# data[].attributes.body
"6 Herbs and Spices for Cancer Prevention." Everyday Health. Everyday Health Media, 21 Jan. 2013. Web.

Fritz, H., Seely, D., Flower, G., Skidmore, B., Fernandes, R., Vadeboncoeur, S., ... & Sabri, E. (2013). Soy, red clover, and isoflavones and breast cancer: a systematic review. PloS one, 8(11), e81968.

"Red Clover." Benefits of Health Foundation. Jon Barron, 2016. Web.

"Red Clover." Memorial Sloan Kettering Cancer Center. Memorial Sloan Kettering Cancer Center, 2016. Web.

"Red Clover." University of Maryland Medical Center. University of Maryland Medical Center, 24 Mar. 2015. Web.

Vadeboncoeur, Sarah. "Red Clover (Trifolium Pratense)." / Herbal Products / CAM-Cancer. Complementary and Alternative Medicine for Cancer, 29 Jan. 2015. Web.

# data[].attributes.attachment_link.description
I found this on Facebook, from herbsandoilsworld.com. And, red clover blossoms taken internally have been found to reduce inflammation of the lungs and liver and can help treat skin conditions such as eczema and psoriasis. You can buy red clover blossoms from this page ( Herbs and Oils) on Mountain Rose Herbs.

# data[].attributes.attachment_link.title
Crimson and Clover - Tommy James & The Shondells

# data[].attributes.attachment_link.readable_text
Heidi Fritz,

Dugald Seely,

Gillian Flower,

Becky Skidmore,

Rochelle Fernandes,

Sarah Vadeboncoeur,

Deborah Kennedy,

Kieran Cooley,

Raimond Wong,

Stephen Sagar

Subject Areas

For more information about PLOS Subject Areas, click here.

We want your feedback. Do these Subject Areas make sense for this article? Click the target next to the incorrect Subject Area and let us know. Thanks for your help!

Citation: Fritz H, Seely D, Flower G, Skidmore B, Fernandes R, Vadeboncoeur S, et al. (2013) Soy, Red Clover, and Isoflavones and Breast Cancer: A Systematic Review. PLoS ONE 8(11): e81968. https://doi.org/10.1371/journal.pone.0081968

Editor: Toshi Shioda, Massachusetts General Hospital, United States of America

Received: September 19, 2013; Accepted: October 18, 2013; Published: November 28, 2013

Copyright: © 2013 Fritz et al. 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 author and source are credited.

Funding: This project was funded by a Knowledge Synthesis grant from the Canadian Institutes of Health Research. DAK was supported by a career grant from the Sickkids Foundation. No other sources of funding were accessed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Breast cancer accounts for almost one third of cancers diagnosed among women. In the United States, there were approximately 288 thousand new cases expected for 2011 [ 1]. Breast cancer is also the second leading cause of cancer death among women, with nearly 40 thousand attributable deaths expected in 2011 in the US [ 1]. Dietary interventions are emerging as increasingly important strategies for reducing risk of developing breast cancer or recurrence [ 2, 3]. Among breast cancer survivors, for instance, the Women's Healthy Eating and Living (WHEL) study found that interventions with a diet high in fruits and vegetables, dietary fibre, and low in saturated fat reduced recurrence by 31% among women without hot flashes compared to the control group [ 4], and that higher vegetable intake, particularly cruciferous vegetables, may have enhanced the effect of tamoxifen, with a 44% reduction in recurrence [ 5]. If shown effective, these and other dietary strategies represent an important way for women to reduce their cancer risk, or for breast cancer patients to reduce recurrence and safely augment the effects of cancer treatment. Soy has emerged as a specific food that may reduce breast cancer risk [ 6], and is among the most commonly used complementary medicines utilized by breast cancer patients seeking to reduce risk of recurrence [ 7, 8]. There remains considerable controversy, however, as to its safety, particularly in breast cancer survivors due to purported estrogenic effects [ 9].

Soy, also known as Glycine max, contains the class of phytoestrogens known as isoflavones, specifically, genistein, daidzein, glycitein, biochanin A, and formononetin [ 9]. Isoflavones resemble 17-beta-estradiol in structure, and as such are able to bind the estrogen receptor (ER) in vitro [ 10], behaving much as a natural selective estrogen receptor modulator (SERM) [ 9]. For instance, the soy isoflavones have been found to exert partial ER agonist and antagonist activity depending on local estrogen concentrations, with antagonist properties in concentrations similar to premenopausal levels, and agonist properties with postmenopausal levels [ 9, 11- 13]. Much of the present debate around soy stems from conflicting in vitro and animal evidence. Some studies show that soy isoflavones can increase tumor cell proliferation [ 14], while other studies show the opposite [ 15- 18]. The effects of isoflavones in human systems promise to be equally complex [ 19]. Furthermore, in about 30% of the population, daidzein can be further metabolized in the gut to equol, a metabolite with higher affinity for ER-ß [ 20- 22]. Finally, in addition to ER mediated activity, soy exerts ER-independent effects, including inhibition of vascular endothelial growth factor (VEGF), and proapoptotic effects; genistein in particular inhibits tyrosine kinase and induces the tumor suppressor protein, PTEN [ 23- 25].

Like soy, red clover contains the isoflavones genistein, daidzein, biochanin A, and formononetin, however, soy contains higher amounts of genistein and daidzein, while the dominant isoflavones in red clover are biochanin A and formononetin [ 26- 29]. In vivo, formononetin is metabolized to daidzein [ 30], which may be metabolized to equol among equol producers [ 31- 33]. There are several commercial extracts of red clover, marketed for the treatment of menopausal symptoms including hot flashes (Promensil®), as well as for menopause related health concerns such as bone loss and dyslipidemia (Rimostil®); Trinovin® is marketed for men's health, specifically for benign prostatic hypertrophy. Promensil® contains 26mg biochanin A, 16mg formononetin, 1mg genistein, and 0.5mg daidzein per tablet (~40mg total) [ 27]. Other commercial products such as Rimostil® and Trinovin® contain slightly varying amounts, but are still largely comprised of biochanin A and formononetin [ 27]. Red clover also contains coumestrol, a coumestan that has been less well characterized, however, it is present in very low amounts such that its net contribution to red clover's purported estrogenic effect is questionable [ 30, 34].

To better elucidate the effect of soy, red clover, or isoflavones from these plants on breast cancer, we conducted a systematic review of soy and red clover as used by breast cancer patients or those at risk of breast cancer, assessing their impact on the risk of primary breast cancer or risk of recurrence. We also assessed the impact of isoflavones on surrogate endpoints for predicting breast cancer risk, including circulating estradiol and effects on estrogen responsive tissues such as the breast, endometrial, and vaginal tissues. Finally, we assessed the efficacy of isoflavones in treating menopausal symptoms in patients who have undergone breast cancer treatment.

Methods

Search strategy

Electronic search strategies were developed and tested through an iterative process by an experienced medical information specialist in consultation with the review team. Using the OVID platform, we searched Ovid MEDLINE®, Ovid MEDLINE®In-Process & Other Non-Indexed Citations, EmbaseClassic+Embase, and AMED (Allied and Complementary Medicine). We also searched the Cochrane Library on Wiley (including CENTRAL, Cochrane Database of Systematic Reviews, DARE, HTA, and NHS EED). The strategy was peer reviewed prior to execution by an experienced information specialist using the PRESS Checklist [ 35]. No amendments were suggested.

Strategies utilized a combination of controlled vocabulary (e.g., Soybeans, Phytoestrogens, Breast Neoplasms) and keywords (soy, plant estrogen, breast cancer). Vocabulary and syntax were adjusted across databases. Searches pertaining to soy and soy isoflavones were performed in May 2011 and updated in March 2013; there were no language or date restrictions on any of the searches. Searches pertaining to red clover and red clover isoflavones were performed in October 2011 and updated in December 2012. Additional references were also sought through hand-searching the bibliographies of relevant items. Specific details regarding the searches appear in Table S1.

Inclusion criteria

For inclusion, evidence had to come from clinical trials or observational studies in humans. Human trials had to: a) assess the safety and/ or efficacy of soy or red clover or isoflavones from these plants in breast cancer patients for the purposes of treatment or secondary prevention, or the reduction of side effects associated with chemo- or radiation- therapy; alternately, human trials had to: b) assess the effect of soy or red clover or isoflavones from these plants on risk of primary breast cancer among women without a history of previous breast cancer. Clinical surrogate studies were included if they examined endpoints directly related to breast cancer risk, pathogenesis, or objective markers assessing healthy bodily function such as hematological function in breast cancer patients. All types of breast cancers (carcinoma in situ, invasive breast cancer) were included.

Observational studies had to report on risk of primary breast cancer or breast cancer recurrence associated with soy or red clover consumption compared with non-consumption, in a prospective or retrospective design. In vitro and in vivo studies were excluded due to the high risk for confounding and previous work on natural health products (vitamin A) showing a lack of correlation between preclinical and clinical results [ 36]. Due to the nature of soy as a commonly consumed food and red clover as a non-dietary item, there were limited observational studies of red clover consumption expected or identified. Therefore these studies focus solely on soy.

Data extraction

We piloted data extraction forms and conducted extraction independently in duplicate to assess inter-researcher reliability (HF, RF, GF, SV). No major inconsistencies in data extraction were found. Both quality and efficacy data were extracted. Extraction sheets were prepared based on the Consolidated Standards of Reporting Trials (CONSORT) statement for clinical trials and the Newcastle-Ottawa scale (NOS) for observational studies [ 37- 39]. RCTs were assessed for bias using the Cochrane Risk of Bias tool[ 40].

Results

A total of 4179 records were screened, and 131 records were included: from the soy literature search, 2867 records were screened, of which 127 were included for full analysis and review. From the red clover search, 1312 records were screened, of which four were included for full review. Figure 1 shows a flowchart of the literature search and study selection. We consider the evidence pertaining to soy and red clover independently below.

Soy

Nested Case Control Studies.

A total of 10 nested case control studies were included pertaining to soy, shown in Figures 2-3. The nested case control studies were embedded within larger cohorts, which are also reported in their entirety as prospective cohort studies below. These were the Shanghai Breast Self-Exam study, various arms of the EPIC study, and the Multiethnic cohort in Hawaii [ 85- 92]. As with case control studies, these studies primarily assessed the risk of developing breast cancer associated with soy intake, with mixed results.

Of eight studies assessing risk of breast cancer, one showed significantly reduced risk associated with higher plasma genistein [ 90]. Grace et al reported a small but significant increase in risk of breast cancer associated with higher serum daidzein and serum and urinary equol levels; this was expressed as a log ₂ odds ratio (associated with doubling of exposure) and as a different measure was not included in the forest plots [ 88]. Nonetheless, these findings indicated a 30-45% increased odds of breast cancer with a doubling of daidzein (equol precursor) and equol levels. In this study, 39% of the population were equol producers. No significant associations were found for other isoflavones including genistein, glycitein, or O-MDA (O-desmethylangolensin, another daidzein metabolite).

Five studies showed no significant effects in either direction. One study examined mammographic density as a surrogate of breast cancer risk, however with mixed findings [ 85]. While cases were shown to have higher breast density at all ages compared with control subjects, and this was significantly associated with soy intake during adulthood (-8.6%, p=0.04), the validity of equating this association with increased risk of breast cancer was undermined by associations between body mass index and breast density. Leaner women with BMI <25 had greater percent density, but lower risk of breast cancer.

Uncontrolled Trials.

We included 11 uncontrolled trials examining the effect of soy on safety outcomes relevant to breast cancer risk and progression [ 118, 119]. These are summarized in Figure 5 alongside the RCTs, with additional details available in Table S4. These studies showed no evidence of harm from consumption of soy.

Randomized Controlled Trials.

Of the 38 randomized controlled trials, five assessed the efficacy of soy for hot flashes among breast cancer survivors [ 127- 131], while 34 assessed safety parameters in healthy women [ 131- 164]; one study assessed both hot flashes and safety parameters and hence is counted twice [ 129]. The five RCTs assessing hot flashes or menopausal symptom scores all found reduced symptoms in both the treatment and the control groups, between 15% to 50% compared to baseline [ 129, 131], however with no significant difference between them.

A total of nine RCTs assessed the impact of soy consumption on estrogen metabolism as measured through urinary excretion of urinary metabolites. Of these, five reports of six studies found no significant change in 2-hydroxyestrone, 16α-hydroxyestrone, or 2:16α hydroxyestrone ratio compared to the control group (p>0.05 for all) [ 148, 149, 156, 161, 164]. The remaining three studies found significant increases in 2-hydroxyestrone or 2:16α hydroxyestrone ratio [ 141, 152, 154], which has been thought to represent an anticancer shift in estrogen metabolism. Nettleton found that consumption of soy protein at a dose of 0.64mg isoflavones/kg daily had no impact on urinary estrogen metabolites in the group as a whole; however there was a significant increase in 2-hydroxyestrone (p=0.01) and 2:16α hydroxyestrone ratio (p=0.04) among subjects who were equol producers [ 141]. Xu reported a significant decrease in 4-hydroxyestrone (p<0.05) and an increase in 2:16α hydroxyestrone ratio (p<0.05) among subjects consuming soy protein isolate (65 or 132g daily) compared to those consuming a low-isoflavone protein isolate (control) [ 152]. Lu reported similar results from consumption of 36oz soymilk delivering 158mg isoflavones daily, with significant increases in 2-hydroxyestrone and 2:16α hydroxyestrone ratio compared to subjects consuming an isoflavone-free soymilk [ 154].

Three studies looked at molecular markers of breast cell proliferation or genetic markers of breast cancer risk. One study found no change in BRCA 1 and 2 mRNA level due to soy consumption, however there was a decrease over time in the placebo group, yielding a significant difference between groups (p<0.001) [ 136]. One study of women with either benign or malignant breast disease, found that consumption of 60g soy protein delivering 45mg isoflavones daily increased in vivo markers of breast epithelial proliferation: thymidine labeling index showing the number of cells in S-phase (p=0.026) and progesterone receptor expression (p=0.04), both increased after two weeks compared to baseline [ 157]. On the other hand, a recent study found that consumption of an isoflavone supplement (235mg daily) for six months had no effect on Ki-67 labeling index compared to placebo, although there was a significant decrease in both treatment and placebo groups compared to baseline [ 160]. There was also no change in the rate of atypical cytology.

Risk of bias.

RCTs were assessed as having a moderate risk of bias, with inadequate reporting of random sequence generation and allocation concealment (selection bias) in 18 and 32 of 43 unique RCTs, respectively. The majority of RCTs showed low risk of performance bias, detection bias, attrition bias, and reporting bias; description of blinding of participants, blinding of outcome assessment, complete outcome data and reporting was adequate in over 88% of the studies. The risk of bias across studies is shown in Figure S5.

Interactions.

Seven studies included in our review investigated the effect of soy in combination with hormonal therapies: tamoxifen and aromatase inhibitors. Four cohort studies [ 100, 103, 106, 112] and three RCTs [ 128, 130, 131] reported no significant differences with respect to treatment outcomes or rates of adverse effects associated with use of soy among women who were receiving tamoxifen therapy. In one RCT, vaginal spotting was reported in four subjects in the soy group compared to one in the placebo group, but with such low numbers, the statistical significance of this finding was not reported [ 130].

Of the cohort studies, the WHEL cohort exhibited relatively low soy isoflavone intake (≥6.3mg total isoflavones) and yielded null results: HR for recurrence among tamoxifen users was 0.59 (0.27-1.29) [ 100]. Guha et al found no significant effects overall associated with intake of either ~13mg genistein, ~9.5mg daidzein, or ~800mg glycitein, however there was a trend toward a protective effect among tamoxifen users, p trend =0.05 [ 103]. Shu et al examined 5042 breast cancer survivors, roughly half of whom were on tamoxifen (n=2262), and found that both soy protein intake (>15.31mg/d) and total isoflavone intake (>62.68mg/d) were protective against recurrence and death, HR recurrence 0.66 (0.52-0.84) for protein, and HR 0.74 (0.59-0.95) for isoflavones [ 112]. When analyzed by receptor status, these associations remained significant only among women with ER+ status [ 112]. Kang et al found decreased risk of recurrence among post-menopausal women consuming >42.3mg isoflavones in a cohort of breast cancer patients, all of whom were on tamoxifen or aromatase inhibitors, AHR 0.67 (0.54-0.85) [ 106].

One cohort study examined soy consumption by post-menopausal women who were on anastrozole, and found decreased risk of recurrence among those with an intake of >42mg/d isoflavones, AHR 0.65 (0.47-0.85) [ 106].

Red Clover

Human trials.

Two RCTs pertaining to red clover were included [ 165, 166]. These investigated the effects of the proprietary red clover extract, Promensil®, on hot flashes or estrogen-responsive tissues as a surrogate of breast cancer risk, among high risk populations.

In brief, Atkinson et al assessed the effect of red clover on mammographic density as well as a panel of other markers of estrogenic activity, in 205 women with an increased risk of breast cancer due to their breast density pattern (Wolfe P2 or DY mammographic breast patterns); there was also a secondary assessment of hot flashes [ 165]. Participants were randomized to receive 40 mg red clover isoflavones (Promensil®) or placebo daily for one year.

Powles et al investigated the effect of red clover in 401 women with a family history of breast cancer (at least one first degree relative affected), assessing circulating FSH, endometrial thickness, mammographic density, and bone density [ 166]. Participants were randomized to receive 40mg red clover isoflavones (Promensil®) or placebo for three years.

Hot flashes.

Results of the study by Atkinson showed no significant changes in hot flash score (p=0.88) or mean number of daily hot flashes (p=0.41) when groups were compared [ 165]. Nonetheless, it should be noted that this was a secondary endpoint, and that this study included women in whom the severity of menopausal symptoms was already low at baseline; menopausal symptoms were not present in all subjects. Baseline mean number of hot flashes per day was 2.1 +/-2.7 for the treatment group and 2.5 +/-3.0 in the control group. There was a comparable decrease of -0.8 +/-2.1 at 12 months for the red clover group and -1.0 +/-1.8 for the control group (p=0.41), which however was not significant. Similar non-statistically significant results were found for the menopausal symptom score, a composite of 21 symptoms including night sweats, palpitations, tension/ nervousness, irritability, insomnia, and mood alterations scored for severity. Adverse events (AE) were not reported.

Risk of breast cancer.

Neither of the RCTs reported on breast cancer incidence rates, however surrogate markers of estrogenic activity were examined. Atkinson reported no significant changes in estradiol, FSH, or LH over the one year period: estradiol increased by 14.0 pmol/L in the red clover group compared to a decrease of 0.9 in the placebo group, however this was not significant, p=0.49); FSH decreased 4.2 IU/L in the red clover group compared to a decrease of 2.9 in the placebo group, p=0.83; LH decreased 4.0 IU/L in the red clover group compared to a decrease of 4.2 IU/L in the placebo group, p=0.71; and tyrosine kinase increased 1.62 units of activity/ µg protein in the red clover group compared to an increase of 0.90 in the placebo group, p=0.16 [ 165].

Neither of the two RCTs reported any significant changes in mammographic density among both pre- and post-menopausal women [ 165, 166], and Powles found no significant changes in endometrial thickness between groups [ 166], though neither of these markers are considered to be highly specific or sensitive predictors of breast cancer risk. Atkinson reported a significant interaction between treatment group allocation and the ESR1 polymorphism with respect to effect on mammographic breast density, with decreases of -3.4 (+/-9.7) and -5.2 (+/- 12.0) percent among the CC and CT genotypes respectively, and a 1.4 (+/-12.3%) increase in the TT group ( P = 0.009) [ 165]. ESR1 codes for the estrogen receptor α, and variations may be associated with risk of breast cancer [ 167]. For instance, CC and CT genotypes may be associated with a small reduction in risk of breast cancer compared to TT: CC vs. TT: OR 0.92, 95%CI 0.86-0.99, and CC/CT vs. TT: OR 0.95, 95%CI 0.89-1.00 [ 167].. It is difficult to predict if or how a possible increase in breast density might affect breast cancer risk among TT carriers.

Adverse Events.

Atkinson did not report adverse events. Powles reported adverse effects, which most commonly included breast abnormality, "skin related symptoms" (not described), and other minor adverse events, however these were equally distributed between red clover and placebo groups [ 166].

Risk of Bias.

According to the Cochrane risk of bias tool, the trial reported by Powles was assessed as having low risk of bias in all of the categories, except one for which information about detection bias was not provided [ 166]. The trial by Atkinson was assessed as having low risk of bias in approximately half of the categories but failed to provide adequate information to assess for selection bias, detection bias, and attrition bias [ 165]. Overall, the RCTs were assessed as having low to moderate risk of bias.

Discussion

Among studies included in our review, case control studies showed a stronger association between soy and reduced risk of breast cancer. As shown in Figures 2 and 3, case control studies were much more likely to report significant protective associations between soy intake and risk of breast cancer, while prospective studies were less likely to do so. The reasons for this are unclear. Although not shown here, we conducted subgroup analysis according to the method of exposure assessment to assess for the possibility of recall bias. Our analysis showed no clear separation however, between studies utilizing food frequency questionnaires, structured interviews, or objective assessments of blood or urinary isoflavone concentrations. It is possible that cohort studies were not long enough in duration to prospectively capture the true effect of long term soy exposure.

Several factors influence the biological activity of soy isoflavones in the body. First, soy isoflavones show selectivity toward ER-ß over ER-α [ 168- 175]. This is important because ERß appears to be associated with antiproliferative, anticarcinogenic effects, while ERα is thought to promote carcinogenesis, and is the form measured clinically in the treatment of breast cancer patients [ 176, 177]. In the breast, ERß is found in ductal and lobular epithelial cells as well as stromal cells, while ERα is found only in epithelial cells and not stroma of the breast [ 177]. Moreover, ERα is the receptor used to classify ER positive breast cancer, and the one through which tamoxifen exerts its antiproliferative effects [ 176, 178]. Some have suggested that ERß functions as a possible tumor suppressor gene, pointing out evidence that ERß may control ERα-induced proliferation, and that expression is lost in many breast tumors [ 177, 179, 180]. If soy preferentially activates ERß, this may explain its chemopreventive effects.

Placed in this context, the clinical data reviewed by our study, which demonstrates a lack of any clear pro-estrogenic effects from use of soy, is quite noteworthy. We found no effects on circulating estradiol, and no measurable effects on estrogen-sensitive target tissues, such as breast tissue (density) and endometrium. In addition, our findings are in agreement with those reported by Hooper et al in a 2009 meta analysis of 47 trials [ 31]. Hooper found that soy did not significantly affect estradiol, estrone, or sex hormone binding globulin (SHBG), although amongst pre-menopausal women there was a significant 20% decrease in LH and FSH (p=0.01, 0.5 respectively) [ 31]. In post-menopausal women there were no significant effects on any hormone including estradiol and estrone, with a small nonsignificant 14% increase in estradiol based on 21 studies (p=0.07) and a nonsignificant decrease in estrone. A second meta analysis similarly found no significant effects from soy on breast density, also considered a surrogate of breast cancer risk [ 183].

Our review revealed no clear modification of soy's effect based on menopausal status or ER+/- status in breast cancer patients in large observational studies. However we did find variation according to geographical locale; studies conducted in Asian countries more often reported chemopreventive effects compared to studies in Western countries, which more often reported null results. We attribute this to a difference in soy consumption between these areas. The traditional Japanese diet contains between 6-11g of soy protein and 25-50mg isoflavones; top percentiles of soy intake in Asian studies consume as much as 20g soy protein or 100mg isoflavones per day [ 188], while the cut off for the top quartiles of intake among Western populations is in the range of a few hundred micrograms (mcg) per day [ 52, 74].

The suggestion has been made that the effect of soy depends on genetic variations present in Asian populations. Nechuta et al conducted an analysis of the effect of soy on breast cancer recurrence, investigating the possibility of ethnic variations [ 189]. They reported that in cohorts of American women, after elimination of women of Asian-American descent, the inverse association between soy and breast cancer recurrence remains, undermining the suggestion that the effect of soy is dependent upon genetic difference between ethnicities [ 189].

Strengths

Our review is broad in its scope, assessing soy in the context of breast cancer from several perspectives, including risk of breast cancer, risk of recurrence, estrogenic effects, and risk of interactions with tamoxifen and other hormonal therapies. Our findings are generally in agreement with those of the American Cancer Society, suggesting that moderate amounts of soy intake (up to 3 servings per day) is likely safe for consumption by women with breast cancer [ 190].

Author Contributions

Conceived and designed the experiments: HF DS DK KC RW SS DF. Performed the experiments: HF GF BS RF SV. Analyzed the data: HF GF KC ES DS DF. Contributed reagents/materials/analysis tools: DF ES. Wrote the manuscript: HF DS GF BS RF SV DK KC RW SS ES DF.

# data[].attributes.attachment_link.title
Soy, Red Clover, and Isoflavones and Breast Cancer: A Systematic Review

# data[].attributes.attachment_link.description
BackgroundSoy and red clover isoflavones are controversial due to purported estrogenic activity and possible effects on breast cancer. We conducted a systematic review of soy and red clover for efficacy in improving menopausal symptoms in women with breast cancer, and for potential impact on risk of breast cancer incidence or recurrence.

# data[].attributes.body
A Systemic Review conducted by Heidi Fritz, Dugald Seely ,Gillian Flower,Becky et. all. done in November 28 did a review of soy and red clover for its effectiveness in improving menopausal symptoms in women with breast cancer, and for impact on the risk of breast cancer incidence or recurrence. The study consisted of 4,179 records included in these were 131 articles: 40 RCTs, 11 uncontrolled trials, and 80 observational studies. There was a small amount of evidence that seem to suggest that red clovers ability to influence breast density may also slightly decrease the women’s risk for cancer. It did not show any evidence to support its effectiveness is treating or curing breast cancer. In respect to the similar isoflavones found in soy the study concluded that they were effective in preventing breast cancer and/ or the reoccurrence of it when consumed in large amounts(Fritz, H., Seely, D., Flower, G., Skidmore, B., Fernandes, R., Vadeboncoeur, S., ... & Sabri, E. (2013)).

# data[].attributes.body
No serious side effects have been noted but it has been shown to induce Nausea, Headaches and rashes. In animals that consumed large amounts it demonstrated its ability to cause infertility.

(University of Maryland Medical Center, 24 Mar. 2015)

# data[].attributes.body
·Do not use if you are pregnant, thinking of becoming pregnant or breast-feeding

·It should be used with caution in patients with thyroid disorders, red clover have demonstrated and increase in thyroid hormone activity.

·PT/PTT should be done prior to initiating therapy and the plant should not be used with patients who have a history or bleeding disorders.

Used with caution in patients with estrogen sensitive breast cancer.

(Vadeboncoeur, Sarah, 2015)

# data[].attributes.body
Drug-to-drug interactions:

·Do not take with anticoagulants; red clover heightens the blood thinning effects of anti-coagulants
·Since Red clover have estrogen-like abilities, It should not be taken with hormone replacement therapies or bill control pills

·Red clover have been shown to interfere with Tamoxifen

·Do not take with Methotrexate

(University of Maryland Medical Center, 24 Mar. 2015)

# data[].attributes.body
Available forms And Dosage:

Dried leaves or capsules, in tincture or extract form, and as a topical treatment: The patient should consume 40-85 mg of the pure extract per day. This can be either orally or topically.

Dried herb: Place 1 to 2 tsp dried flowers per 8 oz. of boiling water let steep and them consumer over a timespan of a 1/2 hour. Do this up to 3 times a day.

Powdered herb (available in capsules): Take 40 to 160 mg per day, or 28 to 85 mg of pure red clover isoflavones

Tincture (1:5, 30% alcohol): 3 to 5 mL taken 3 times per day. This can be added to water or in tea.

Fluid Extract (1:1): 1 mL taken 3 times per day. This too can be added to water or tea

Topical treatment: Apply an infusion, liquid extract, or ointment containing 10 to 15% flower heads; apply as needed unless irritation develops.

(University of Maryland Medical Center, 24 Mar. 2015)

# data[].attributes.body
In laboratory studies, the extract from Red clover has demonstrated its ability to fight prostate cancer through its Isoflavones. The isoflavones in the extract inhibits the production normal prostate cells and increase the resistance of prostate cancer cells. Although there is conflicting evidence in whether or not Red clover aides In fighting breast cancer or progressing it, Red clover does induce apoptosis. It does this by use of Formononetin, an isoflavone that activates the Ras-p38 mitogen-activated protein kinase in ER-positive breast cancer cells. Since there is such conflicting evidence it is not a recommended treatment (Memorial Sloan Kettering Cancer Center 2016). The plant also contains genistein, a biochemical that prevents tumors from getting the nutrients in the blood necessary for survival. Additionally Red Clover contains coumestans in the form of biochanin which aide in its cancer fighting abilities (Jon Barron 2016).

# data[].attributes.body
Scientific name: Trifolium pretense

Common names: red clover, cow clover, meadow clover, wild clover

History:

·         a wild plant belonging to the legume family

·         Grows in the wild meadows of Asia and Europe

·         Used to treat whooping cough, prostate cancer, non-estrogen sensitive breast cancer, psoriasis, respiratory problems and menstrual symptoms

(University of Maryland Medical Center, 24 Mar. 2015)

# data[].attributes.attachment_link.title
Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. - PubMed - NCBI

# data[].attributes.body
List of Questions to Ask Prior to Beginning Herbal Medicinal Treatments that may indicate counterfeit treatment:

1. Does the treatment promise a cure for ALL cancers?

2. Are you advised to not use recommended or standard treatment

3.  Is the treatment or drug offered by only one person or clinic?

4. Does the treatment require you to travel to another country?

5.  Are these terms used? o “Miracle Cure o “Scientific Breakthrough o “Secret Ingredient o “Ancient Remedy”

6.  Do the promoters attack the medical or scientific community?

7.  Are there personal miracle stories of amazing results, but no actual scientific evidence?

8.  Is the promotion of the product done by mass media and not by scientific journal?

# data[].attributes.body
American Cancer Society recommended websites for RELIABLE information on CAM therapies:

National Cancer Institute provides information on CAM therapies: www.cancer.gov/cancertopics/treatment/cam

NCCAM – National Center for Complementary and Alternative Medicine provides information on CAM therapies and clinical trials: http://nccam.nim.gov

Memorial Sloan Kettering Cancer Center provides information on herbs and botanicals useful for cancer patients: www.mskcc.org/mskcc/html/11570.cfm

US Dept. of Agriculture, Food and Nutrition information center for information on supplements, vitamins and minerals for cancer patients: http://fnic.nal.usda.gov

National Council Against Health Fraud information on health related fraud and quackery: www.ncahf.org

US FDA provides information and safe use of herbs and dietary supplements, side effects of dietary supplements: www.fda.gov/Food/DietarySupplements/default.htm

United States Federal Trade Commission informs the public about scams and unproven treatments and also helps people log complaints that can lead to further investigation: www.ftc.gov/curious

Quack Watch is an international network of people concerned about health related frauds, myths and misconduct: www.quackwatch.com

retrieved from:

http://www.cancer.org/treatmentsandsideefects/complementaryandalternativemedicine/complementary-and-alternative-methods-for-cancer-management

# data[].attributes.body
Power Point

# data[].attributes.body
Banu, M. (2014). Cancer fighting herbs. Journal of Pharmaceutical Sciences and Research. 6(1), pp. 38-40.

Braun, L. & Cohen, M. (2015). Herbs and Natural Supplements, an evidence-based guide, volume 2. Elsevier, Australia.

Parvez, A., Sanjita, D., Basu, S., & Saumya, D. (2012). Herbs against cancer: an update, Journal of Drug Delivery & Therapeutics, 2(2), pp. 34-38.

Sakarkar, D. & Deshmukh, V. (2011). Ethnopharmacological review of traditional  Medicinal plants for anticancer activity, International Journal of PharmTech Research, 3(1), pp. 298-308.

# data[].attributes.body
Saffron- Fun Facts - uwlax.edu. (n.d.). Retrieved October 23, 2016, from https://bioweb.uwlax.edu/bio203/s2012/geenen_rach/facts.htm

Tarvand Saffron (2005), “What is Saffron?”, Tarvand Saffron Company [January 10, 2006].

What information can you give me on growing saffron ... (n.d.). Retrieved October 23, 2016, from https://attra.ncat.org

Bolhassani, A., Khavari, A., & Bathaie, S. Z. (2014). Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochimica Et Biophysica Acta (BBA) - Reviews on Cancer, 1845(1), 20-30. doi:10.1016/j.bbcan.2013.11.001

What's the right dose of saffron? | NutritionFacts.org. (n.d.). Retrieved October 23, 2016, from http://nutritionfacts.org/questions/how-do-you-dose-saffron/

# data[].attributes.body
According to the study, “Saffron and natural carotenoids: Biochemical activities and anti-tumor effects”, the carotenoids in saffron reduce the potential growth of certain cancer cells in the body.

# data[].attributes.body
A key ingredient in bouillabaisse is saffron. Saffron has the reputation of being the most expensive spice by weight in the world. The stigma of a little purple perennial crocus flower, it must be gathered by hand during a harvest that lasts just a couple of weeks in the fall, and there are only three stigmas per blossom. It takes about 75,000 flowers to yield a pound of saffron. Fortunately, a pinch (about 20 threads) is usually all it takes to impart saffron's distinctive yellow color and vaguely metallic, dried alfalfa hay and bittersweet wildflower-honey flavor. Saffron is featured in Spanish and Indian cooking; it's often a major component of curry powders; Iran, Greece, Morocco, and Italy also harvest and use saffron, too.

# data[].attributes.attachment_link.readable_text
A classic French shellfish and fish stew recipe, prepared with the freshest possible seafood, caught and served the same day. Served with a spicy sauce rouille.

Photography Credit: Elise Bauer

Bouillabaisse is a classic French dish from southern France, in particular, of the port town Marseille. It requires many different varieties of fish, and traditionally was made with whatever the fishermen hadn't sold that morning.

There are plenty of variations of bouillabaisse, and even in Marseille you'll find strong debates over the proper way to make it.

The most important thing is that you should use several varieties of fish, and the fish should be very fresh. In Provence you would use a variety of Mediterranean fish, but here in the states where we can't get those fish fresh, we have to make substitutions.

The distinctive flavors of a bouillabaisse broth include saffron, which also gives it its orange color, orange zest, and fennel.

Use firm fish for fillets such as sea bass, red mullet, haddock, halibut, cod, conger, or red porgy. Small whole fish can be added as well. Also traditional are mussels, squid, and crab.

Bouillabaisse Recipe

Print

Ingredients

3 pounds of at least 3 different kinds of fish fillets

1 pound mussels or clams

1 pound squid or crab

1/4 cup olive oil

1 cup thinly sliced onions

2 leeks, white and light green parts only, thinly sliced

1/4 fennel bulb, thinly sliced, or 1 teaspoon of fennel seeds

2 cloves garlic, crushed

3 large tomatoes, roughly chopped

1 bay leaf

3 sprigs fresh thyme

1/2 teaspoon saffron threads

2 teaspoons salt

One long, wide strip of orange zest

8 ounces clam juice or fish stock

1/4 teaspoon freshly ground black pepper

Sliced rustic French bread, plain or toasted

Directions for Sauce Rouille:

1 Tbsp hot fish stock or clam broth

2 cloves peeled garlic

1 small red hot pepper

1/2 teaspoon salt

1/4 cup soft white bread, pulled into bits

1/2 cup olive oil

Put hot fish stock or clam broth into the bottom of a blender. Add garlic and red hot pepper, salt and bread. Blend until very smooth. With the blender still running, add olive oil slowly and stop the blending as soon as the oil disappears.

Method

1 Cook the onions, leeks, fennel: Heat 1/4 cup of olive oil in a large (6-8 quart) pot on medium high heat. When the oil is hot, add the onions, leeks, and fennel. Stir to coat the vegetables with the olive oil. Cook on medium heat until softened but not browned, about 10-15 minutes.

2 Add garlic, tomatoes, bay leaf, thyme saffron, salt, orange zest: Add the crushed garlic, chopped tomatoes, bay leaf, thyme, saffron, salt, and orange zest.

Cook until the tomatoes are soft and broken down, about 10 more minutes.

3 Add fish fillets, cover with water and stock, boil: Cut fish fillets into 2-inch pieces. Bring 2 cups of water to a boil. Lay the fish pieces over the vegetable mixture and pour over with 2 cups of boiling water. Add clam juice or fish stock. Bring everything to a boil, and cook, uncovered, for about 5 minutes.

4 Add shellfish: Add the mussels, squid, and/or crab, pushing aside the fish so that the shellfish is now covered in liquid, and boil for 10 minutes more, uncovered.

Add freshly ground black pepper, and more salt to taste. Remove the bay leaves, sprigs of thyme, and orange zest from the broth.

5 Place slice of bread in bowl, top with rouille, then broth, then fish and shellfish: To serve, remove the fish and shellfish to a platter to keep warm. Place a thick slice of crusty French bread (plain or lightly toasted) in each bowl and put a dollop of the rouille sauce on top of the bread.

Ladle the soup broth over the bread, and then portion out fish and shellfish onto the bowls.

Hello! All photos and content are copyright protected. Please do not use our photos without prior written permission. If you wish to republish this recipe, please rewrite the recipe in your own unique words and link back to Bouillabaisse on Simply Recipes. Thank you!

Print

Elise Bauer is the Founder and President of Simply Recipes. A blogging pioneer, Elise launched Simply Recipes in 2003 as a way to keep track of her family's recipes, and along the way grew it into one of the most popular cooking websites in the world. In 2012 Elise was named "one of America's 50 Most Powerful People in Food" by The Daily Meal. Elise is dedicated to helping home cooks be successful in the kitchen. Elise is a graduate of Stanford University. She lives in Carmichael, California.
More from Elise

# data[].attributes.attachment_link.description
Photography Credit: Elise Bauer Bouillabaisse is a classic French dish from southern France, in particular, of the port town Marseille. It requires many different varieties of fish, and traditionally was made with whatever the fishermen hadn't sold that morning. There are plenty of variations of bouillabaisse, and even in Marseille you'll find strong debates over the proper way to make it.

# data[].attributes.body
Economic Facts:

Saffron is the most expensive spice in the world.

It takes about 75,000 crocus flowers to make one pound of saffron spice.

It takes about 4,500 crocus flowers to make up one ounce of saffron spice.

Saffron can cost up to $315/oz. to $5,040/lb.

A few pinches of saffron can be enough to color and flavor an entire meal.

One Saffron corm can multiply to five corms in three years.

# data[].attributes.body
Saffron Production:

Saffron spice is mainly produced and exported from countries such an Iran and India with almost 90% of the world production in 2007 coming from Iran (ncat.org). The life of the flower that it produces is less than a day long and they bloom at different points in the season, not all at the same time (ncat.org). Elderly women were usually given the job of removing the saffron stigmas from the crocus flowers, a very strenuous job, because there are no machines that can separate the three delicate stigmas from the flower to produce the spice (uwlax.edu).

# data[].attributes.attachment_link.description
this is just the best song ever made....

# data[].attributes.body
Saffron (C. sativus L.) contains two main carotenoids: crocin and crocetin. These carotenoids, which are responsible for the color of saffron, have potent anti-tumor effects. “Epidemiological evidence showed that a high dietary intake of fruits and vegetables rich in carotenoids is associated with a reduced risk for cancer (Azam Bolhassani a, , Afshin Khavari a, S. Zahra Bathaie b)”. “In general, carotenoids possess potent cancer chemo-preventive properties. The mechanisms underlying cancer chemo-preventive activities of carotenoids include modulation of carcinogen metabolism; regulation of cell growth and cell cycle progression [e.g., cell-cycle delay in the G1 phase, reduction in cyclin D1 protein levels, inhibition of both Cdk4 and Cdk2 kinase activity, hypo-phosphorylation of pRb], inhibition of cell proliferation; anti-oxidant activity; immune modulation; enhancement of cell differentiation [e.g., activation of differentiation-related proteins: retinoid-like receptors (RAR), nuclear receptors effective in the differentiation of adipocytes (PPARγ), xenobiotic and orphan nuclear receptors such as the steroid/xenobiotic receptor/pregnane X receptor (SXR/PXR)/or the constitutive androstane receptor (CAR), reduction of growth factor-induced stimulation of AP-1 transcriptional activity by altering the composition of AP-1 complexes bound to DNA, inhibition of Wnt/β-catenin signaling via the connection along the Akt/GSK3β/β-catenin]; hormone and growth factor signaling [e.g., inhibition of insulin growth factor (IGF)-1]; stimulation of cell-to-cell gap junction communication; apoptosis and retinoid-dependent signaling (Azam Bolhassani a, , Afshin Khavari a, S. Zahra Bathaie b)”.

# data[].attributes.body
Saffron can be used in small increments of around 30mg (about 13 threads) per day. 5 grams of Saffron may cause serious adverse effects, and 20 grams can be fatal. It can be take in a tablet form, steeped in soup or tea, or eaten raw.

There do not seem to be any adverse interactions with saffron and other drugs.

Effective for Alzheimer’s Disease, Depression, and Cancer prevention.

# data[].attributes.body
Saffron has been analyzed extensively. It contains, approximately, these common plant components:

| Substance | Proportion
| Simple sugars | 12–15%
| Water | 9–14%
| Proteins, amino acids, other nitrogen compounds | 11–13%
| Cellulose (fiber) | 4–7%
| Fats | 3–8%
| Minerals (measured as acid soluble ash) | 1–1.5%
| Other non-nitrogen (mainly complex sugars) | about 40%

# data[].attributes.body
Scientific Name(s): Crocus sativus,

Common Name(s): saffron crocus, autumn crocus

History of Saffron:

Although it was initially cultivated in Greece, Saffron is native to Southwest Asia, used for medicine, dye, seasoning, and, fragrance. “The color that it produces when clothing is dyed is a golden yellow that the Buddhist monks adopted as their color after the legend of the Buddhist missionary Madhyantikawas sent to Kashmir in 5th century B.C. and sowed a crop of saffron which spread all throughout the Indian subcontinent (Tarvand, 2005)”.

# data[].attributes.attachment_link.readable_text
Christine Daniel faces 150 years in federal prison and $5.5 million in fines for selling phony cures for cancer, diabetes and Alzheimer's, preying mostly on evangelical Christians.

A San Fernando Valley doctor and evangelical minister who federal prosecutors said used bogus herbal medications to offer false hope to dozens of people suffering from diseases such as cancer and Alzheimer's was found guilty Tuesday of nearly a dozen federal charges.

Twenty-eight victims or family members of victims who died while taking the products testified against Christine Daniel, 57, who was found guilty Tuesday on four counts of mail and wire fraud, six counts of tax evasion related to income tax filings as well as one count of witness tampering.

Daniel faces a maximum sentence of 150 years in federal prison and fines totaling $5.5 million. She is scheduled to be sentenced Dec. 5 in federal court in Los Angeles.

Federal prosecutors successfully argued that Daniel leveraged her position of trust among evangelical Christians and through a program on the Trinity Broadcasting Network to push the phony treatments, which were marketed under the names C-Extract, "the natural treatment" and "the herbal treatment."

Some of the medications, prosecutors said, contained nothing more exotic than sunscreen preservatives and beef extract.

Daniel charged her customers up to $4,270 for a week's worth of the product while a six-month treatment program retailed for $120,000 to $150,000, prosecutors said. The high price for the product was justified because she and her employees alleged that it was "made with herbs from around the world and was manufactured in a laboratory according to the needs of each patient."

According to federal prosecutors, Daniel fraudulently marketed and sold a medical treatment that she and her employees claimed had a 60% to 100% rate in curing Stage 4 "metastatic or terminal cancers." She also claimed to be able to reverse conditions such as multiple sclerosis, stroke, Parkinson's disease, diabetes and hepatitis.

"These are some of the most vulnerable victims in society. Most of these victims were dying, most of them were terminal cancer patients. Most of them only had been given a few months to live. Some had small children," Assistant U.S. Atty. Joseph Johns said.

Daniel "stepped into the breach and took everything they had, including their time. Instead of spending their final days with their families, they spent it some flea-ridden motel drinking her foul treatment," Johns said.

Neither Daniel nor her attorneys could be reached for comment.

The pitch of a unique formula of herbs from around the world convinced about 55 people to pay $1 million to Daniel's Sonrise clinic in Granada Hills, prosecutors said. A chemical analysis showed most of the products would have been ineffective against cancer or other ailments.

One victim who was diagnosed with breast cancer that was spreading through her body contacted Daniel and was told that chemotherapy would not help. The woman traveled to Southern California and was told that her herbal treatment program "would shrink her tumors and kill her cancer cells."

The victim and her husband paid Daniel thousands of dollars for the herbal product. The victim took the "cure" and at one point prosecutors said Daniel pronounced her "cancer-free" at a party held for patients. In fact, the cancer continued to spread and the woman died in four months.

Prosecutors also presented evidence that, under the guise of a nonprofit organization, Daniel instructed patients to classify their medical service payments as donations. From 2002 through 2004, Daniel failed to report nearly $1.3 million on corporate income tax returns - or about $438,809 in taxable income for Christine Daniel, M.D., Inc.

Daniel failed to report about $315,109 on her personal income tax returns for the same time period, resulting in a tax loss to the government of $73,895.

Prosecutors also presented evidence that Daniel attempted to influence the testimony of at least two witnesses who were called before the grand jury. Daniel was acquitted of one count of witness tampering.

andrew.blankstein@latimes.com

# data[].attributes.attachment_link.title
Doctor convicted in fake cancer treatments

# data[].attributes.attachment_link.description
Christine Daniel faces 150 years in federal prison and $5.5 million in fines for selling phony cures for cancer, diabetes and Alzheimer's, preying mostly on evangelical Christians. A San Fernando Valley doctor and evangelical minister who federal prosecutors said used bogus herbal medications to offer false hope to dozens of people suffering from diseases such as cancer and Alzheimer's was found guilty Tuesday of nearly a dozen federal charges.

# data[].attributes.attachment_link.readable_text
INTRODUCTION

Cancer has become a prominent cause of death throughout the world which has caused about 7.6 million deaths in 2008 and will probably rise to over 11 million in 2030.[ 1] Apart from death, it is indicated that cancer pain significantly decreases the global quality of life scores of patients.[ 2] In spite of the ongoing progress in the field of treatment, we still face the problem of drug resistance and side effects with the present chemotherapeutic compounds; therefore, the trend towards scientific exploring in natural compounds has significantly increased recently.[ 3]

Moringa oleifera Lam, also known as the 'drumstick tree' or the 'horse radish tree', is the most widely distributed and cultivated member of Moringaceae family. This highly valued plant is native to western and sub-Himalayan tracts, India, Pakistan, Asia Minor, Bangladesh, Africa and Arabia and has become naturalized in many tropical and subtropical areas. M. oleifera is considered as 'natural nutrition of the tropics', since almost all parts of this tree possess high nutritional value and even have been used to resist malnutrition in breast-feeding mothers.[ 4, 5]

Furthermore, this tree is traditionally believed and scientifically proven to have numerous medicinal traits which have drawn more attention towards it. Some examples of such traits include antibacterial, antitrypanosomal, hypotensive, antispasmodic, antiulcer, anti-inflammatory, hypo-cholesterolemic, and hypoglycemic activities.[ 4] Besides, M. oleifera leaves are found to have antitumor activities which is probably due to the presence of O-Ethyl-4-(α-L-rhamnosyloxy) benzyl carbamate, (α-L-rhamnosyloxy)-benzyl isothiocyanate, niazimicin, and 3- O-(6'- O-oleoyl-β-D-glucopyranosyl)-β-sitosterol together with some natural antioxidants such as phenolic compounds.[ 5, 6]

With regard to the problems of whole plant culture including environmental demands and variation in the production of secondary metabolites, plant cell culture is an agreeable alternate. The advantages of this procedure include cost-effective downstream process and being independent of geographical, environmental, and seasonal factors which can affect both type and amount of the metabolites.[ 7]

In this study we investigated the cytotoxic activity of the calli extract gained through plant cell culture of M. oleifera leaves in comparison to the leaf extract of this plant against Hela cell line.

MATERIALS AND METHODS

Callus establishment

M. oleifera leaves were collected from Bushehr Jahad Keshavarzi farm in September 2010. Young leaves of M. oleifera were sterilized by ethanol 70% and sodium hypochlorite, for one and five minutes, respectively; then, washed with sterile distilled water and transferred into culture jars containing sterile solid medium. The whole procedure was done under aseptic condition using class II laminar air flow hood.

Solid culture medium containing 30 g/l of sugar, 4.4 g/l of MS basal medium, 8 g/l of agar and also growth regulators including 0.25 mg/l of kinitine, 0.25 mg/l of 2,4-dichlorophenoxy acetic acid and 0.5 mg/l of α-naphthyl acetic acid was used.

The pH of the medium was adjusted at 5.7 ± 0.1. It was sterilized by autoclave at the condition of 121°C with 1/8 barr pressure for 15 minutes.[ 8] Culture jars were incubated at 25 ± 2°C in a dark place to establish the callus. Approximately every 1-2 months, when the jars were fully occupied, subculture was done. Each time, the calli were collected and freeze dried.

Extraction of cultured cells

Five gram of leaves or 5 g of freeze dried callus was ground and extracted through maceration in 160 ml and 240 ml of methanol:water 80:20, respectively. After three days the extracts were shaken, filtered and methanol was evaporated under reduced pressure using a rotary evaporator at 40°C.[ 9] Afterwards, the remained water content was removed by two days of freeze drying.

Measurement of phenolic compounds

Folin Ciocalteu method was applied to measure and compare the amount of the phenolic compounds in the callus and leaf extracts. Using pre-defined concentrations of gallic acid, as the standard solutions, a calibration curve was plotted and based on it, the concentrations of the phenolic compounds in the leaf and callus samples were measured spectrophotometrically at 765 nm.[ 10] The test was performed three times.

Maintenance of human cell lines

Human cervix carcinoma cell line (Hela) obtained from the Pasture Institute, Tehran, Iran. The medium used for maintenance of this cell line was roswell park memorial institute (RPMI) 1640 supplemented with 10% fetal calf serum and combination of penicillin/streptomycin (100 IU/ml and 100 μg/ml). Incubation was performed at 37°C in a humidified atmosphere with 5% CO ₂ condition.[ 11] The growth curve was plotted over five days.

Preparation of different concentrations of extracts

Stoke solutions of the leaf and callus extract of M. oleifera were obtained by dissolving the powder in dimethyl sulfoxide (DMSO) and then adding culture media. The maximum amount of DMSO was 10%, so that the final concentration of DMSO in the culture medium did not exceeded 1% in the wells. Based on the results of a pilot study using 1, 5, 10, 20, 50, 100, 200 and 500 μg/ml of leaf and callus extracts, it was decided to apply 1000, 1500, 2000, 2500 and 3000 μg/ml of callus extract and 100, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 and 500 μg/ml of leaf extracts for the main test. The test procedure was performed in tetraplicate and repeated three times different days.

MTT cell viability assay

The cytotoxic effect of the extracts of M. oleifera leaf and callus against Hela human tumor cell line was determined by a rapid colorimetric assay, using Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).[ 12] This assay is based on the reaction between mitochondrial dehydrogenase enzymes in viable cells and the tetrazolium rings of a soluble reagent which results in the production of violet formazan crystals. Crystals are solubilized in DMSO. The number of surviving cells is proportional to the amount of formazan and is represented by the color of the solution. The color is quantified using multi-well scanning spectrophotometer (ELISA reader). Briefly, 180 μl of cell suspension (2 × 10 ⁴ cell/ml) was seeded in 96-well microplates except for the first row which contained only 180 μl of Roswell Park Memorial Institute medium (RPMI) 1640 which was considered as the blank. After 24 h of incubation in the previously mentioned conditions, 20 μl of specific concentrations of the leaf and callus extracts, were added. For the positive and negative controls doxorubicin 20 μg/ml and DMSO 10% were added instead of extracts, respectively. All plates were incubated for 48 h and using the ELISA reader the absorbance was measured. To evaluate cell survival, 20 μl of MTT solution (5 mg/ml in phosphate buffer solution) was added to each well and incubated for 3 h. Then gently 150 μl of old medium containing MTT was replaced by DMSO and pipetted to dissolve any formed formazan crystals. Absorbance was then determined at 540 nm by an ELISA plate reader. Each extract concentration was assayed in 4 wells and repeated 3-times. Standard curve (absorbance against the number of cells) was plotted. Intraday and interday variations were determined. Based on the standard curve percent cell survival was calculated. Percent of cell survival in the negative control was assumed 100.

Statistical analysis

SIGMASTAT ™ (Jandel Software, San Raphael, CA) was used to perform statistical tests. Analyze-of-variance followed by the Student-Newman-Keuls test was used to see the differences among the groups. Significance was assumed at the 5% level.

RESULTS

Extract yeilds

The yield of the extracts was 19.6% and 18.2% for leaf and callus, respectively.

Callus formation

Fresh healthy creamy calli was established. The callus was compact in appearances.

Quantification of phenolic compounds

The standard curve for gallic acid, as indicator for phenolic compounds is shown in . There was a linear relationship between the absorbance and gallic acid concentration.

Based on the standard curve, the percent of phenolic compounds in dry powder of callus and leaf extracts was estimated as 0.237 ± 0.04% and 0.746 ± 0.09%, respectively.

Cytotoxic effect of extracts of M. oleifera

A good relationship between absorbance and the number of cells was observed for Hela cells, (r ² = 0.974) []. Intraday and interday variations for all standard curves were acceptable (%CV<15).

To evaluate appropriate time of incubation of Hela cells with tested compounds, the growth curve of Hela cells over 120 h was plotted []. Based on the data, 48 h of incubation was used for further cytotoxicity tests.

Doxorubicin, a known cytotoxic antibiotic at concentration of 20 μg/ml, as a positive control significantly reduced the cell viability to less than 25%. Extracts were considered cytotoxic when they reduced cell viability to less than 50%. Callus extract of M. oleifera significantly and concentration-dependently decreased the viability of Hela cells at concentrations above 2000 μg/ml []. Also, leaf extract of M. oleifera significantly and concentration-dependently decreased the viability of Hela cells at concentrations above 260 μg/ml [].

DISCUSSION

Cancer is one of the most important diseases of the 21 st century. The incidence of cancer is increasing during the last decades in most countries. In spite of numerous efforts to combat this disease, world medicine is facing with the lack of perfect cure for it. In the last decades there has been a great emphasis towards the researches on complementary and alternative medicine for cancer management.[ 6] Several studies have been conducted on herbs to find new anti-tumor compounds. It has been shown that about 3000 plants, which possess anticancer properties, subsequently have been used as anticancer drugs.[ 13, 14]

It has been shown that plants from Moringaceae family including M. oleifera possess antitumor activities.[ 5, 6] Using MTT assay, we sought to determine if the extracts of callus and leaves of M. oleifera have cytotoxic activity against Hela cells. Doxorubicin, a known cytotoxic drug, significantly decreased the number of Hela cells indicating accuracy of the method used in this study. Our findings showed that leaf and callus extracts of M. oleifera significantly reduced viability of Hela cells in a concentration-dependent manner. However, the IC50 of leaf extract of M. oleifera was lower than that of the callus extract. Several studies have been shown that phenolic compounds are involved in cytotoxic activity of plants.[ 15, 16, 17, 18, 19, 20]

It has been shown by Dehshahri et al. that leaf extract of M. peregrina possess antioxidant activity. They concluded that phenolic compounds in this plant were involved in its antioxidant activity.[ 21] As the content of phenolic compounds in the leaf extract of M. oleifera was higher than that of the callus extract it may be concluded that at least in part they are involved in the cytotoxicity of the extracts. However, the differences between the ratio of cytotoxicity and phenolic constituents of callus and leaf extracts of M. oleifera indicates that the cytotoxic effect of this plant is not merely due to the phenolic compounds. Studying on the volatile glucosinolate content of seed and leaf of M. peregrina showed that isobutyl isothiocyanate was the main glucosinolate degradation product of aerial parts of this plant, while no isothiocyanates were found in cell cultures of M. peregrina.[ 8] The difference in the constituents of plant and callus of M. peregrina may explain the difference in the cytotoxicity of their extracts. More investigation is needed to find out which components are responsible for cytotoxicity of M. oleifera. M. oleifera has several therapeutic and nutritional applications. Although its callus did not have desirable cytotoxic activity, it can be applied for other medical applications. Regarding to the numerous advantages of plant cell culture, it worth's to investigate the callus for further useful compounds and traits.

# data[].attributes.attachment_link.title
Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells

# data[].attributes.attachment_link.description
Cancer has become a prominent cause of death throughout the world which has caused about 7.6 million deaths in 2008 and will probably rise to over 11 million in 2030.[ 1] Apart from death, it is indicated that cancer pain significantly decreases the global quality of life scores of patients.[

# data[].attributes.body
How to Roast Garlic:
• Prep Time: 5 mins
• Total Time: 40 mins
• Servings: 2

# data[].attributes.attachment_link.title
How To Roast Garlic Recipe - Genius Kitchen

# data[].attributes.attachment_link.description
This is a simple how to recipe for roasting garlic in the oven. Super easy and the flavor of roast garlic is so sweet and creamy. Our favorite way to serve it up is to dump the roasted garlic in some good olive oil seasoned with salt/pepper, and serve it atop french bread.

# data[].attributes.body
The cancer fighting properties of garlic have been trusted since ancient times. Hippocrates recommended garlic for cancerous growths.  In the literature review, the authors of this study cite a 1958 study by Weisberger& Pensky that first described garlic’s effectiveness inhibiting the growth of cancer cells. Organosulfur compounds in garlic have been credited as the primary anti-cancer compounds, which are either oil soluble (diallyl sulfide and trisulfide, dithiins, and ajoene)  or water soluble (S-allylcysteine or S-allylmercaptocysteine).  Previous studies have shown that these compounds have been effective in fighting esophageal, stomach, intestinal and pulmonary cancers.

This quantitative experimental study aimed to answer the question: What effect will water soluble garlic compounds have on cultured cervical cancer cells when tested in differing concentrations?

Garlic was purchased and ground to a paste. Using water distillation method, the water soluble compounds (AGE) were extracted.

Cultured cervical cancer cells were measured and incorporated with a control solution, containing no AGE, and then concentrations of 100, 200, 300, 400, and 500ul of AGE, and incubated for 24 hours.  Afterward, percentage of cell death was measured at each concentration.

Results showed that the groups containing the garlic compound AGE had measurable and quite significant cell death.  As the concentration of garlic extract became stronger, the cancer cells had higher percentages of cell death. In the 500ul concentration, only 5% of cells survived. This study showed the significance of the potential for the use of garlic in the fight against cancer.

Islam, M., Kusumoto, Y. & Al-Mamun, M.  (2011).  Cytotoxicity and cancer (HeLa)  Cell killing efficacy of aqueous garlic (Allium sativum) extract.  Journal of Scientific Research, 3(2), pp375-382.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202505/

# data[].attributes.body
Do not take over 7g/daily if using Anticoagulants: may cause bleeding

Do not take higher doses if using Antihypertensives: may cause low BP and may raise serum levels of Lisinopril.

# data[].attributes.body
when taken in normal dosage. Effects may be decreased if Garlic is taken in the morning, with breakfast.

·        Mild Gastro-Intestinal effects: bloating, gas, reflux, diarrhea

·        Garlic odor and Garlic breath

·        Not recommended above usual dietary intake during first trimester of pregnancy.
·        Not recommended for patients with bleeding disorders.

# data[].attributes.body
Fresh Garlic:  2 – 5 g daily (cloves must be crushed)

Dried Powder: 0.4 – 1.2 g daily

Oil:  2 – 5 mg daily

Pill:  300 to 1,000 mg

# data[].attributes.headline
How Garlic Works to Prevent and Fight Cancer

# data[].attributes.subject
How Garlic Works to Prevent and Fight Cancer

# data[].attributes.body
Garlic has anti-tumor properties due to its high content of organic sulfides and polysulfides.

Detoxifying compounds are less toxic, more water soluble, and can be excreted more easily than chemotherapeutic agents.

Mutagenesis, or mutating/abnormal cells, is stopped by a sulphur-containing compound:  Ajoene.

Garlic’s compounds make the body’s own cancer fighting cells even MORE toxic to cancer cells.

Garlic contains compounds that amplify the activity of the body’s immune cells…..this promotes the body’s own production of Cytokines (molecules that signal inflammation and cancer fighting cells to where they need to be to heal the body).

Cancer fighting compounds are released when Garlic’s bulbs are crushed – compounds, including Allin and diallyldisulphide, protect healthy cells’ DNA from the damaging effects of carcinogens.

Garlic’s compounds have been shown, both in vitro and in vivo testing, to inhibit metastasis in lung carcinoma cells and colon cancer cells by disrupting abnormal cells at the tubule level.

Japan’s National Medical Centre conducted research that showed that Garlic extract reduced side effects in patients undergoing chemotherapy and radiation.

# data[].attributes.body
Scientific Name(s): Allium sativum L. Family: Liliaceae (lilies

Common Name(s): Garlic , allium , stinking rose , rustic treacle ,
nectar of the gods , camphor of the poor , poor man's treacle

Garlic has been used medicinally for thousands of years.

Garlic is a member of the onion family, easily grown in most areas. The bulb is used in cooking and medicine.

Hippocrates was first to recommend Garlic for its effectiveness in fighting cancer.

Garlic is especially effective in fighting cancers of the lungs, bladder, esophagus, stomach and intestines.