healthy cells [20]. Acetogenin 1 was reported to exhibit cytotoxic activities against the human pancreatic tumor cell line (PACA-2), human prostate adenocarcinoma (PC-3) and human lung carcinoma (A-549), while Acetogenin 2 was reported to exhibit cytotoxicity against human hepatoma carcinoma cell line (Hep G2) [21]. Seven isoquinoline alkaloids including reticuline, coclaurine, coreximine, atherosperminine, stepharine, anomurine and anomuricine have been isolated from the leaves, root and stem barks of A. muricata[22]. The essential oil of the fresh fruit pulp of A. muricata contains 2-hexenoic acid methyl ester (23.9%), 2-hexenoic acid ethyl ester (8.6%), 2-octenoic acid methyl ester (5.4%), 2-butenoic acid methyl ester (2.4%), β-caryophyllene (12.7%), 1,8-cineole (9.9%), linalool (7.8%), α-terpineol (2.8%), lialyl propionate (2.2%) and calarence (2.2%) [23]. Therefore, we attempted to investigate the growth-inhibitory and apoptotic effects of extracts from leaf, twigs and roots from A. muricata against Human promyelocytic leukemia (HL-60 cells). Methods Preparation of extracts

The plant material including leaf, twigs and roots of were collected in Yaounde, capital city of Cameroon and authentified by Mr NANA, a botanist of the National Herbarium of Cameroon in comparison to the voucher specimens under the reference number of 3289/HNC. They were then shade dried and grounded using a blender. The obtained powder was cold macerated with ethanol 95°C with occasional stirring for 3 days. After 3 days, the suspension was filtered through filtered paper and the filtrate was evaporated to dryness at low temperature, using a rotary evaporator. The procedure was repeated 3 times and until total decoloration of the mixture observed. The obtained extracts were stored and used for further analysis. Determination of phenolic composition of the extracts Total phenol determination

The total phenol was determined by the Folin–Ciocalteu method, the reaction mixture contains: 200 μL of diluted spice extract, 800 μL of freshly prepared diluted Folin Ciocalteu reagent and 2 mL of 7.5% sodium carbonate. The final mixture will be diluted to 7 mL with deionized water. Mixture was kept in the dark at ambient conditions for 2 h to complete the reaction. The absorbance at 765 nm will be measured. Garlic acid was used as standard and the results were expressed as mg garlic acid (GAE)/g of dried material. Determination of total flavonoid content

Total flavonoid content was determined using aluminium chloride (AlCl3) according to a known method using quercetin as a standard. The spice extract (0.1 mL) was added to 0.3 mL distilled water followed by 5% NaNO2 (0.03 mL). After 5 min at 25°C, AlCl3 (0.03 mL, 10%) was added. After further 5 min, the reaction mixture was treated with 0.2 mL of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 mL with water and the absorbance was measured at 510 nm. The results will be expressed as mg of quercetin (QE)/g of dried material Determination of total flavonols

Total flavonols in the plant extracts were estimated using the method of Kumaran and Karunakaran [24]. To 2.0 mL of sample (standard), 2.0 mL of 2% AlCl3 ethanol and 3.0 mL (50 g/L) sodium acetate solutions were added. The absorption at 440 nm was read after 2.5 h at 20°C. Extract samples were evaluated at a final concentration of 0.1 mg/mL. Total flavonoid content was calculated as mg of quercetin (mg/g) using the following equation y = 5.3911x + 0.0313, R2 = 0.9967 based on the calibration curve, where x was the absorbance and y was the concentration of quercetin (mg/g). Antitumor activity of the extracts Cell culture

The Human promyelocytic leukemia (HL-60 cells) was obtained from European Collection of Cells Culture (ECCC), Sigma–Aldrich, India. They were grown in RPMI-1640 medium containing 10% Fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL medium). The cells suspension was kept in the incubator (Thermo Electron Corporation, USA) at 37°C, 5% CO2; 95% humidity. Cells were used for different assays during logarithmic growth phase while the untreated control cultures received only the vehicle (dimethyl sulfoxide [DMSO] < 0.1%). Cells viability and treatments

HL-60 cells were seeded in 96 different well plates containing 15 × 103 μL/well, respectively. The cultured cells were then treated (triplicate wells per condition) by adding of 100 μL of serial dilutions of the three extracts in DMSO to give a final concentration of 100, 30, 10 and 1 μg/mL. In addition, the DMSO alone was added to another set of cells as the solvent control (DMSO < 0.1%). The cells were then incubated for another 48 h prior to the addition of 20 μL of 2.5 mg/mL solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) into each well. The incubation was continued for 3 h before the media was removed. A mixture of DMSO (150 μL) was added to each well and mixed to ensure dissolving of the crystal formazan before the absorbance at 570 nm was measured. Three replications of each experiment were performed and fifty percent of inhibitory concentration (IC50) of each extract was calculated. Hoechst 33258 staining of cells for nuclear morphology

HL-60 cells (2×106 cells/3 mL/well) were treated with different extracts at different concentrations of extract for 24 h. They were collected, centrifuged at 400 g and washed once with PBS. A solution of Hoechst (Hoechst, 10 μg/mL; citric 10 mM; Na2HPO4 0.45 M; Tween-20 0.05%) was added in each tube and kept in the dark at room temperature for 30 min. The mixture was washed with PBS and the pellet suspended in 100 μL of PBS/glycerol (1:1). The solution (10 μL) was poured into the slide and nuclear morphology alterations observed under fluorescence microscope (Olympus X 70, magnification 20 X) [25]. Reactive oxygen species (ROS) assay

ROS production was monitored by flow cytometry using 2’ , 7’- dichlorodihydrofluorescin diacetate (DCFH2-DA). This dye is a stable non polar compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield 2’ ,7’ dichlorodihydrofluorescin (DCFH2), which is trapped within the cells. Hydrogen peroxide or low molecular weight peroxides produced by the cells oxidizes DCFH2 to a highly fluorescent compound 2’ ,7’-dichlorofluorescein (DCF). Thus, the fluorescence intensity was proportional to the amount of hydrogen peroxide produced by the cells. Briefly, HL-60 cells (1 × 106 cells/2 mL/well) were treated with extracts of leaf, roots and twigs of A. muricata at different concentrations for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with DCFH2-DA (50 μM) and kept in the dark. Cells were then collected, centrifuged (200 g; 4°C; 5 min) and the pellet was washed with 1 mL of PBS and centrifuged as mentioned earlier. The pellet was suspended in 500 μL of PBS and the fluorescence was assessed by comparing two fluorescence emission 480 nm/530 nm using a flow-cytometer (BD-LSR). Mitochondrial membrane potential (MMP) assay

HL-60 cells (1×106 cells/2 mL/well) were treated with the three extracts at different concentrations for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with Rhodamine-123 (200nM) and kept in the dark for 30 mn. Cells were collected, centrifuged (400 g; 4°C; 5 min), the pellet was washed with 1 mL of PBS and centrifuged as mentioned earlier. The depolarization of mitochondrial membrane was examined by measuring the fluorescence emission shift (red to green) of the Δψm sensitive cationic Rh-123 dye. The fluorescence intensity of 10,000 events was analyzed in FL-1 channel on BD FACS Calibur (Becton Dickinson, USA) flow cytometer. The decrease in fluorescence intensity because of MMP loss was analyzed in FL-1 channel and the change of potential membrane (Δψm) was assessed by comparing fluorescence. DNA content and cell cycle phase distribution

HL-60 cells (1×106 cells/2 mL/well) were treated with extracts at 20, 50, 100 μg/mL for 24 h. They were harvested and washed with 1 mL of PBS, then centrifuged at 400 g for 5 min at 4°C. The pellet was suspended in 100 μL of PBS and 900 μL of hypertonic buffer (PI-25 μg/mL, RNAase- 40 μg/mL, sodium citrate-0.1% and Triton-100X-0.03%) and incubated at 37°C in dark for 20 min. Finally, cells were analyzed immediately on flow cytometer FACS Calibur (Becton Dickinson, USA). The data were collected in list mode on 10,000 events and illustrated in a histogram, where the number of cells (counts) is plotted against the relative fluorescence intensity of PI (FL-2; λem: 585 nm; red fluorescence). The resulting DNA distributions were analyzed by Modfit (Verity Software House Inc., Topsham, ME) for the proportions of cells in G0/G1, S phase, and G2/M phases of the cell cycle [26]. Statistical analysis

The viability experiments were done in triplicates and each data point represents the average of at least 3 independent experiments. The distributions of the data are abnormal. The data was expressed as mean ± SD. In order to carry out statistical analysis, the data was analyzed using SPSS (Version 11.5; SPSS Inc.,) and M.S. Office, Excel software. One way analysis of variance technique was applied to observe the significance between the groups. The post hoc test Duncan’s multiple range test was performed to know the significant difference among the groups. Entire statistical analysis was carried out at p < 0.05. Results Phenolic contents of A. muricata

The results of this study showed that the level of polyphenols, flavonoids and flavonols varied depending on the part of the plant (Table 1). The concentration of these three groups of molecules is higher in the leaves compare other parts of plant extract. The lowest concentration of flavonoids and flavonols was found on the stem barks of A. muricata while its roots show the lower phenols content.

Table 1. Phenolic composition and fifty percent inhibition of extracts of A. murica In vitro anticancer activity Effects of A. muricata extracts on the proliferation of HL-60

In this study we used a microculture assay based on metabolic reduction of MTT to evaluate the cytotoxic effect extracts of A. muricata on HL-60 cells. This technique permitted us to evaluate dose-dependent effect, by linear regression analysis showing acceptable R2 values and correlation coefficients. As shown in the Figure 1, the addition of extracts at different concentration to the cultured cells inhibited dramatically and significantly the proliferation of the cells in a dose-dependent manner. The values of IC50 of the extract after 48 h was between 6–12 μg/mL which is lower than 20 μg/mL (Table 1).

thumbnailFigure 1. Viability of HL-60 cells after 48 h treatment with extracts of A. muricata; (n = 3); (A) HL-60 cells; L (Leaf); R (Roots); T (twigs). Morphological changes of apoptotic treated HL-60 cells with A. muricata extracts

To investigate of A. muricata extracts on the nuclear modification on HL-60 cells, the Hoechst 33258 staining test was performed at different concentrations (20, 50 and 100 μg/mL) after 24 h of treatment. Hoechst 33258 reagent is a membrane-permeable blue fluorescent dye which stained cell nucleus. As observed in Figure 2, the control or untreated cells present the characteristics of healthy cells. They appeared to be intact oval shape and the uniform nuclei were stained with a less bright blue fluorescence. Cells treated with tested extracts exhibited a bright blue color when the concentration of extract increases. These results demonstrated typical features of apoptosis such as cell shrinkage, chromatin condensation, and fragmentation to multiple aggregate of apoptotic bodies and cell decrement (Figure 2). The apoptotic nuclei clearly showed highly condensed or fragmented chromatin. At 100 μg/mL, most of the cells undergo apoptosis and the number of apoptotic bodies increases.

thumbnailFigure 2. Effect of extracts A. muricata on nuclear morphological changes of HL-60 cells. After 24 h of treatment cells staining with Hoechst 33258 incubated for 30 min, and observed under fluorescence microscope. Olympus,Tokyo, Japan; magnification 200×. Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly. Apoptotic cells gradually increased in a dose-dependent manner; (A): Leaf; (B): Roots; (C): twigs. A. muricata extracts induce apoptosis by generation of ROS

To investigate whether extracts of A. muricata inhibit the HL-60 cells by through the generation of ROS, we monitored the redox status of the HL-60 treated cells using the oxidation sensitive fluorescent dye DCFDA. As shown in Figure 3A, B and C, the ROS levels generated after 24 h treatment of HL-60 cells varied with the concentration of extract and the part of plant used in the study. However, the higher ROS levels were found at the concentration of 20 μg/mL (9.99% for twigs), 50 μg/mL (4.08% for Leaf) and 100 μg/mL (3.07% for the twigs) (Figure 3D). At the higher concentration (100 μg/mL), the ROS produced by cells with the extract of the roots and leaves were lower than that of untreated control cells (Figure 3D).

thumbnailFigure 3. Effects of extracts A. muricata on ROS production on HL-60 cells; cells were treated with extract for 24 h followed by staining with DCHFH2-DA (50 μM), incubated for 30 min and the fluorescence in the cells was immediately analyzed using flow cytometry. Data are presented the fluorescence intensity; (A): Leaf; (B): Roots; (C): twigs: (D); Variation of ROS production of extracts of A. muricata; values are expressed as means ± standard error (n = 3). Values affected with different letters are significantly different (p < 0.05) from the control. L: Leaf; R: Roots; T: twigs, Ctl: control; app: apoptic phase. A. muricata extracts disrupt mitochondrial membrane potential in HL-60 cells

After treatment of cells with extracts at different concentration, we observed an increase of fluorescence intensity indicating the mitochondrial membrane depolarization as Figure 4A, B & C. The depolarization of mitochondrial membrane varied with concentration and the extracts. A dose-dependent increase of fluorescence observed is ranging from 17.34% to 98.91% (Figure 4B). Among the extract tested, the twigs extract demonstrated a higher depolarization of mitochondrial membrane (21.75%) at 20 μg/mL while those of leaf and roots showed a maximum depolarization at 100 μg/mL (98.29 and 98.91% respectively). All the extracts at 50 μg/mL exhibited more than 50% of depolarization of mitochondrial membrane (Figure 4D). These results show that extracts induced apoptosis on HL-60 cells after 24 h through the disruption of mitochondrial membrane.

thumbnailFigure 4. Effects of extracts of A. muricata on the integrity of mitochondrial membrane, HL-60 cells were treated different concentrations of extracts, incubated 1 h with 200 nM of Rh-123 and then analyzed by flow cytometry. Data are presented the fluorescence intensity: (A): Leaf; (B): Roots; (C): twigs: (D); Variation of the integrity of mitochondrial membrane by extracts of A. muricata; values are expressed as means ± standard error (n = 3). Values affected with different letters are significantly different (p < 0.05) from the control. L: Leaf; R: Roots; T: twigs; Ctl: control. A. muricata extracts induce a G0/G1 cell cycle arrest in HL-60 cells

The changes in the cell cycle distribution were shown in Figure 5A and the apoptotic cells were counted based on G0 DNA contents. The results show no changes in the cell cycle distribution of the control group, however, the accumulation of cells was found in apoptotic (G0) with significant modification of G2/M and S phases when the concentration of all the tested extracts increase (Figure 5A, B & C). The cell population of G0 phase significantly increased from 2.51 – 96% (leaf), 2.83 – 95% (roots) and 5.01 – 98% (twigs) and in the meantime the proportion G0/G1drastically increased (Figure 5D). The results demonstrated that all the tested extracts induced apoptosis on HL-60 cells through G0/G1 phase cell cycle arrest.

thumbnailFigure 5. Cell cycle analysis of extracts of A. muricata on HL-60 cells. After 24 h, treated cells were incubated with RNAse (40 μg/mL), stained with propidium iodide (25 μg/mL), and analyzed by BD-FACS Caliber flow-cytometer; (A): Leaf; (B): Roots; (C): twigs; Cells distribution after treatment with extracts of A. muricata; values are expressed as means ± standard error (n = 3). Values affected with different letters are significantly different (p < 0.05) from the control. L: Leaf; R: Roots; T: twigs; Ctl: control; app: apoptic phase. Discussion

Natural polyphenols are secondary metabolites produced by plants for their defense against different types of stress, e.g. ultraviolet radiation, aggression of pathogens, low soil fertility, changes of environmental temperature, severe drought, and grazing pressure [27]. The interest on plant phenols is increasing in the recent decade because of their health promoting potential. It is widely known that diets containing an abundance of phenols have protective effects against a variety of diseases, particularly cardiovascular disease and cancer. Phenols from herbal extract are raising great interest as powerful and safe anticancer strategy for their broad range targeting capability and low side effects. Depending on the chemical structure, several beneficial effects of polyphenols and their implications in the human health have been identified including in cancer [28], neuroprotection [6,29], cardiovascular system dysfunction and damage, the metabolic syndrome, diabetes, aging, and different inflammation-related pathologies [27,30,31]. Chemotherapy drugs from polyphenols could improve the survival of cancer patients, with low side effects. Thus, it is urgent to develop novel drugs which are more effective [32]. Novel strategies for determination of natural products with biological activity require the implementation of large-scale screening programs.

The antiproliferative activities of A. muricata have been carried out on several cancer cells with significant positive results [15,21]. Plant extracts with IC50 values ≤ 30 μg/mL are considered pharmaceutically active [33]. Our MTT results (Table 1) indicated that extracts of A. muricata inhibited significantly the HL-60 cells in vitro and can be considered as active according to the suggestions from the National Cancer Institute (NCI) which stated that the IC50 lower or equal to 20 μg/mL can be used as a benchmark for suitable screening cancer drugs from plants and herbs [34]. A dose- dependent inhibitory effect of extracts was also observed in HL-60 treated cells (Figure 1A). Among these extracts the roots exhibited the higher cytotoxic effects than other extracts (Table 1). Our results demonstrated that A. muricata extracts have significant cytotoxic potential on HL-60 cells.

Apoptosis is a crucial mode of programmed cell death, which is an active physiological process to eliminate selectively unnecessary cells [35]. Induction of cell apoptosis in tumor tissue is the best stage for cancer therapy [35]. Apoptosis is a common mode of action of chemotherapeutic agents, including the natural product-derived drugs. Furthermore, induction of apoptosis is recognized as an efficient strategy for cancer chemotherapy and a useful indicator for cancer treatment and prevention. Hence several researchers nowadays have performed apoptotic screening of natural products from herbal extracts in Cameroon [36,37]. Apoptosis involves specific morphological and biochemical changes such as chromatin condensation, membrane blebbing, cell shrinkage, DNA fragmentation, etc. Induction of apoptosis is the key to success of plant products as anticancer agents [38,39]. After 24 h of treatment with A. muricata extracts, the characteristics of apoptotic cells, including the increase of bright blue color of Hoechst 33258 staining was observed (Figure 2A, B & C) as well as evident DNA fragmentations at 100 μg/mL, which are the important hallmarks of apoptosis [40]. The results indicated that all the extracts from A. muricata induce apoptosis of HL-60 cells.

Mitochondrion is an integral part of apoptotic machinery and events such as loss of mitochondrial membrane potential is classical evidence for apoptosis [35]. Usually, the decrease in MMP occurs during the early stage of apoptosis before the cell morphology changes. The sharp decrease in the membrane potential indicates the irreversible occurrence of early apoptosis due to an increase in the permeability of the mitochondrial membrane follow by the release of apoptotic factors, including cytochrome c [41]. Based on the current research, we propose that the following mechanism is responsible for extracts-induced HL-60 cell apoptosis. Briefly, extracts of A. muricata acts through the disruption of membrane mitochondrial to arrest cells in the G0/G1 phase and inhibit cell proliferation. Cell cycle checkpoints are control mechanisms that ensure the proper progression of cell cycle events [42]. In vitro apoptosis assay study presented here (Figure 5A, B, C), showed that extracts from A. muricata induced apoptosis in HL-60 tumor cells in a dose-dependent manner, compared to untreated cells. In this experiment, the extracts of A. muricata induced G0/G1 cell cycle arrest in HL-60 cells at different concentration after 24 h of treatment (Figure 5A, B, C). Therefore, we can suggest that the anticancer effects of A. muricata extract is associated with G0/G1 cell cycle arrest and cell differentiation. The cell cycle analysis revealed that all the extracts of A. muricata can markedly induce a G0/G1 phase arrest in HL-60 cells, but they have low effects on the G2/M phase. Although the induction mechanism of cell differentiation by A. muricata extract is not clear, a block of cell cycle progression at the G0/G1 phase may be closely related to the differentiation.

Most chemopreventive agents known today from plant extracts are subdivided into two categories: (i) blocking agents which inhibit the initiation step by preventing carcinogen activation and (ii) suppressing agents, which inhibit malignant cell proliferation during promotion and progression steps of carcinogenesis [43]. Natural products, including medicinal plants, herbs and spices provide rich resources for anticancer drug discovery [44]. Phytochemical analysis of the extracts of A. muricata was performed in order to identify the chemical nature of the active principles. Quantitative analysis of the extracts revealed the presence of phenolics, flavonoids and trace amounts of flavonols (Table 1). Previous studies, demonstrated the presence of a number of phytochemicals, including phenols as flavonoids, tannins, anthraquinones and steroids in the leaf extracts of A. muricata[45]. Cinnamic acid derivative, Coumaric acid hexose, 5-Caffeoylquinic acid, Dihydrokaempferol-hexoside, p-Coumaric acid, Caffeic acid derivative and Dicaffeoylquinic acid represented phenolics acids group were isolated in its fruits [46]. Intensive chemical investigations of the leaves and seeds of A. muricata have resulted in the isolation and identification of a great number of acetogenins with interesting biological or pharmacological activities, such as antitumoral, cytotoxicity and apoptosis on HCT-116 and HT-29 cancer cells, [16,47]. Hence, previous studies revealed that several acetogenins act as a DNA topoisomerase I poison, arrested cancer cells at the G1 phase and induced apoptotic cell death in a Bax-and caspase-3- related pathways, and inhibited NADH-ubiquinone oxidoreductase (complex I) in mitochondria [21]. It can be suggested that the synergistic effects of phytochemicals present in this plant extracts including acetogenins may be the underlying principle behind the chemotherapy potential agents of A. muricata. These groups of molecules found in a large quantity in A. muricata extracts may act in synergy and might be responsible for their antiproliferation activity through the significant decrease in the mitochondrial membrane potential induction of apoptosis. Conclusions

In conclusion, A. muricata exhibit antiproliferative effects of HL-60 cells by inducing loss of cell viability, morphology changes, loss of membrane mitochondrial potential and G0/G1 phase cell arrest. Our data confirm the potential of A. muricata as an agent of chemotherapeutic and cytostatic activity in HL-60 cells. These data suggest that these extracts have potential for cancer chemotherapy. However, the complete mechanisms underlying the therapeutic effects of the extracts need to be investigated as well as the identification of the active molecules present in the different parts of the plant. Competing interests

The authors declared no potential conflicts of interest with respect to the research authorship and/or publication of this article.

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Food chemistry analysis on annonacae family  Has there been any food chemistry HPLC or mass spec data comparing the quantity of annonacin and other neurotoxins to compare and gauge potential neurtoxic compounds? Infact these compounds could exist in such 27 December 2014

Food chemistry analysis on annonacae family has there been any food chemistry hplc or mass spec data comparing the quantity of annonacin and other neurotoxins to compare and gauge potential neurtoxic compounds? infact these compounds could exist in such

Food chemistry analysis on annonacae family.

Has there been any food chemistry HPLC or mass spec data comparing the quantity of annonacin and other neurotoxins to compare and gauge potential neurtoxic compounds? Infact these compounds could exist in such small quantity in some members of the family that it may not be significant.

I looked on Pubmed and could not find any of this data. What is known in terms of quantity and potential in vitro testing. I mean we could go crazy over all this without really talking numbers. Apples have seeds containing cyanide but that hasnt seemed to keep us from that old analogy "an apple a day......." \

Posted by J 14 May 2012 | 18h22

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10 Amazing Benefits Of Soursop / Graviola Leaves For Skin, Hair And Health 18 December 2014

10 amazing benefits of soursop / graviola leaves for skin, hair and health

Soursop also known as Graviola is a fruit that has its origin in the forests of South America, Caribbean, Africa and Southeast Asia. This is an evergreen broad leaved tree whose every part is useful and has medicinal properties. This fruit is extremely delicious with a sharp aroma and a sweet-sour taste which is basically a combination of the taste of pineapple and strawberry. Recently, it has gained attention and popularity due to its natural cancer cell killing properties. Apart from its anti- cancer properties, it has several other medical benefits.


Soursop leaves are the most beneficial parts of this tree. They have the Acetoginin containing compounds namely bulatacin, asimisin and squamosin. Acetoginin acts as an anti- feedent. Thus, they are often used in killing insects and pests which die by consuming these leaves even in small amounts. Scientific research conducted by The National Cancer Institute has proved that Soursop leaves can effectively attack and destroy cancer cells. In addition to this, they are also used in the treatment of several other diseases.


Health Benefits of Soursop Leaves:

Soursop leaves are rich in several compounds including protein, calcium, fructose, fat, vitamins A and B and the like. Thus, the leaves have excellent medicinal properties making them usable as an ingredient in several herbal health products. The health benefits of soursop leaves are as follows.

  1. Treatment of Cancer:


Soursop leaves can inhibit cancer cells and cure cancer more quickly and effectively than chemotherapy which results in several side effects besides being expensive. In fact, research has proved that soursop has an active ingredient that is 10000 times stronger than chemotherapy in fighting cancer cells. Thus, soursop leaves can treat different types of cancers including prostate, lung and breast cancers. For treatment, boil 10 soursop leaves in 3 cups of water until only one cup of water remains, strain and cool it and drink this concoction every morning for 3-4 weeks to determine improvement in the condition. Soursop leaves cancer treatment is one of the most potent cures till date.

  1. Treatment of Uric Acid:


Eating soursop leaves can greatly help in treating gout. In fact, many alternative medicines use soursop leaves for the treatment of gout. For this purpose, take 6 to 10 soursop leaves which are old but still green and wash them clean. Boil the leaves in 2 cups of water and simmer until one cup of water remains. This concoction should be taken twice a day i.e. morning and evening for maximum benefits.

  1. Treatment of Back Pain:


Back pain is commonly experienced these days, particularly while exercising. Using chemical drugs for back pain can cause side effects. Soursop leaves are an effective herbal remedy for treating back pain without any negative effect. You can boil 20 pieces of soursop leaves in 5 cups of water until only 3 cups of water are left. Drink ¾ cup of this concoction once in a day for relief.

  1. Treatment of Eczema and Rheumatism:

Rheumatic diseases are commonly observed in elderly people, causing great pain. Soursop leaves are a natural treatment for arthritis pain. For this purpose, mashes the soursop leaves until they become smooth and apply on the areas of the body affected by pain due to arthritis and eczema, regularly twice a day.

  1. Treatment of Diabetes:


The limit of normal sugar levels ranges from 70 mg to 120 mg. The nutrients in soursop leaves are believed to stabilize blood sugar levels in the normal range. Besides, the extracts of soursop leaves can be used as one of the natural diabetes remedies. All this makes these leaves beneficial for diabetics.

  1. Boosts the Immune System and Prevents Infections:


The nutrient content of soursop leaves is believed to boost the immune system and avoid infections in the body. Boil 4/5 soursop leaves in 4 cups of water until one cup water remains and drink this concoction regularly once in a day for beneficial results.

  1. Other Benefits:


In addition to the benefits mentioned above, soursop leaves are extremely effective in inhibiting the growth of bacteria, virus, parasites and tumor development. Their healing properties make them capable of being used as an anti-seizure medication. They are also capable of reducing fever and lowering high blood pressure. They help in treating inflammation and swollen feet. They aid in digestion and improve appetite. Soursop leaf consumption on a regular basis helps in improving stamina and facilitating quick recovery from diseases.

Skin Benefits of Soursop Leaves:


Due to their medicinal properties soursop leaves are extremely beneficial for health. As pointed out earlier, they are used in the treatment of some of the deadliest diseases. The leaves offer some skin benefits as well.

  1. Treatment of Boils:

Ulcer is a skin disorder that is characterized by immense pain and even has the risk of catching infection. Boils can occur on the body or on the face, thus interfering with your skin health and beauty. Soursop leaves are a natural remedy to cure ulcers. You can pick some young soursop leaves and place them on the body affected by ulcers.

  1. Treatment of Eczema:

As already stated earlier, soursop leaves can treat eczema in a natural way. You can mash a few soursop leaves and apply it on the affected areas twice a day regularly. This will help in alleviating the pain caused by eczema besides treating it.

A pulp made with fresh soursop leaves and rose water when applied on the skin can be very useful in preventing the occurrence of blackheads and other skin problems too.

Hair Benefits of Soursop Leaves:


  1. Get rid of Lice:

All of us long for healthy and damage free hair. But unfortunately, the unhealthy lifestyle coupled with exposure to harmful chemicals and environmental pollutants is responsible for several hair problems like dandruff, split ends, hair loss, pre mature greying etc. Natural ingredients and herbal products can be very effective in combating these problems. As far as soursop leaves are concerned, much is not known about their benefits for hair. However, soursop leaves have the capability to inhibit the growth of parasites, besides other medicinal properties. Thus, applying a soursop leaf decoction on your hair can help you to get rid of head lice.

Finding soursop leaves in Inida is not easy but if you do find it, pick it up right away!

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Fighting Cancer with Science and Nature 24 November 2014

Fighting cancer with science and nature

Fighting Cancer with Science and Nature Why natural agents that kill cancer cells warrant further investigation. Published on July 10, 2011 by Christopher Lane, Ph.D. in Side Effects 9 inShare email

In the U.S. (and across the West, more generally), we are understandably suspicious of medical hoaxes and scams, given the damage they can cause. Our media and pundits try to weed out medical assertions that are unsupported by science. The Internet, in particular, abounds with dubious products whose untested, often wildly oversold effects can easily persuade the unthinking and the credulous to part with their cash.

Yet as someone who follows and comments on developments in especially psychiatry, I frequently am struck by the faith we place in products that come with FDA approval, but a litany of unpleasant, sometimes risky side effects. When, for example, millions of men across America are willing to "double their risk of hearing loss" and jeopardize their eyesight for an erection, as recent studies warn about those routinely taking Viagra, you know that potency is something we take very seriously. Enough to tune out such warnings and opt unthinkingly for a blue pill, rather than a wealth of natural aphrodisiacs whose effect is basically identical—minus, of course, the nasty health risks. See All Stories In The Wisdom of Nature

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When our knowledge of natural products is also limited, even impoverished, by cultural biases that skew toward pharmaceuticals, our information-base shrinks accordingly. We ignore the well-known medicinal properties of vast amounts of natural products and end up placing enormous faith in pills whose very advertisements are forced to devote significant amounts of time and space to a laundry list of side effects. (The U.S. shares with New Zealand the dubious distinction of being the only Western countries to allow direct-to-consumer advertising of psychotropic pharmaceuticals.)

Because I also spend large amounts of time in South America, in particular Peru, from where I'm blogging this summer, cultural differences in medicine and public health can be especially striking. From here, over the last few weeks, I've been able to follow with intense interest the arguments both for and against naturally existing cancer-fighting agents, such as the leaves of the guanabana tree (hereafter known as "graviola"). From all that I've been able to investigate, from PubMed to the National Cancer Institute at the National Institutes of Health, my sense is that the leaves of this remarkably promising fruit should be getting more rather than less attention from the scientific community. As the studies below indicate, there is already strong evidence that acetogenins in the plant’s leaves target tumors in what scientists call “apoptosis,” or programmed cell death. Unlike in chemotherapy, however, that targeting, according to Zeng et al (1996b), is “relatively nontoxic to noncancerous cells.” In Peru, graviola is routinely part of a cancer-fighting arsenal, along with chemotherapy. In the States the latter prevails, eclipsing most other options.

First, some key background. About three years ago, word spread rapidly on the web about the cancer-fighting properties of "graviola," the leaves of the guanabana tree (Annona muricata), also known as "soursop," "cherimoya," "custard apple," and "Brazilian paw paw." The tree grows in Peru, Colombia, and Brazil, as well as countries in sub-Saharan Africa and South-East Asia with similarly temperate climates. In all of these countries, the fruit is eaten widely; it is often put in shakes and fruit salads, because it is so delicious. Additionally, the leaves and fruit are frequently used to treat viruses, infections, and depression. There is also, apparently, limited production of the fruit in southern Florida.

As millions of people suffer from all kinds of cancers, for which we have treatments such as radiation and chemotherapy (generally, with terrible side effects) but no cure, interest in and demand for graviola, in particular, skyrocketed around 2008 when reports of its efficacy started to rise. At the same time, some small businesses and a few hucksters, generally selling graviola with a host of other products (essiac tea, burdock root, sheep sorrel, blue-green algae, and so on), overstated their medicinal effects, whose accuracy had also been exaggerated in all the hubbub and excitement. Very quickly, a treatment known to kill some cancer cells and tumors (see below) morphed inaccurately into assurance about cancer's cure.

As rapidly as excitement had skyrocketed, skeptics rushed to denounce the "cancer cure" as a fraud and scam afflicting the needy, the desperate, and the gullible. The Federal Trade Commission (FTC) swooped in to fine and close various businesses that had unwisely reported an ability to cure cancer. And in September 2008, Medical News Today publicized the FTC's actions, quoting its director of the Bureau of Consumer Protection, Lydia Parkes, as saying: "There is no credible scientific evidence that any of the products marketed by these companies can prevent, cure, or treat cancer of any kind" (my emphases).

The only problem with Parkes's blanket dismissal of all these elements is that, in the case of graviola at least, her claim wasn't in fact true.

In 1976, as Richard D. McCarthy, MD, reports on the encyclopedic cancer website U.S. Cancer Centers: Cancer Center Information and Research, "The NCI or the National Cancer Institute did some research on the guanabana cancer cure [sic] and came up with some interesting results. The study . . . showed that the leaves and stems of the plant were incredibly efficient at destroying certain cancerous cells in the body."

"Inexplicably," another site notes, "the results [of the NCI research] were published in an internal report and never released to the public. Since 1976, guanabana has proven to be an immensely potent cancer killer in 20 independent laboratory tests, but as of now, no double-blind clinical trials. [However], a study published in the Journal of Natural Products, following a recent study conducted at Catholic University of South Korea, stated that one chemical in guanabana was found to selectively kill colon cancer cells at '10,000 times the potency of (the commonly used chemotherapy drug) Adriamycin. '... The most significant part of the Catholic University of South Korea report is that guanabana was shown to selectively target the cancer cells, leaving healthy cells untouched."

Adds the same site, "A study at Purdue University, Indiana, recently found that leaves from the guanabana tree killed cancer cells among six human cell lines and were especially effective against prostate, pancreatic and lung cancers." The article, "Paw Paw and Cancer: Annonaceous Acetogenins from Discovery to Commercial Products," appearing in Journal of Natural Products 71.7 in 2008, was authored by Dr. Jerry L. McLaughlin of Purdue's Department of Medicinal Chemistry and Molecular Pharmacology. The journal is published by the American Chemical Society and American Society of Pharmacognosy.

In response to the predictably enormous rise in public interest in graviola, Cancer Research UK also released a statement about the alleged cancer "cure" that included these sentences:

"In laboratory studies, graviola extracts can kill some types of liver and breast cancer cells that are resistant to particular chemotherapy drugs. But there haven't been any large-scale studies in humans. So we don't know yet whether it can work as a cancer treatment or not. Overall, there is no evidence to show that graviola works as a cure for cancer."

On the basis of the laboratory studies quoted here, with Cancer Research UK acknowledging that "graviola extracts can kill some types of liver and breast cancer cells," you might think that such preliminary but hopeful results would at least warrant further investigation. As the site states, "we don't know yet," implying a desire for further research, but also essentially acknowledging ignorance because of the rather astonishing absence of "large-scale studies in humans."

What's startling about the reaction in the United States, by contrast, was that the FTC's action overrode the initial findings of the National Cancer Institute. Even more puzzling, the NCI, after establishing those findings, decided against publicizing them and, for reasons unclear to me, against pursuing them further.

Here in Peru, by contrast, the leaves of the guanabana tree—in short, the very parts of it that both the NCI and CR determined do in fact have cancer-fighting properties—are widely considered part of a parallel set of treatments against cancer, along with chemotherapy and the removal of tumors through operations. In the case of one woman I interviewed, the former mayor of a small town (Supe) three hours north of Lima, graviola was noted by her doctor both for helping to kill cancer cells and for greatly boosting the immune system, itself a common victim of chemo, which destroys more than just cancer cells. In both cases, science supports his recommendation.

After an operation to remove a cancerous tumor in one of her ovaries, the woman (whose information I am relaying with her permission) was given a combined treatment of chemotherapy and a dietary supplement of graviola, taken with meals three times a day. (It's taken either in capsule form or as a tea with boiling water; in either case, that should always be in consultation with a doctor or oncologist). One year later, after she had battled intense side effects from chemo—including hairloss, skin damage, and nausea—she wasn't just in remission. Her cancer had completely disappeared. It remains gone to this day, two years later.

Although it is impossible in this case to distinguish clearly where the effects of chemo ended and those of graviola began, a carefully administered large-scale human trial could control for that variable. My point, then, is not to state or even imply that graviola is a "cure" for cancer. Following the science, I am instead urging that the States and other countries fund and advance such research, owing precisely to the initial findings of the National Cancer Institute and, more recently, of Purdue's Cancer Center.

In Peru, to point up the differences and give readers more information, a major chain, Bionaturista, with stores across the capital and surrounding country, grinds the guanabana leaves into powder under laboratory conditions, with all equipment carefully sterilized, and exports it to a client-base around the world. The chain is one of Peru's largest exporters—not bad for a country whose enviable economic boom is owing partly to its vast natural reserves of minerals and natural gas.

Nor, I should add, is graviola alone among natural products in having anticancer properties. In 2006, to cite Wikipedia, "a research team from the Ben Gurion University in Israel found that lemongrass (Cymbopogon citratus) also caused apoptosis (programmed cell death) in cancer cells." A key ingredient in especially Thai and Vietnamese cuisine, lemongrass—which is widely and inexpensively available across South-East Asia, South America, and in certain Western stores and supermarkets—is also routinely used as a delicious, healthful tea. According to the published studies below, its potency in killing cancer cells is dwarfed by that of graviola.

In urging that a lot more funding and research go into investigating why graviola kills certain kinds of malignant cancer cells, apparently I risk the ire of the FTC's Bureau of Consumer Protection. But rather than give a blanket dismissal of graviola and guanabana, perhaps the FTC should heed the findings of both the NCI and Cancer Research UK and look into this further. The health of millions of cancer sufferers and survivors around the world quite clearly depends on it.

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Curing Cancer: Nobel Laureate Otto Warburg 15 November 2014

Curing cancer: nobel laureate otto warburg

As SparkFriends and regular readers of my blog know, my sister lost her battle with cancer on Dec 2, 2010 in a hospice, while I held her hand. She was only 51, & left behind a 12 year old son, an 11 year old daughter, husband and other family.

Otto Heinrich Warburg, with a doctorate of chemistry, and a second doctorate in medicine, was a physiologist and noted biochemist born in 1883 in Freiburg, Baden, Germany. Dr. Warburg won the Nobel Prize in Physiology or Medicine in 1931, and died in Berlin in 1970. He believed in eating organic.

Dr. Warburg was awarded the Nobel prize for his discovery that cancer is caused by weakened cell respiration due to lack of oxygen at the cellular level, and proving cancer thrives in anaerobic (without oxygen), or acidic, conditions. In other words, the main cause for cancer is acidity of the human body. In his Nobel Prize winning study, Dr. Warburg illustrated the environment of the cancer cell. According to Warburg, damaged cell respiration causes fermentation, resulting in low pH (acidity) at the cellular level. Warburg also wrote about oxygen's relationship to the pH of cancer cells internal environment. Since fermentation was a major metabolic pathway of cancer cells, Warburg reported that cancer cells maintain a lower pH, as low as 6.0, due to lactic acid production and elevated CO2. HE PROVED CANCER CANNOT GROW NOR DEVELOP IN BODY ALKALINITY OF 7.36. He firmly believed that there was a direct relationship between pH and oxygen. Higher pH means higher concentration of oxygen molecules while lower pH means lower concentrations of oxygen. A normal healthy cell undergoes an adverse change when it can no longer take in oxygen to convert glucose into energy. In the absence of oxygen, the cell reverts to a primal nutritional program to nourish itself by converting glucose through the process of fermentation. The lactic acid produced by fermentation lowers the cell pH (acid/alkaline balance) and destroys the ability of DNA and RNA to control cell division. The cancer cells then begin to multiply. The lactic acid simultaneously causes severe local pain as it destroys cell enzymes. Cancer appears as a rapidly growing external cell covering, with a core of dead cells.

So we have known the proven cause, prevention & cure of cancer since 1931.

It is rumoured Dr. Warburg was awarded a second Nobel prize but was unable to receive it because he was Jewish and Hitler prevented it from being awarded; the Nobel Prize authorities deny this however. The reason given was that Hitler had prevented all Nobel Prizes from being accepted; however several scientists in Dr. Warburg's laboratory were awarded and did receive the Nobel Prize. In my opinion it's clear to me Dr. Warburg was awarded at least two Nobel Prizes.

Dr. Otto Warburg finished one of his most famous speeches, "The Prime Cause and Prevention of Cancer", with the following statement: “…nobody today can say that one does not know what cancer and its prime cause is. On the contrary, there is no disease whose prime cause is better known, so that today ignorance is no longer an excuse that one cannot do more about prevention.”

Considering fats to be the main contributor to weight gain is a popular misconception that leads to massive confusion and explains why so many overweight people are not succeeding in losing weight. Many people would be shocked to find out that we may gain weight from eating cheese not only because it is rich in fat, but mostly due to its high acidic level. In response to high pH acid, the body creates fat cells to store the acid. Almonds have 70% fat, and pork has only 58%. However, pork has one of the highest acid values, -38, while almonds are alkaline forming, +3. Cucumbers and watermelons are so alkalizing that they can neutralize the acidifying effect of eating beef. This is why it is very important to know the pH index of all foods, showing the food's ability to alkalize the body. Please see the reference links to pH Food Indexes.

The so-called "bad" cholesterol, lipoprotein (LDL) is made by our own liver in order to bind the toxins and deactivate the acidic waste that came from certain food, not to cause arteriosclerosis (3).

Food, stress, mood, and music all alter our pH balance. Anything that is stimulating could leave an acidic residue in our body; any activities that are calming and relaxing could make us more alkaline. Dr. Coldwell believes 80+% of cancers are initiated by or caused by STRESS, which alters our pH balance, which makes us acidic.

A lack of education about dietary pH balance results in confusion among people who are trying to eat healthy and stay alkaline to lose weight and to avoid cancer. Test your pH with litmus paper and you will discover on days you eat leafy greens (kale, collards, Swiss chard, etc) you will be healthily alkaline; on the days you don't, you won't be alkaline, even if you're eating raw. Here is the pH test strip paper I use: TRM009/ItemDetail?n=0

Once cancer has gained a foothold, it does not survive well in the presence of an alkaline cellular pH level, nor in the presence of highly oxygenated cells. As Nobel Laureate Otto Warburg discovered, low cellular oxygen is a primary causal factor for cancer. Perhaps you have heard the name, Dr. Joanna Budwig, or, The Budwig Protocol? Dr. Johanna Budwig of Germany, who passed away in 2003, was Dr. Warburg's protegé in 1951. She continued Dr. Warburg's work and found that IN ORDER FOR PROPER CELLULAR UTILIZATION OF OXYGEN TO TAKE PLACE, OUR DIETS MUST CONTAIN ADEQUATE AMOUNTS OF UNSATURATED FATTY ACIDS. Over a 50 year period, Johanna Budwig had a MEDICALLY DOCUMENTED 90% plus cancer cure rate in Germany with 4500 patients. Some cancer victims that she cured with her protocol were considered terminal. See reference link #5 below if interested in more information on The Budwig Protocol.

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