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Music Medicine: Sound At A Cellular Level | Dr. Lee Bartel | TEDxCollingwood

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The healing power of music: Robin Spielberg at TEDxLancaster

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What You Need to Know About HYDROGEN with Dr. Shigeo Ohta

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Why raising your vibration increases serendipity. | Joanna McEwen | TEDxUniversityofBrighton

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The excitement surrounding molecular hydrogen/hydrogen water and its medical/scientific research is full-blown in Asia (Japan, Korea, China). Since in 2010, the hydrogen industry (relating to hydrogen products or hydrogen water devices) was a zero-dollar industry.

Now in 2017, this industry is estimated to be close to $1 billion. 1 The hydrogen industry has absolutely exploded in the light of the promising scientific research about molecular hydrogen and it administration methods.

First, let's look at how molecular hydrogen has been administered in research studies. Then we'll look at convenient ways for individuals to consume molecular hydrogen daily.

Administration methods seen in scientific studies

Drinking Hydrogen-Rich Water (produced by multiple methods)

Hydrogen-rich water is water infused with molecular hydrogen. In research studies, it has been demonstrated that hydrogen infused water containing a dissolved hydrogen concentration of 0.5-1.6 ppm+ can be therapeutic for human consumption. 3

Hydrogen Inhalation

Hydrogen inhalation uses the process of water electrolysis to produce molecular hydrogen and then mixes the hydrogen gas (H2) with oxygen (O2). Most of these devices keep the hydrogen concentration under 4 percent. This ensures the safety of the person receiving treatment, as molecular hydrogen is noncombustible under 4 percent. The application of molecular hydrogen via inhalation has shown great benefit in the scientific literature, as it allows larger amounts of H2 gas to enter the bloodstream to be readily distributed throughout the body.

Hydrogen Saline (via intravenous drip i.e. IV)

This method of administration has yielded great results within the scientific literature and will most likely be seen used in a clinical and medical setting, such as hospitals.

Drinking ERW (Electrolyzed Reduced Water)

ERW is hydrogen water generated using the process of electrolysis. This method produces water with an alkaline pH as a by-product due to the hydrogen gas production. 4

Hydrogen Baths

Hydrogen baths are used mainly for topical purposes to administer molecular hydrogen to the body via the skin. Since molecular hydrogen has the highest diffusion rate out of any molecule it is perfect for topical application. Research studies have demonstrated this method to be therapeutic for multiple skin disorders. 5,6

Hydrogen Saline Eye Drops

Molecular hydrogen appears to be therapeutic for multiple eye diseases (cataract, glaucoma, etc). This method enhances saline solution for the eye with molecular hydrogen. This method is simple and practical for researching molecular hydrogen's therapeutic effect for the eye. 7

Intraperitoneal Injection (IP injection)

Hydrogen Intraperitoneal injection or Hydrogen-rich IP injection is when a hydrogen infused solution is injected into the peritoneum (abdominal cavity). Typically, this solution is hydrogen saline. This method is applied more towards animals in animal studies than humans in human studies.

Convenient ways to consume molecular hydrogen

Molecular hydrogen is approved by the FDA as a supplement that can be dissolved in water and safe to consume. 8 This is one of the first signs that hydrogen water is about to make a major "splash." The market for hydrogen products is small but growing, as hydrogen water awareness grows and individuals start to understand what hydrogen water really is and the potential it carries. I have no doubt molecular hydrogen/hydrogen water is and will become more mainstream. As for now, there are limited but effective options and technologies individuals can look into to start their own journey with hydrogen-rich water. Let take a brief overview of some technologies:

Hydrogen-Rich Water
Hydrogen Water Generators

HIM (Hydrogen Infusion Machines) 9 - these home units use a special membrane called a Nafion membrane or PEM (proton exchange membrane). The units are specifically designed for producing and dissolving molecular hydrogen into highly-filtered water.

H2 Portable Units (HIMs or ERW) - portable water bottles that produce and dissolve H2 into drinking water. These devices can generate neutral pH or alkaline pH hydrogen-rich water depending on the type. They are convenient and can produce hydrogen-rich water within 5 to 10 mins.

Water Ionizers (ERW) 10 - these devices produce alkaline hydrogen water. Water ionizers are not optimally designed for producing hydrogen drinking water. These devices were originally designed for and intended to produce alkaline water, not for dissolving molecular hydrogen into drinking water effectively.

Hydrogen Tablets 11 - use elemental magnesium to produce molecular hydrogen. These metals are called alkali metals; they naturally react with water to produce molecular hydrogen. These tablets can be ingested or dropped into water to produce hydrogen rich water. They are a great way of producing higher concentrations of dissolved hydrogen in water, or to drink hydrogen water on the go.

Hydrogen Sticks 12 - these are like hydrogen tablets, they utilize elemental magnesium to produce molecular hydrogen in drinking water. However, hydrogen magnesium sticks need consistent maintenance to ensure its ability to produce the therapeutic concentration of dissolved molecular hydrogen in water.

Hydrogen Inhalation

There is one company that offers a quality hydrogen inhalation device. These devices allow individuals to breath molecular hydrogen through a nasal cannula.

Not all hydrogen water devices are created equal

One must remember not all hydrogen water products are created equal. Different technologies can offer higher dissolved hydrogen concentrations and have better build quality. The object is the find an effective means of obtaining great filtered hydrogen-rich water with minimal maintenance that will help you reach the hydrogen milligram mark that appears to be therapeutic in human studies, which is 1-3 milligram a day. 13

Here is a simple formula to help you understand how much hydrogen water to drink per day:

Molecular hydrogen dissolved into water is measured in parts per million (or mg/L). 14

Hydrogen water measuring at 0.5 ppm in 33 oz. or 1 liter is equal to 0.5 mg of H2.

Hydrogen water measuring at 1 ppm in 33 oz. or 1 liter is equal to 1 mg of H2.

Hydrogen water measuring at 2 ppm in 33 oz. or 1 liter is equal to 2 mg of H2.

This simple knowledge is a key element to help you determine which type of hydrogen water device will help you reach your daily molecular hydrogen intake goals. The best part about all of this is you can consume one of the world's greatest antioxidant while hydrating.

To learn more about molecular hydrogen or hydrogen water's potential health benefits and to see supporting scientific research visit:

Interested in experiencing molecular hydrogen? Tywon's professional recommendations

More about H2:

* Molecular hydrogen as a preventative measure
* Molecular hydrogen offers remarkable medical potential
* Therapeutic effects of molecular hydrogen against cancer?
Check out Tywon Hubbard's educational websites - H2HUBB.com and on YouTube - to learn more about hydrogen and surrounding sciences in a simple and entertaining manner.

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Which hydrogen water product will help you achieve daily molecular hydrogen intake goals? - Cancer Tutor

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The excitement surrounding molecular hydrogen/hydrogen water and its medical/scientific research is full-blown in Asia (Japan, Korea, China). Since in 2010, the hydrogen industry (relating to hydrogen products or hydrogen water devices) was a zero-dollar industry. Now in 2017, this industry is estimated to be close to $1 billion.

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Vitamin C for cancer? ‘Miracle man’ Anton Kuraia's highly controversial treatment

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Abstract

Nigella sativa has been used as traditional medicine for centuries. The crude oil and thymoquinone (TQ) extracted from its seeds and oil are effective against many diseases like cancer, cardiovascular complications, diabetes, asthma, kidney disease etc. It is effective against cancer in blood system, lung, kidney, liver, prostate, breast, cervix, skin with much safety. The molecular mechanisms behind its anticancer role is still not clearly understood, however, some studies showed that TQ has antioxidant role and improves body's defense system, induces apoptosis and controls Akt pathway. Although the anti-cancer activity of N. sativa components was recognized thousands of years ago but proper scientific research with this important traditional medicine is a history of last 2∼3 decades. There are not so many research works done with this important traditional medicine and very few reports exist in the scientific database. In this article, we have summarized the actions of TQ and crude oil of N. sativa against different cancers with their molecular mechanisms.

Traditional medicine, Keywords: Nigella sativa, Thymoquinone, Antioxidant, Anti-cancer mechanism

Introduction

Cancer is one of the major threats of modern life, which is considered as the second cause of death after myocardial infarction ( Grundy, 1991). Millions of people die every year in different types of cancer despite tremendous efforts to find methods of control and cure. In the last century, great advances were made in modern medical science to control disease. But many diseases like cancers are not yet curable fully. To find out new and authentic therapies, scientists are working with traditional or folk medicines in parallel of modern medicine. Nigella sativa has been used for medicinal purposes for centuries. It originated from Southeastern Asia and also used in ancient Egypt, Greece, Middle East and Africa. In Islam, it is regarded as one of the greatest forms of healing medicine available ( Nigella-sativa-research.com, 2010; Wikipedia, 2010). It is a flowering plant, of which seed is used as a spice. The seed is called black cumin in English, while in old Latin it was called 'Panacea' meaning 'cure all'; in Arabic it is termed as 'Habbah Sawda' or 'Habbat el Baraka' translated as 'Seeds of blessing'. It is also known as 'Kalo jeera' (in Bangladesh), 'Kalonji' (in India) and 'Hak Jung Chou' in (China) ( Aggarwal et al., 2008). Both seeds and oil extracted from this plant are used in medicinal purposes.

The active ingredients of N. sativa have beneficial effects against many diseases, including cancers. For example, it is effective in the diminishing the risk of atherosclerosis by decreasing the serum low density lipoprotein cholesterol level and increasing the serum high density lipoprotein cholesterol levels ( Dahri et al., 2005; Nader et al., 2010); it exerts therapeutic and protective effect in diabetes by decreasing morphological changes and preserving pancreatic beta-cell integrity ( Kanter et al., 2009) and by beneficially changing the hepatic enzyme activities ( Pari and Sankaranarayanan, 2009); it is effective against hypertension ( Khattab and Nagi, 2007; Dehkordi and Kamkhah, 2008); it has a potent antihistaminic effect on airways of asthmatic patients ( Boskabady et al., 2010); its components are promising agents to complement schistosomiasis specific treatment ( El Shenawy et al., 2008); its oil protects kidney tissue against oxygen free radicals, preventing renal dysfunction and morphological abnormalities ( Bayrak et al., 2008; Uz et al., 2008; Ragheb et al., 2009). For thousands of year, the seeds, oils and extracts of N. sativa have been used as an anticancer agent by Unani, Ayurveda and the Chinese system of medicine that have originated from the Arab, Ind-Bangla and China, respectively. The modern scientific research with the investigation of anticancer activity of N. sativa is a comparatively recent affair (for the last 2∼3 decades). There are not so many research works done in this field and very few review articles exist in this area. We have searched the scientific databases like Pubmed, Web of Science and Google scholar and summarized the current scientific information about the anticancer activities of N. sativa with mechanisms of action.

Role of N. sativa as an anticancer agent

Many active ingredients have been found in the seeds of N. sativa. The seeds contain both fixed and essential oils, proteins, alkaloids and saponin ( Ali and Blunden, 2003). Ghosheh et al. (1999) described the quantification of four pharmacologically important components: thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (THY), in the oil of N. sativa seed by HPLC. Much of the biological activities of the seeds have been shown to be due to thymoquinone, the major component of the essential oil, which is also present in the fixed oil ( Ali and Blunden, 2003). TQ is considered as potent anti-oxidant ( Badary et al., 2003), anti-carcinogenic and anti-mutagenic agent ( Bourgou et al., 2008; Khader et al., 2010) (structure of thymoquinone is shown in ). Moreover, TQ is a relatively safe compound, particularly when given orally to experimental animals ( Al-Ali et al., 2008). Alpha (α)-hederin, a pentacyclic triterpene saponin (structure: ) isolated from the seeds of N. sativa, was also reported to have potent in vivo antitumor activity ( Swamy and Huat, 2003).

N. sativa seeds or oils or its active ingredients like TQ are effective against different cancers:

Blood Cancer

El-Mahdy et al. (2005) reported that TQ exhibits anti-proliferative effect in human myeloblastic leukemia HL-60 cells. Derivatives of TQ bearing terpene-terminated 6-alkyl residues were tested in HL-60 cells and 518A2 melanoma by Effenberger et al. (2010). They found the derivatives induce apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species. Swamy and Huat (2003) observed that α-hederin also induced death of murine leukemia P388 cells by a dose- and time-dependent increase in apoptosis.

Breast Cancer

Aqueous and alcohol extracts of N. sativa were found to be effective in vitro in inactivating MCF-7 breast cancer cells ( Farah and Begum, 2003). N. sativa, in combination with melatonin and retinoic acid reduced the carcinogenic effects of DMBA (7, 12-di-methylbenz(a)anthracene) in mammary carcinoma of rats ( El-Aziz et al., 2005). Terpene-terminated 6-alkyl residues of TQ were tested in MCF-7/Topo breast carcinoma by Effenberger et al. (2010). They found the derivatives inducing cell death by apoptosis.

Colon Cancer

Gali-Muhtasib et al. (2004) suggested that TQ is anti-neoplastic and pro-apoptotic against colon cancer cell line HCT116. Salim and Fukushima (2003) demonstrated that the volatile oil of N. sativa has the ability to inhibit colon carcinogenesis of rats in the post-initiation stage, with no evident adverse side effects. Norwood et al. (2006) suggested TQ as chemotherapeutic agent on SW-626 colon cancer cells, in potency, which is similar to 5-flurouracil in action. However, on HT-29 (colon adenocarcinoma) cell, no effect of TQ was found ( Rooney and Ryan, 2005).

Pancreatic Cancer

Chehl et al. (2009) showed that TQ, the major constituent of N. sativa oil extract, induced apoptosis and inhibited proliferation in PDA (pancreatic ductal adenocarcinoma) cells. They also suggested TQ as a novel inhibitor of pro-inflammatory pathways, which provides a promising strategy that combines anti-inflammatory and proapoptotic modes of action. TQ also can abrogate gemcitabine- or oxaliplatin-induced activation of NF-kappa B, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics ( Banerjee et al., 2009). The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. Torres et al. (2010) evaluated the down-regulatory effect of TQ on MUC4 in pancreatic cancer cells. But in a study, Rooney and Ryan (2005) did not find any preventive role of TQ on MIA PaCa-2 (pancreas carcinoma) cells.

Hepatic Cancer

The cytotoxic activity of N. sativa seed was tested on the human hepatoma HepG2 cell line by Thabrew et al. (2005), and 88% inhibitory effect on HepG2 was found after 24-hr incubation with different concentrations (0-50 mg/ml) of the N. sativa extract. Nagi and Almakki (2009) reported that oral administration of TQ is effective in increasing the activities of quinone reductase and glutathione transferase and makes TQ a promising prophylactic agent against chemical carcinogenesis and toxicity in hepatic cancer

Lung Cancer

Swamy and Huat (2003) mentioned the antitumor activity of α-hederin from N. sativa against LL/2 (Lewis Lung carcinoma) in BDF1 mice. Also, Mabrouk et al. (2002) showed that supplementation of diet with honey and N. sativa has a protective effect against MNU (methylnitrosourea)-induced oxidative stress, inflammatory response and carcinogenesis in lung, skin and colon. However, Rooney and Ryan (2005) reported that α-hederin and TQ, the two principal bioactive constituents of N. sativa enhance neither cytotoxicity nor apoptosis in A549 (lung carcinoma), HEp-2 (larynx epidermoid carcinoma) cells.

Skin cancer

Topical application of N. sativa extract inhibited two-stage initiation/promotion [dimethylbenz[a]anthracene (DMBA)/croton oil] skin carcinogenesis in mice. Again, intraperitoneal administration of N. sativa (100 mg/kg body wt) 30 days after subcutaneous administration of MCA (20-methylcholanthrene) restricted soft tissue sarcomas to 33.3% compared with 100% in MCA-treated controls ( Salomi et al., 1991).

Fibrosarcoma

TQ from N. sativa was administrated (0.01% in drinking water) one week before and after MCA treatment significantly inhibited the tumor incidence (fibrosarcoma) and tumor burden by 43% and 34%, respectively, compared with the results in the group receiving MCA alone. Moreover, TQ delayed the onset of MCA-induced fibrosarcoma tumors. Also in vitro studies showed that TQ inhibited the survival of fibrosarcoma cells with IC50 of 15 mM ( Badary and Gamal, 2001). Oil of N. sativa also decreased the fibrinolytic potential of the human fibrosarcoma cell line (HT1080) in vitro ( Awad, 2005).

Renal Cancer

Khan and Sultana (2005) reported the chemo-preventive effect of N. sativa against ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, hyper-proliferative response and renal carcinogenesis. Treatment of rats orally with N. sativa (50 100 mg/kg body wt) resulted in significant decrease in H ₂O ₂ generation, DNA synthesis and incidence of tumors.

Prostate Cancer

TQ, from N. sativa, inhibited DNA synthesis, proliferation, and viability of cancerous (LNCaP, C4-B, DU145, and PC-3) but not non-cancerous (BPH-1) prostate epithelial cells by down-regulating AR (androgen receptor) and E2F-1 (a transcription factor) ( Kaseb et al., 2007). In this study, they suggested TQ as effective in treating hormone-sensitive as well as hormone-refractory prostate cancer. Yi et al. (2008) found that TQ blocked angiogenesis in vitro and in vivo, prevented tumor angiogenesis in a xenograft human prostate cancer (PC3) model in mouse, and inhibited human prostate tumor growth at low dosage with almost no chemotoxic side effects. Furthermore, they observed that endothelial cells were more sensitive to TQ-induced cell apoptosis, cell proliferation, and migration inhibition compared with PC3 cancer cells. TQ also inhibited vascular endothelial growth factor-induced extracellular signal-regulated kinase activation but showed no inhibitory effects on vascular endothelial growth factor receptor 2 activation.

Cervical Cancer

Shafi et al. (2009) reported that methanol, n-Hexane and chloroform extracts of N. sativa effectively killed HeLa (human epithelial cervical cancer) cells by inducing apoptosis. Effenberger et al. (2010) tested terpene-terminated 6-alkyl residues of TQ on multidrug-resistant KB-V1/Vb1 cervical carcinoma and found the derivatives inducing cell death by apoptosis.

Molecular mechanisms of N. sativa action against cancer

Cancers are the abnormal cell growth caused by genetic alteration. So, any agent which has anti-cancer activity, either protect genetic material from alteration or kill the genetically altered cancer cells. The active ingredients (mainly TQ) from N. sativa act on cancer cell to help to kill them by several molecular pathways ().

El-Mahdy et al. (2005) suggested the apoptotic mechanisms behind the anti-proliferative effect of TQ (from N. sativa) on myeloblastic leukemia HL-60 cells. They reported that TQ induces apoptosis, disrupts mitochondrial membrane potential and triggers the activation of caspases 8, 9 and 3 in HL-60 cells. The apoptosis induced by TQ was inhibited by a general caspase inhibitor, z-VAD-FMK; a caspase-3-specific inhibitor, z-DEVD-FMK; as well as a caspase-8-specific inhibitor, z-IETD-FMK. Moreover, the caspase-8 inhibitor blocked the TQ-induced activation of caspase-3, PARP cleavage and the release of cytochrome c from mitochondria into the cytoplasm. In addition, TQ treatment of HL-60 cells caused a marked increase in Bax/Bcl2 ratios due to upregulation of Bax and downregulation of Bcl2 proteins. Their results indicated that TQ-induced apoptosis is associated with the activation of caspases 8, 9 and 3, with caspase-8 acting as an upstream activator and activated caspase-8 initiates the release of cytochrome c during TQ-induced apoptosis. TQ action was also found as pro-apoptotic against colon cancer cell line HCT116 ( Gali-Muhtasib et al., 2004). It was showed that the apoptotic effects of TQ are modulated by Bcl-2 protein and are linked to and dependent on p53. TQ also down-regulates the expression of NF-kappa B-regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin) gene products ( Sethi et al., 2008). Torres et al. (2010) found TQ inducing apoptosis by the activation of c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways in pancreatic cancer cell.

TQ has also been reported to be active in controlling Akt pathway. Yi et al. (2008) found that TQ effectively inhibited human umbilical vein endothelial cell migration, invasion, and tube formation by suppressing the activation of AKT and extracellular signal-regulated kinase. Xuan et al. (2010) found that LPS (lipopolysaccharides: a bacterial component)-induced phosphorylation of prosurvival kinases Akt and ERK1/2 was abrogated by TQ in dendritic cells.

NF-kappa B plays a key role in regulating the immune response, and incorrect regulation of NF-kappa B has been found to be linked to cancer ( Albensi and Mattson, 2000). Sethi et al. (2008) found that TQ suppressed tumor necrosis factor-induced NF-kappa B activation in a dose- and time-dependent manner and inhibited NF-kappa B activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-kappa B activation is correlated with sequential inhibition of the activation of I kappa B alpha kinase, I kappa B alpha phosphorylation, I-kappa-B-alpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappa B-dependent reporter gene expression. Also Oberg et al. (2009) reported that a herbal melanin (HM) from N. sativa modulates cytokine production and suggested it as a ligand for TLR4 (toll-like receptor 4). They investigated the possibility that the HM-induced cytokine production is via an NF-kappa B signaling pathway and found that HM induced the degradation of I kappa B-alpha, a key step in the activation of NF-kappa B. Moreover, addition of I kappa B kinase (IKK) specific inhibitors effectively inhibited the observed HM-induced production of IL-8 and IL-6 by TLR4-transfected HEK293 (embryonic kidney 293) cells and THP-1 (Human acute monocytic leukemia) cells ( Oberg et al., 2009).

Many studies showed that N. sativa oil or TQ has antioxidant activity and increases the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) etc ( Ebru et al., 2008; Barron et al., 2008; Ismail et al., 2010). And antioxidant enzymes are clearly related to cancer- mostly their increased activities are beneficial against different types of cancer ( Khan et al., 2010). Administration of N. sativa oil or TQ can lower the toxicity of other anticancer drugs (for example, cyclophosphamide) by an up-regulation of antioxidant mechanisms, indicating a potential clinical application for these agents to minimize the toxic effects of treatment with anticancer drugs ( Alenzi et al., 2010).

In addition to these cancer inhibiting properties, components of N. sativa have cancer protective roles. Ibrahim et al. (2008) reported that N. sativa oil administration has a protective effect against the CCl ₄-mediated suppression of CYP (drug-metabolizing cytochrome P450 enzymes). And genetic abnormalities and polymorphisms of CYP enzymes are associated with cancer ( Sim and Ingelman-Sundberg, 2006; Chen et al., 2008). Radiotherapy is one of the most common strategies for treating human cancers but this treatment is somehow risky for normal tissue. Cemek et al. (2006) showed that N. sativa and glutathione treatment significantly antagonize the effects of radiation. Therefore, N. sativa may be a beneficial agent in protection against ionizing radiation-related tissue injury. Assayed (2010) investigated the radio-protective potential of N. sativa crude oil against hemopoietic adverse effects of gamma irradiation. He found that irradiation resulted in significant reduction in hemolysin antibodies titers and delayed type hypersensitivity reaction of irradiated rats, in addition to significant leukopenia and significant decrease in plasma total protein and globulin concentration and depletion of lymphoid follicles of spleen and thymus gland. Furthermore, there was a significant increase in malondialdehyde concentration with a significant decrease in plasma GPx, CAT and erythrocyte SOD activities. But oral administration of N. sativa oil before irradiation considerably normalized all the above-mentioned criteria; and produced significant regeneration in spleen and thymus lymphoid follicles. Thus N. sativa oil is recognized as a promising natural radioprotective agent against immunosuppressive and oxidative effects of ionizing radiation.

Concluding remarks

The anti-cancer activities of N. sativa components were recognized thousands of years ago but proper scientific research with this important traditional medicine is a very recent story. More research works should be emphasized behind this because it is a safe and promising anticancer agent. Specially, researchers should investigate the active ingredients more broadly, because, there is very few authentic reports about the chemical composition of seeds or oil of N. sativa exist. Also, the exact molecular mechanisms of thymoquinone and other components on different cancers should be investigated with more emphasis because current understandings are mostly unclear. For example, it is reported that N. sativa oil can protect cells from radiation, but the molecular mechanisms behind this is not properly understood. Currently, in some parts of the world, there is a renaissance of interest in traditional remedies. Many investigators now believe that traditional medicine is a promising source of new therapeutics against cancer. Extensive research with N. sativa may contribute to the discovery of new anticancer strategies.

References

14. Boskabady MH, Mohsenpoor N, Takaloo L. Antiasthmatic effect of Nigella sativa in airways of asthmatic patients. Phytomedicine. 2010 Epub ahead of print. [[ PubMed] Google Scholar]

16. Chehl N, Chipitsyna G, Gong Q, Yeo CJ, Arafat HA. Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB (Oxford) 2009;11:373-381. [[ PMC free article] [ PubMed] Google Scholar]

22. El-Aziz MA, Hassan HA, Mohamed MH, Meki AR, Abdel-Ghaffar SK, Hussein MR. The biochemical and morphological alterations following administration of melatonin, retinoic acid and [[ Nigella sativa in mammary carcinoma: an animal model. Int J Exp Pathol. 2005;86:383-396. PMC free article] [ PubMed] Google Scholar]

34. Khan MA, Tania M, Zhang DZ, Chen HC. Antioxidant enzymes and cancer. Chin J Cancer Res. 2010;22:87-92. [ Google Scholar]

49. Shafi G, Munshi A, Hasan TN, Alshatwi AA, Jyothy A, Lei DK. Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of [[ Nigella sativa. Cancer Cell Int. 2009;9:29-36. PMC free article] [ PubMed] Google Scholar]

53. Torres MP, Ponnusamy MP, Chakraborty S, Smith LM, Das S, Arafat HA, Batra SK. Effects of Thymoquinone in the Expression of Mucin 4 in Pancreatic Cancer Cells: Implications for the Development of Novel Cancer Therapies. Mol Cancer Ther. 2010;9:1419-1431. [[ PMC free article] [ PubMed] Google Scholar]

57. Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, Sethi G, Aggarwal BB, Liu M. Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Therr. 2008;7:1789-1796. [[ PMC free article] [ PubMed] Google Scholar]

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Anticancer Activities of Nigella Sativa (Black Cumin)

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Nigella sativa has been used as traditional medicine for centuries. The crude oil and thymoquinone (TQ) extracted from its seeds and oil are effective against many diseases like cancer, cardiovascular complications, diabetes, asthma, kidney disease etc. It is effective against cancer in blood system, lung, kidney, liver, prostate, breast, cervix, skin with much safety.

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Top 10 Toxic Foods and Top 10 Healing Foods | Dr. Josh Axe

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Boswellia serrata is an ancient herb used in traditional medicine for arthritis and other inflammatory diseases. Its ability to relieve inflammation-related pain and symptoms is well-documented in medical studies. Read on to learn how to take Boswellia serrata, the dosages for different medical conditions, and the necessary precautions to keep in mind.

Different types of Boswellia serrata supplement

When we talk about "how to take boswellia", it all depends on which boswellia supplement you are taking.

There are 3 main types of Boswellia supplements:

1. Supplements with dried Boswellia gum resin

2. Boswellia extracts, standardized to a high percentage of boswellic acids

3. Boswellia extracts that are high in AKBA, the most important boswellic acid

The main difference between these supplements is how potent there are.

Supplements with dried Boswellia gum resin are the least potent as they contain little amounts of the active ingredients in Boswellia.

Next in potency are standardized Boswellia extracts. These extracts contain a certain percentage of boswellic acids, which are the active ingredients in Boswellia resin. Typically, the extracts have somewhere between 30% and 65% of Boswellic acids. The higher the percentage, the more potent is the supplement.

Finally, there are special, patented formulations that are high in AKBA - which is the most important and beneficial type of boswellic acid.
>>> Read more on how to choose the right Boswellia supplement.
Boswellia serrata dosage

General dosage recommendations for Boswellia

There are no general guidelines for Boswellia dosage. The dosages that we represent below are taken from manufacturers' suggested use and studies made on Boswellia and various medical conditions.

Dosage for supplement that contain Boswellia gum resin (so-called whole herb supplements)

Typical dosage is 800 mg, taken twice or three times per day.

Dosage for standardized Boswellia serrata extracts

Standardized supplements usually contain 60-65% standardized boswellic acids. For these, the recommended dosage ranges from 300-500mg taken thrice a day. They are typically used for alleviating inflammatory symptoms and pain.

Dosage for AKBA-enriched solutions

These are patented technologies that are generally more powerful due to higher concentration of AKBA. They contain 10-30% of AKBA as opposed to 1-3% in normal standardized extracts.

Supplements with 20 to 30% AKBA can be taken in much lower dosage - 100mg to 300 mg taken once per day.

For products with 10% AKBA, the suggested dosage is up to 500mg taken once or twice daily.

Boswellia serrata dosages for specific medical conditions

For osteoarthritis

For best results, go for special Boswellia serrata formulations with 20-30% AKBA. You'll only need 100-300mg a day.

If you're taking regular standardized extracts with 60-65% boswellic acids, raise the dosage to 300-400mg three times daily.

For rheumatoid arthritis

AKBA-concentrated formulations are believed to be more effective than normal standardized extracts at just 100-300mg per day dosage.

On the other hand, standardized Boswellia serrata has been successfully taken at a total of 1,200-3,600mg a day for improved joint swelling in rheumatoid arthritis ( 1) ( 2).

For bronchial asthma

German study has shown that taking 3 capsules of Boswellia resin daily (each with 300mg of gum resin) eased the symptoms of bronchial asthma ( 3).

For ulcerative colitis

Two studies ( 4), ( 5) examined the effects of Boswellia resin on ulcerative colitis. One used 350mg and the other 300mg of resin; taken three times daily in both studies.

The results have shown that Boswellia is more successful than the drug sulfasalazine in treating ulcerative colitis and triggers remission of the disease in larger percentage of patients.

For Crohn's disease

400mg of standardized Boswellia serrata extracts taken thrice daily is clinically proven effective and well-tolerated in Crohn's disease patients ( 6).

For diabetes

One study shows that a total of 900mg Boswellia serrata gum resin taken daily for 6 weeks increased levels of blood HDL, lowered cholesterol, LDL, fructosamine, and other blood parameters in type 2 diabetes patients ( 7).

For cancer

Studies on cerebral edema and glioma have used a total daily intake of 3,600-4,200mg of standardized Boswellia serrata extracts.

Clinical trials on Boswellia treatment for cancer

Patients receiving irradiation of the brain for primary brain tumors or brain metastases of solid tumors were administered 4,200mg a day of Boswellia serrata extracts during radiotherapy. 60% of patients had more than 75% decrease in the edema by the end of the treatment (8) (9).

A study on glioma patients who took 3,600mg a day for 7 days prior to surgery ended up with greatly reduced perifocal edema plus improvement in clinical symptoms. Those who took 2,400mg per day had a lower degree of perifocal edema reduction (10) (11).

In a smaller trial with 12 patients with brain tumors and progressive cerebral edema, 3 of the 7 people with glioblastoma and tumor progression plus 3 of the 5 patients with radiochemotherapy-related leukoencephalopathy had reduced edema after taking 3,600mg daily for 12 weeks. All leukoencephalopathy patients also reported a clinical improvement (12).

A 39-year-old woman with symptomatic multiple brain metastases of a mammacarcinoma took 2,400mg per day of standardized Boswellia serrata for 10 weeks. At the same time, she began receiving brain radiation therapy and capecitabine chemotherapy. Her brain metastases were no longer visible in the CT scan by the treatment's conclusion (13).

When and how to take Boswellia serrata supplement

Most Boswellia extracts are packaged in capsule or softgel form. Take your supplement with a glass of water after having a high fat meal. Boswellia is fat-soluble so it will be better absorbed this way ( 14).

Boswellia serrata for colon conditions

If using Boswellia serrata for inflammatory bowel disease (ulcerative colitis or Crohn's disease), it is thought to be best to take the supplement before meals.

Recommended time of day to take Boswellia serrata supplement

AKBA-rich Boswellia serrata supplements like ApresFlex and 5-LOXIN can be taken during the first meal of the day.

For the less potent standardized extracts, take one capsule every 8 hours. This is how long the substance stays in the body after taken orally ( 15).

Boswellia side effects and safety

Boswellia serrata is regarded as generally safe and well-tolerated ( 16), ( 17), ( 18). On rare occasions, it may induce side effects such as:

Nausea

Diarrhea

Acid reflux

Skin rash

Allergic reactions-if you experience symptoms like chest pain, difficulty in breathing, swelling in different areas of the body, stop taking Boswellia serrata. Consult your doctor to make sure you don't have allergic reactions to the herb.

There has been one case of a woman who ended up with a bezoar, a solid mass of indigestible material that builds up in the digestive tract ( 19). However, it has been speculated that her celiac disease might have been the culprit behind this complication and not Boswellia serrata.

How can you minimize the side effects of Boswellia serrata?

There is no definite way to avoid side effects. However, it has been suggested that if Boswellia serrata causes you to suffer from heartburn, taking esomeprazole (Nexium) alongside it might help.

Boswellia serrata and long-term supplementation

Majority of studies on Boswellia lasted somewhere between 2-12 weeks. However, there have been a few studies that lasted 6, 9, and even 12 months.

Boswellia serrata's has been well tolerated with few side effects; mostly gastrointestinal distress. The dosages of Boswellia used in these studies ranged between 1,200mg and 3,600mg daily ( 20) ( 21).

People who shouldn't take Boswellia supplements

Pregnant and breastfeeding women

Boswellia serrata can induce menstruation, birth-related problems and even miscarriage. If you are pregnant, lactating or planning to have a baby, stay away from Boswellia serrata.

Patients with liver failure

Those who are suffering from liver failure are at greater risk of the side effects because the damaged liver will not be able to process Boswellia serrata properly. This might result in higher levels of Boswellia in blood and increased side effects.

Patients with stomach ulcer or GERD

Steer clear of Boswellia serrata if you have stomach ulcer or GERD as it might worsen your symptoms.

Patients with degenerative lung disease

Boswellia serrata can potentially cause more harm to your lung tissues. Further investigation is needed to confirm this.

Children

Boswellia serrata's safety in children is not well-researched. It is best if you don't give this herb and its extracts to children.

Boswellia serrata: interactions with food, drugs or medications

Blood sugar medications

Boswellia serrata has been found to stabilize blood sugar levels. If you are taking medications for blood glucose regulation, don't take Boswellia serrata extracts as your blood sugar might fall to low.

Other pain relievers (ibuprofen, aspirin, or naproxen)

Refrain from taking Boswellia supplements with non-steroidal anti-inflammatory drugs (NSAIDs) of similar purpose. The combination could cause damage to the liver.

Warfarin

Taking Boswellia serrata with warfarin, a medication that inhibits blood clotting, can trigger excessive bleeding ( 22).

Boswellia serrata may also increase the effects of:

Anti-cancer medications

Asthma medications like Singulair

Glucosamine or chondroitin (supplements for joint health)

Garlic and red yeast (allergic reaction)

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Different types of Boswellia serrata supplementWhen we talk about "how to take boswellia", it all depends on which boswellia supplement you are taking.There are 3 main types of Boswellia supplements:Supplements with dried Boswellia gum resinBoswellia extracts, standardized to a high percentage of boswellic acidsBoswellia extracts that are high in AKBA, the most important boswellic acidThe main difference between these supplements is how potent there are.

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Could Boswellia Prevent 1.5 Million Deaths in the U.S.? | Life Extension

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Abstract

Numerous cancer therapeutics were originally identified from natural products used in traditional medicine. One such agent is acetyl-11-keto-beta-boswellic acid (AKBA), derived from the gum resin of the Boswellia serrata known as Salai guggal or Indian frankincense. Traditionally it has been used in Ayurvedic medicine to treat proinflammatory conditions. In the present report, we hypothesized that AKBA can affect the growth and metastasis of colorectal cancer (CRC) in orthotopically-implanted tumors in nude mice. We found that the oral administration of AKBA (50-200 mg/kg) dose-dependently inhibited the growth of CRC tumors in mice, resulting in decrease in tumor volumes than those seen in vehicle-treated mice without significant decreases in body weight. In addition, we observed that AKBA was highly effective in suppressing ascites and distant metastasis to the liver, lungs, and spleen in orthotopically-implanted tumors in nude mice. When examined for the mechanism, we found that markers of tumor proliferation index Ki-67 and the microvessel density CD31; were significantly downregulated by AKBA treatment. We also found that AKBA significantly suppressed NF-κB activation in the tumor tissue and expression of pro-inflammatory (COX2), tumor survival (bcl-2, bcl-xL, IAP-1, survivin), proliferative (cyclin D1), invasive (ICAM-1, MMP-9) and angiogenic (CXCR4 and VEGF) biomarkers. When examined for serum and tissue levels of AKBA, a dose-dependent increase in the levels of the drug was detected, indicating its bioavailability. Thus, our findings suggest that this boswellic acid analogue can inhibit the growth and metastasis of human CRC in vivo through downregulation of cancer-associated biomarkers.

Keywords: AKBA, colorectal cancer, NF-κB, growth, metastasis

Introduction

Colorectal cancer (CRC) is the third most-common malignancy and the fourth most-frequent cause of cancer death in the United States. Lifestyle factors such as diet, exercise, and obesity have been linked to CRC risk. It seemed a bit broad to say it is primarily a disease of lifestyle . Although surgical excision, chemotherapy, and radiotherapy are available, survival is still very poor when the disease is diagnosed at an advanced stage and better treatments are desperately needed. Thus, novel therapeutic agents, multitargeted to several mechanisms of carcinogenesis, are needed.

One increasingly promising source of therapeutic agents is traditional medicine from natural compounds. Numerous cancer therapeutics, including vincristine, vinblastine, camptothecin, doxorubicin, and paclitaxel, had their roots in plants thought to have medicinal value in traditional medicine. Several of these compounds have proved to be safe, effective, and multitargeted. One promising compound is boswellic acid, a pentacyclic triterpene derived from the fragrant gum resin of the bark of the Boswellia serrata tree. This resin is known as Salai guggul or Indian frankincense and is thought to be related to Biblical frankincense, which has been used for thousands of years for medicinal purposes. Boswellia has been used for centuries in traditional Ayurvedic medicine as a treatment for a wide variety of proinflammatory disorders. More recently, acetyl-11-keto-beta-boswellic acid (AKBA) has been tested in a number of such disorders, including rheumatoid arthritis , ulcerative colitis , and trinitrobenzene sulphonic acid-induced colitis in rats . In humans, Boswellia extracts have been shown to exhibit activity against osteoarthritis , inflammatory bowel disease , chronic colitis , and collagenous colitis .

AKBA has been shown to inhibit leukotriene biosynthesis from endogenous arachidonic acid in intact peripheral mononuclear neutrophils through the inhibition of 5-lipooxygenase (LOX), yet has no effect on 12-LOX and cyclooxygenase (COX)-1 observed. Boswellic acid can directly inhibit 5-LOX with a half maximal inhibitory concentration as low as 1.5 μM . Other pentacyclic triterpenes (amyrin and ursolic acid) lack this effect on 5-LOX. Further studies have revealed that the pentacyclic triterpene ring structure, hydrophilic group on C4 ring A, and 11-keto function are all essential for this 5-LOX inhibitory activity . It has been shown by photoaffinity labeling that AKBA binds to 5-LOX at a site distinct from its substrate binding site . Unlike other pentacyclic triterpenes, boswellic acid inhibits the leukocyte elastase with a half maximal inhibitory concentration of 15 μM . In addition to 5-LOX and elastase, AKBA was also found to bind and inhibit topoisomerase I and II α . Its affinity constant for topo I and II α were 70.6 nM and 7.6 nM, respectively. Amazingly, this triterpene are "more potent inhibitors of human topoisomerases I and IIalpha than camptothecin, and amsacrine or etoposide, respectively."

AKBA has been shown to inhibit the growth of a wide variety of tumor cells, including glioma , colon cancer , leukemia , human melanoma , hepatocellular carcinoma , and prostate cancer . Although AKBA can inhibit the growth of CRC cells and has shown activity against inflammatory bowel disease , whether it can inhibit the growth and metastasis of CRC in an orthotopic mouse model is not known. In the present report, we evaluated the activity of the orally administered boswellic acid analogue AKBA in a mouse xenograft model of human CRC. Our results indicate that AKBA is bioavailable and inhibits the growth and metastasis of human CRC in vivo through downregulation of inflammatory, proliferative, invasive, and angiogenic biomarkers.

Material and Methods

Materials

AKBA was kindly supplied by Sabinsa Corp (Piscataway, NJ). Polyclonal antibodies against intercellular adhesion molecule 1 (ICAM-1), cyclin D1, matrix metalloproteinase-9 (MMP-9), and IAP-1, and monoclonal antibodies against, Bcl-2, COX-2 and Bcl-xL were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Survivin was obtained from R&D system (Minneapolis, MN) and vascular endothelial growth factor (VEGF) was obtained from Thermo Fisher Scientific (Waltham, MA). Polyclonal antibodies against CXC receptor 4 (CXCR4) were obtained from Abcam (Cambridge, MA). The liquid DAB 1 substrate chromogen system with horseradish peroxidase that was used for immunocytochemical (IHC) analysis was obtained from DakoCytomation Ltd (Carpinteria, CA).

Animals

Male athymic nu/nu mice (4 weeks old) were obtained from the breeding colony of the Department of Experimental Radiation Oncology at The University of Texas MD Anderson Cancer Center (Houston, TX). The animals were housed 5 per cage in standard acrylic cages in a room maintained at constant temperature and humidity. Mice were housed under 12-h light and darkness cycles and were fed a regular autoclave chow diet with water ad libitum. Before initiating the experiment, we acclimatized all mice to a pulverized diet for 3 days. Mice were checked to ensure they were lesion-free and pathogen-free. Our experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at MD Anderson Cancer Center.

Orthotopic human CRC mouse model

Human CRC cell line HCT116 was stably transfected with luciferase as previously described . The luciferase-transfected HCT116 cells were then harvested from subconfluent cultures after brief exposures to 0.25% trypsin and 0.2% ethylenediaminetetraacetic acid. Trypsinization was stopped with a medium containing 10% fetal bovine serum. The cells were washed once in serum-free medium and resuspended in PBS. Only suspensions consisting of single cells with >90% viability were used for injections. Mice were anesthetized with a ketamine/xylazine solution, a small left abdominal flank incision was made, and 1.5 × 10 ⁶ HCT116 cells suspended in 50 μL PBS were injected into each mouse's cecum using a 30-gauge needle and a calibrated push button-controlled dispensing device (Hamilton Syringe Company; Reno, NV). To prevent leakage, a cotton swab was held cautiously for 1 min over the injection site. The abdominal wound was closed in one layer with wound clips (Braintree Scientific, Braintree, MA).

Experimental protocol

One-and-a-half weeks after implantation, mice were randomly assigned to one of the following treatment groups (n = 6): (i) corn-oil vehicle only (100 μL daily) as the control; (ii) AKBA (50 mg/kg once daily, orally); (iii) AKBA (100 mg/kg once daily, orally); and (iv) AKBA (200 mg/kg once daily, orally by gavage) (). Tumor volumes were monitored weekly using the IVIS 200 bioluminescence imaging system and Living Image software (Caliper Life Sciences, Hopkinton, MA). Before imaging, animals were anesthetized in an acrylic chamber with a 2.5% isoflurane/air mixture and injected intraperitoneally with 40 mg/mL of D-luciferin potassium salts suspended in PBS at a dose of 150-mg/kg. After 10 min of incubation with the luciferin, mice were placed in a right lateral decubitus position and a digital gray-scale image was acquired, followed by the acquisition and overlay of a pseudocolor image representing the spatial distribution of detected photons emerging from the active luciferase within the animal. Signal intensity was quantified as the sum of all detected photons within the region of interest per second per steradian.

Mice were imaged on days 0, 7, 14, 21, and 28 of treatment. Therapy was continued for 4 weeks and blood was collected by cardiac puncture after 2 h of AKBA treatment. Animals were sacrificed after 28 days. Blood plasma was separated and stored at -80°C. Primary tumors in the cecum were excised, and the final tumor volume was measured as V= 2/3 πr ³, where r is the mean of the three dimensions (length, width, and depth). Metastatic colonies were counted in the liver, intestines, lungs, rectum, and spleen. The final tumor volume and the number of metastases were initially subjected to 1-way analysis of variance (ANOVA) and then later compared among groups using an unpaired Student t-test. Each excised tumor was divided into three parts. The first part of the tumor tissue was formalin-fixed and paraffin-embedded for routine hematoxylin-and-eosin staining and immunohistochemical (IHC) analysis. The second part was fixed in OCT for IHC analysis of Ki-67 and CD31. The third part was snap frozen in liquid nitrogen and stored at -80°C for archiving. Hematoxylin-and-eosin staining confirmed the presence of tumors in each cecum.

Measurement of NF-κB in CRC tissues

CRC tumor tissues (75-100 mg per mouse) from both control and experimental mice were minced and incubated on ice for 30 min in 0.5 mL of ice-cold buffer A consisting of 10 mM HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl ₂, 0.5 mM dithiothreitol, 0.1% IGEPAL CA-630 in the paragraph about materials. Also, please tell what the 0.1% was dissolved in.], and 0.5 mM phenylmethylsulfonyl fluoride. The minced tissue was homogenized using a dounce homogenizer and centrifuged at 16,000 g at 4°C for 10 min. The resulting nuclear pellets were suspended in 0.2 mL of buffer B consisting of 20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl ₂, 420 mM NaCl, 0.5 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride, and 2-μg/mL leupeptin and were incubated on ice for 2 h with intermittent mixing. The suspension was then centrifuged at 16,000 g at 4°C for 30 min. The supernatant (nuclear extract) was collected and stored at -70°C until use. Protein concentration was measured by the Bradford protein assay with bovine serum albumin as the standard.

To assess NF-κB activation, we isolated nuclei from CRC tissue and carried out electrophoretic mobility shift assays as previously described . In brief, nuclear extracts prepared from CRC tissue (as described in the previous step) were incubated with ³²P-end-labeled 45-mer double-stranded NF-κB oligonucleotides (4 μg of protein with 16 fmol of DNA) from the human immunodeficiency virus long-terminal repeat (5′-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGA GGCGT GG-3' boldface indicates NF-κB binding sites) for 15 min at 37°C. The resulting DNA-protein complex was separated from free oligonucleotides on 6.6% native polyacrylamide gels. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (GE Healthcare, Sunnyvale, CA) using ImageQuant software.

Immunolocalization of VEGF, COX-2, MMP-9, and cyclin D1 in CRC tissues

The expression of VEGF, COX-2, MMP-9, and cyclin D1 were evaluated using an IHC method described previously . In brief, CRC tumor samples were fixed with paraformaldehyde and embedded in paraffin. After being washed in PBS, the slides were blocked with protein block solution (DakoCytomation) for 20 min and incubated overnight with mouse monoclonal antihuman VEGF, COX-2, MMP-9, and cyclin D1 antibodies (1:100, 1:200, 1:100, and 1:200, respectively). Slides were washed and incubated with biotinylated link universal antiserum, followed by horseradish peroxidase-streptavidin conjugate. The slides were rinsed and their color was developed using 3,3-diaminobenzidine hydrochloride as a chromogen. Finally, sections were rinsed in distilled water, counterstained with Mayer's hematoxylin solution, and mounted with DPX mounting medium for evaluation. Pictures were captured with a Photometrics CoolSnap CF color camera (Nikon, Lewisville, TX) and were analyzed with MetaMorph version 4.6.5 software (Universal Imaging; Downingtown, PA).

IHC analysis of Ki-67 in CRC tissues

Frozen sections (5 μm) were stained with anti-Ki-67 antibody (rabbit monoclonal clone SP6; NeoMarkers; Fremont, CA) as previously described . Results were expressed as the percentage of Ki-67-positive cells ± the standard error per 40x magnification. Ten 40x magnification fields were examined and counted for each of three tumors from each treatment group. The values were initially analyzed using a 1-way ANOVA and then compared among groups using an unpaired Student t-test.

Microvessel density in CRC tissues

Ethanol-fixed, paraffin-embedded sections (5 μm) were stained with rat antimouse CD31 monoclonal antibodies (Pharmingen, San Diego, CA) as previously described . Areas of highest microvessel density were then examined under high magnification (100x) and counted. Any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean number of vessels ± the standard error per high-power field (100x magnification). Twenty high-power fields were examined and counted for each of three tumors from each treatment group. The values were subjected to a 1-way ANOVA and then compared among groups using an unpaired Student t-test.

Western blot analysis of CRC biomarkers

Tumor tissues (75-100 mg per mouse) from control and experimental mice were minced and incubated on ice for 30 min in 0.5 mL of ice-cold whole-cell lysate buffer consisting of 10% NP-40, 5 M NaCl, 1 M HEPES, 0.1 M ethyleneglycoltetraacetic acid, 0.5 M ethylenediaminetetraacetic acid, 0.1 M phenylmethylsulfonyl fluoride, 0.2 sodium orthovanadate, 1 M NaF, 2 μg/mL aprotinin, and 2 μg/mL leupeptin. Minced tissue was homogenized using a dounce homogenizer and centrifuged at 16,000 g at 4°C for 10 min. Proteins were then fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membranes, blotted with each antibody, and then detected by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ).

Measurement of serum and tissue levels of AKBA

AKBA was determined by reverse-phase high performance liquid chromatography (HPLC) on a high-pressure chromatograph (Waters, Milford, Massachusetts) equipped with an ultraviolet detector and a 2.0 × 150-mm column (Gemini C18; particle size, 5 μm, 110A ⁰). The column was eluted in an isocratic flow with a component acetonitrile:methanol:acetic acid (1%) ratio of 70:20:10 at an eluent flow rate of 0.35 mL/min. The sample volume was 10 μL. The target compounds were detected at 250 nM. The solution components were identified and quantitatively analyzed using the external standard method with linear regression equations, reliability of approximation (R ²), and detection thresholds for AKBA by HPLC.

The pharmacokinetics of AKBA was studied in a treatment group of 24 mice that weighed 25-30 g. The AKBA was administered by mouth as a corn oil suspension in a dose of vehicle, 50, 100, or 200 mg/kg. The blood samples were taken from animals 3 h after the administration of AKBA or vehicle. The samples were centrifuged for 20 min at 2000 rpm to obtain the blood plasma, treated with acetone (1.5:1) for deproteination, and centrifuged again for 15 min at 3000 rpm. The supernatant fraction was separated and extracted with chloroform (2:1). The chloroform extract was separated, the solvent was removed, and the dry residue was dissolved in methanol and analyzed by HPLC.

Statistical analysis

Statistical comparisons between drug-treated groups and the untreated control group were performed using 1-way ANOVAs. Data were presented as means ± standard deviations. P values of < 0.05 were considered statistically significant.

Results

The goal of our study was to determine whether AKBA can be used as a potential therapeutic agent against advanced CRC. To determine this, a nude mouse model with orthotopically transplanted human CRC was employed. The mechanisms by which AKBA manifests its effects were investigated in vivo at different dose concentrations. To facilitate tumor imaging in mice, luciferase-transfected human CRC cells were used.

AKBA inhibits growth of human CRC in mice

We used an orthotopically implanted human CRC xenograft nude mouse model to investigate the effects of AKBA on tumor growth. The protocol employed was outlined in . We found a much more rapid increase in tumor volume in the vehicle group than in the three treatment groups. AKBA significantly suppressed tumor volume as determined by bioluminescence imaging in vivo () and by autopsy on the last day of the experiment using Vernier calipers (). The tumor volume in the group receiving the highest dose of AKBA (200 mg/kg) was significantly lower than that in the control group on day 28 after treatment ( P < 0.001 vs. vehicle) (, right panel). In our present tumor inhibition model, the average tumor volume in the control mice increased from 2.49 ± 0.39 mm ³ to 8.91 ± 0.41 mm ³ after 28 days, whereas the average tumor volume in the AKBA-treated mice (200 mg/kg) decreased from 8.91 ± 0.41 mm ³ to 4.16 ± 0.19 mm ³, indicating that the proliferation rate of tumor cells in treated mice was greatly inhibited by AKBA. The results showed that AKBA 200 mg/kg ( P < 0.001 vs. control) significantly decreased CRC growth; growth was further decreased in a dose-dependent manner ( P < 0.034 vs. AKBA [100 mg/kg]; , left panel and right panel). Additionally, there was no significant change in average animal weight between the control group and treated groups (). Although we did not use survival as an endpoint, enhanced survival might have been expected in the treatment group.

AKBA inhibits distant organ metastasis and ascites in mice

Whether AKBA can inhibit the formation of ascites and distant organ metastasis was also investigated. We monitored the animals every day for ascites. Vehicle-treated mice developed ascites 21 days after tumor cell implantation, and this presence of ascites was confirmed at the time of sacrifice. AKBA significantly decreased the incidence of ascites in nude mice at 100 mg/kg, and at 200 mg/kg, the decrease was profound ().

On day 35, mice were sacrificed and examined for the presence of metastasis. The results showed that CRC had metastasized to liver, lungs, and spleen in all control mice. However, AKBA inhibited metastasis to most organs when compared with the control vehicle ().

AKBA downregulates proliferative and angiogenic biomarkers in CRC tissue

To determine how AKBA decreases CRC growth and metastasis, we first examined markers of proliferation and angiogenesis, Ki-67 and CD31, respectively. Our results showed that AKBA significantly decreased the expression of Ki-67 (, left panel), and the largest decrease occurred at a dose of 200 mg/kg (, right panel). AKBA also significantly decreased the expression of CD31, a marker of microvessel density (, left panel), and again maximum decrease occurred at a dose of 200 mg/kg (, right panel).

AKBA inhibits NF-κB activation in human CRC in mice

Because NF-κB has been linked to the growth and metastasis of CRC, we examined whether AKBA affects NF-κB activation in CRC tumor tissue. Electrophoretic mobility shift assay analysis for NF-κB in nuclear extracts from tumor samples showed that AKBA inhibited NF-κB activation in a dose-dependent manner. Maximum inhibition was found at a dose of 200 mg/kg ().

AKBA downregulates the expression of inflammatory, survival, proliferative, invasive, and angiogenic proteins

NF-κB has been shown to regulate the expression of genes involved in inflammation, survival, proliferation, invasion, and angiogenesis . Therefore, we evaluated the expression of these molecules in CRC tumor tissue from control and treated mice. A Western blot analysis revealed that AKBA decreased the expression of genes involved in inflammation (COX-2), proliferation (cyclin D1), invasion (MMP-9 and ICAM-1), angiogenesis (VEGF), and metastasis (CXCR4) (). The expression of the antiapoptotic gene products Bcl-2, Bcl-xL, survivin, and IAP-1 was also downregulated at the dose of 200 mg/kg ().

We also analyzed the expression of COX-2, VEGF, MMP-9, and cyclin D1 by IHC. We found that the expression of all these proteins was downregulated by AKBA in a dose-dependent manner in CRC tissue from orthotopically transplanted nude mice (). These results were in agreement with those of the Western blot analysis.

Bioavailability of AKBA

Serum and CRC tissue levels of AKBA were examined by HPLC compared with pure AKBA (). Serum levels of AKBA were determined 2 h after oral administration of 50, 100, or 200 mg/kg of the drug in mice (). Levels of 138.8 ± 55.24, 477.6 ± 76, and 594.5 ± 40.36 ng/mL of the drug were detected at 50, 100, and 200 mg/kg doses, respectively. Levels in CRC tissues were 291.8 ± 14.4, 370.6 ± 3.6, and 405.0 ± 3.9 ng/g tissue at 50, 100, and 200 mg/kg doses, respectively (). These results indicate that there is a strong correlation between tumor regression and the amount of AKBA in the blood plasma.

Discussion

CRC is one of the most lethal cancers, especially when it reaches the advanced stages. Because many cancer therapeutics have their roots in natural products, we investigated the effect of a boswellic acid analogue derived from a fragrant gum resin commonly used in Ayurvedic medicine for centuries. We investigated the effects of this triterpene on the growth and metastasis of CRC tumors in orthotopically transplanted nude mice. We found that AKBA alone suppressed the proliferation and metastasis of human CRC. This correlated with the downregulation of various biomarkers linked to inflammation, cell proliferation, cell survival, invasion, and angiogenesis ().

We found that AKBA at 50 mg/kg significantly inhibited CRC growth in an orthotopic mouse model, but when the dose was increased to 200 mg/kg, it further enhanced the antitumor effects. This is the first report, to the best of our knowledge, wherein an orthotopic CRC model was applied to evaluate the preventive effects of oral administration of AKBA on CRC growth, metastasis, and CRC-induced ascites. More than 70% of tumor growth was inhibited by 200 mg/kg AKBA, and this level of inhibition was also seen in dose-dependent manner. The animals tolerated AKBA very well, as indicated by the lack of significant weight differences between treated and untreated mice from the start of the study.

The mechanisms by which AKBA acted in vivo were also investigated in detail. We found that oral administration of this triterpene decreases the levels of the proliferation marker Ki-67 and the microvessel density marker CD31. Additionally, the observed downregulation of various cell survival proteins and cyclin D1 may also contribute to the mechanism of action of AKBA against CRC. Numerous reports have suggested that cyclin D1 is closely associated with the proliferation of cancer cells . Dai et al found that abnormal expression of cyclin D1 exists in CRC and may play an important role in its carcinogenesis . Our findings suggested that AKBA downregulated cyclin D1 expression in CRC tumor tissues. Overexpression of COX-2 has been linked to the proliferation of CRC . The suppression of COX-2 expression by AKBA in CRC tissue may also account for its antitumor activity. We found that NF-κB in CRC tissue was downregulated in mice given AKBA compare with the controls. Since NF-κB activation has been linked with the growth of CRC, this may be another mechanism of action in this triterpene.

We found that the metastasis of CRC tumor to the lungs, liver, and spleen was also significantly suppressed by AKBA. The mechanism by which suppression of metastasis occurs was also investigated in detail. Although COX-2 expression has been associated with CRC metastasis , VEGF is the primary and most potent inducer of angiogenesis . VEGF activates cellular signaling pathways by binding to its receptor tyrosine kinase, which promotes several events required for angiogenesis, including endothelial cell survival, proliferation, and migration, and vascular permeability . In the present study, AKBA downregulated the expression of VEGF in tumor tissues, indicating that it has antiangiogenic effects. Our results are in agreement with those of Pang et al, which suggested that AKBA inhibits prostate tumor growth by suppressing VEGF receptor 2-mediated angiogenesis in mice We also found that AKBA downregulates CXCR4. The overexpression of CXCR4 and its role in CRC metastasis has been reported . Kim et al showed that CXCR4 expression in CRC patients increases the risk of recurrence, liver metastasis, and poor survival . AKBA also suppressed the expression of the invasive gene ICAM-1 in CRC tissue. AKBA inhibited metastasis-associated MMP-9 expression in CRC tumors as well . This study suggested that AKBA suppressed the expression of various biomarkers important to the progression and metastasis of CRC tumors.

A very important aspect of an agent's ability to suppress tumors is the agent's bioavailability, which serves primarily a protective regulatory function constraining diffusion across capillaries in relation to lipid solubility and molecular weight. For a successful chemotherapeutic treatment, especially that involving tumor cell clusters invading adjacent tissues; the chemotherapy must be able to reach the blood. AKBA shows high lipid solubility , and we detected it in concentrations of up to 405 ng/gm in CRC tumors, which corresponded to concentrations of up to 494.5 ng/mL in the plasma at a dose of 200 mg/kg. The concentration of AKBA was a little higher in the plasma than in the CRC tissues. These results agree with those reported by Reising and colleagues in a previous study of AKBA pharmacokinetics. Thus, these data demonstrate substantial levels of AKBA in CRC tumor tissue after a single oral dose and confirm the bioavailability of AKBA. These results are further correlated with the tumor volume suppression and decrease in metastases to the lungs, liver, and spleen that we found with AKBA treatments.

Although our studies are the first to show the effect of AKBA against orthotopically implanted tumors, there are some reports about the chemopreventive effects of this agent against CRC. Previously, Boswellia extracts have been shown to be effective in animal models of chronic colonic inflammation-associated fibrosis in rats. Latella et al showed that 2,4,5-trinitrobenzene sulphonic acid-induced colitis in rats is prevented by oral administration of Boswellia extracts (50 mg/kg/day) . Another study showed that AKBA can confer protection to mice with experimental ulcerative colitis induced by dextran sodium sulfate .

Boswellia extracts (350 mg thrice daily for 6 weeks) showed impressive effects in patients suffering from ulcerative colitis grades II and III compared with controls treated with sulfasalazine (1 g thrice daily). All parameters tested improved after treatment with Boswellia serrata gum resin, with the results being similar to those of the controls: 82% of treated patients went into remission, as did 75% in the sulfasalazine group. Another study investigated the effects of AKBA in patients with chronic colitis . Out of 20 patients treated with the agent, 18 showed an improvement in one or more study parameters; 14 went into remission compared to four of 10 in the sulfasalazine group. Boswellia serrata extracts have also been tested for the treatment of collagenous colitis in a double-blind, randomized, placebo-controlled, multicenter trial . They concluded that Boswellia serrata extracts might be clinically effective in patients with collagenous colitis and suggested larger trials were needed to establish clinical efficacy.

Overall, our study showed that the natural compound AKBA acts as a promising agent against CRC tumors in vivo. In our xenograft mouse model, AKBA not only reduced tumor growth, but also suppressed distant metastasis and ascites, which strongly correlated with the inhibition of numerous biomarkers linked to inflammation, proliferation, invasion, angiogenesis, and metastasis.

Acknowledgments

We thank Walter P agel, Department of Scientific Publications for carefully editing the manuscript. Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research. This work was supported by supported by a core grant (CA-16672), and a program project grant from National Institutes of Health (NIH CA-124787-01A2), and a grant from the Center for Targeted Therapy of MD Anderson Cancer Center.

Footnotes

Conflict of Interest Authors declare no conflicts of interest.

References

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Boswellic Acid Inhibits Growth and Metastasis of Human Colorectal Cancer in Orthotopic Mouse Model By Downregulating Inflammatory, Proliferative, Invasive, and Angiogenic Biomarkers

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Numerous cancer therapeutics were originally identified from natural products used in traditional medicine. One such agent is acetyl-11-keto-beta-boswellic acid (AKBA), derived from the gum resin of the Boswellia serrata known as Salai guggal or Indian frankincense. Traditionally it has been used in Ayurvedic medicine to treat proinflammatory conditions.

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An article published in the Guardian:

A heart disease and cancer-fighting "superbroccoli" developed by British scientists goes on sale in the UK today.
The vegetable looks the same as normal broccoli but contains boosted levels of glucoraphanin, which may protect the body against heart disease and some types of cancer .

The new broccoli , called Beneforte, contains two to three times more glucoraphanin than standard broccoli . It will be sold at Marks & Spencer stores from and make an appearance on the shelves of other supermarkets next year.

Beneforte was developed by British scientists using conventional breeding techniques rather than genetic engineering. Work on the project began after a wild broccoli variety was discovered in 1983 with naturally raised levels of glucoraphanin.

The nutrient is converted in the gut into the bioactive compound sulphoraphane, which circulates in the bloodstream.

Evidence indicates that sulphoraphane has beneficial effects such as reducing chronic inflammation , stopping uncontrolled cell division associated with early-stage cancer , and boosting the body's anti oxidants .

Compared with normal broccoli , eating Beneforte broccoli raises sulphoraphane levels two to four times.

Broccoli is believed to protect against some cancers, especially bowel and prostate. Studies have shown that men with broccoli-rich diets have a reduced risk of aggressive prostate cancer .

Other research suggests broccoli can help prevent heart attacks and strokes by reducing inflammation and keeping blood flowing freely through arteries.Beneforte broccoli was developed at the Institute of Food Research (IFR) and John Innes Centre, both based in Norwich.

Professor Richard Mithen, from the IFR, said: "Our research has given new insights into the role of broccoli and other similar vegetables in promoting health , and has shown how this understanding can lead to the development of potentially more nutritious varieties of our familiar vegetables .

"Now there will also be something brand new for consumers to eat as a result of the discoveries we have made."

The science minister, David Willetts, praised the "fantastic achievement" of the scientists.

He added: "This excellent work has led to the development of a highly commercial food product that will be both grown and sold in the UK, giving a real boost to agriculture, our personal health and the economy."

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Cancer Fighting 'Super Broccoli' on Sale in UK

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An article published in the Guardian: A heart disease and cancer-fighting "superbroccoli" developed by British scientists goes on sale in the UK today. The vegetable looks the same as normal broccoli but contains boosted levels of glucoraphanin, which may protect the body against heart disease and some types of cancer .

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What is Tualang honey?

We all know that honey is good for you and it has been eaten for thousands of years for its nutritional and therapeutic qualities. In recent times, the scientific community has started to look into the therapeutic value of honey and has focused largely on New Zealand's manuka honey which has acquired a reputation for its curative properties.

Tualang honey is probably not as well-known as manuka but has recently come under the microscope with its many health benefits being examined. Tualang honey is harvested from the hives of wild honey bees on the Tualang trees native to the Malaysian jungle. While it may be relatively unfamiliar in the west, it has a long history of traditional use in Southeast Asia for a variety of health purposes.

Many of the health benefits of tualang honey can be attributed to its potent antioxidant and antibacterial properties. According to the published literature, tualang honey also exhibits anti-inflammatory, antidiabetic and antitumor activities. These varied therapeutic properties give tualang honey a wide variety of medicinal applications that have been studied fairly extensively.

A thorough review of the medicinal effects of tualang honey was published in 2013 (1) and this article will take a look at some of the medicinal benefits of this remarkable product.

Therapeutic Benefits of Tualang honey

1. Antioxidant Activity

Most honey is known to possess antioxidant activity but tualang honey is an especially rich source of antioxidants that include phenolic acids, flavonoids, amino acids and proteins. Indeed tualang honey compares favorably with other well-known therapeutic grade honeys such as manuka and Slovenian honeys.
Antioxidants are essential to good health; they protect against cellular damage caused by free radicals, help repair damaged cells and prevent future disease.
Comparative studies have shown that tualang honey has an extremely high phenolic and flavonoid content which gives it more than the normal antioxidant value of other honeys. Research has also demonstrated that tualang honey shielded the body from oxidative stress in diabetic rats. (2)

2. Antibacterial activity

Nearly all varieties of honey have antimicrobial effects but it is important to note that not all honey is equally effective. Manuka honey is especially famous for its antibacterial qualities but research has demonstrated that tualang honey is equally effective against several strains of bacteria.

Tualang honey protects against the growth of common skin bacteria as well as inhibiting the spread of dangerous strains such as streptococcus, salmonella and staphylococcus aureus. Some of these strains of bacteria are caused by hospital infections and have become drug resistant meaning that researchers have been looking for safe, alternative remedies to treat them.

Tualang honey has proven especially effective in preventing bacteria in burn wounds because of its high flavonoid and phenolic content. One study concluded that it could be used as an alternative wound dressing and by comparison it proved less sticky than manuka. (3)

3. Diabetes

Contrary to still popular belief, honey is not always bad for diabetic patients. Many people are concerned that the high sugar content of honeys will have adverse effects on diabetes but certain honey like tualang are high in fructose which is low on the glycemic index and is therefore recommended for sufferers of the disease. Studies conducted on diabetic rats demonstrated that in combination with hypoglycemic agents, tualang honey caused a significant reduction in blood sugar levels. (4)

4. Cancer

Tualang honey has great potential in the treatment of certain forms of cancer and can help improve the effectiveness of more conventional cancer treatments like chemotherapy and oral medications. Studies have shown that because of its abundant flavonoid and phenolic acid content, tualang honey has a significant impact on breast cancer cells which compares favorably with the anticancer activity of tamoxifen. (5)

5. Wound healing

As I mentioned earlier in the article, tualang honey has antibacterial qualities that give it major wound healing potential. Tualang honey works on wounds and burns by easing the inflammation and fluid build-up in the wounded area while stimulating the growth of new cells.

A number of studies have been conducted into the wound healing abilities of tualang honey with the results proving largely positive. One study published in 2010 was conducted into the effects of tualang honey as a wound dressing in full thickness burn wounds that typically require skin graft surgery.

The results showed that wounds treated with tualang honey exhibited a very fast 32% decrease in wound size as early as the sixth day of treatment as well as a significant reduction on bacterial growth. (6)

6. Heart health

Animal studies have also indicated that tualang honey has the potential to keep your heart healthy and protect against cardiovascular disease. One very recent study published earlier this year concluded that tualang honey demonstrated protective effects in rats induced with myorcardial infraction. Its effect on heart health was attributed to improved antioxidant levels in the heart tissue as well as reduced lipid peroxidation levels. (7)

Other studies have also revealed that tualang honey may reduce blood pressure and decrease cholesterol levels which have a major impact on the heart's health.

7. Postmenopausal symptoms

For the millions of women suffering from postmenopausal symptoms, tualang honey may provide a natural and safe alternative to conventional HRT treatments. Despite the many adverse effects caused by the menopause, many women are reluctant to undergo HRT therapy because of the wide range of potential adverse side effects.

A comprehensive study was conducted by the University of Science in Malaysia to examine the effects of tualang honey on a range of postmenopausal symptoms including hormonal deficiency and bone density. (8) Postmenopausal women treated with tualang honey experienced a reduction in menopausal symptoms and an in increase in their bone strength and mineral density.

Indeed the effect of tualang honey on bone density was comparable with the effects of HRT when taken at a dose of 20mg a day. Researchers found that a dose of 20mg a day was safe and concluded that tualang honey could be used to protect against bone weakening conditions without the risk of side effects.

8. Sperm count

Tualang honey has also demonstrated the ability to increase sperm count and also improve sperm motility in men. Experiments on rats have shown that tualang honey can help repair some of the toxic effects of tobacco smoke on sperm production as well as increasing testosterone levels.

One recent study published in 2009showed that tualang honey was as effective as Tribestan in improving sperm count parameter when consumed for a 12 week period. (9) Furthermore the study showed that the honey was safe and no adverse effects were reported.

With all of these amazing health benefits, I am sure that you are eager to try it out for yourself. Depending on where you live, tualang honey may be available in your local health food store or supermarket.

If you cannot find it locally, it is available online from a variety of websites. Like manuka honey, it does not come cheap but we feel the benefits make it well worth the money.

You can eat it directly or add a spoonful to sweeten your drinks, juices or add it to your smoothies to give it an even healthier kick.
(1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743976/
(2)http://www.ncbi.nlm.nih.gov/pubmed/20398890
(3)http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908556/
(4)http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065678/
(5)http://www.ncbi.nlm.nih.gov/pubmed/23476711
(6)http://www.ncbi.nlm.nih.gov/pubmed/20815896
(7)http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433628/
(8)https://clinicaltrials.gov/ct2/show/NCT01300676
(9)http://www.ncbi.nlm.nih.gov/pubmed/25505918

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8 Amazing Tualang Honey Health Benefits - Healthy Focus

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We all know that honey is good for you and it has been eaten for thousands of years for its nutritional and therapeutic qualities. In recent times, the scientific community has started to look into the therapeutic value of honey and has focused largely on New Zealand's manuka honey which has acquired a reputation for its curative properties.

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Research has established that exposure to the sun, with its damaging ultraviolet wavelengths, is responsible for most skin . Skin cancer development due to exposure to destructive light wavelengths is called "photocarcinogenesis." Ultraviolet B (UVB) rays damage the skin's surface, while Ultraviolet A (UVA) penetrates more deeply into the dermis.

Researchers studied the effects of Tualang honey on mouse tissue with early-stage cancer markers after exposure to UVA and UVB radiation. They discovered that the honey, "Inhibited UVB-induced DNA damage and afforded substantial protection from the adverse effects of UBV radiation." Another demonstrated that by using the honey on that burn wounds prior to skin graft surgery, infection was avoided and the size of burns decreased 32 percent.

Tualang Honey and Gamma Irradiation

Gamma radiation is considered the most harmful form of natural radiation. The other types of natural radiation, alpha and beta, take the form of particles, but gamma rays are a form of photon wave. Gamma radiation is, according to dictionary.com, " A photon of penetrating electromagnetic radiation emitted from an atomic nucleus." And gamma radiation, including that which is emitted by a nuclear explosion, is toxic to life, causing immediate, significant damage on the cellular level. Long-term, low-level exposure can cause serious health issues.

According to , we are exposed to, "Natural background radiation from cosmic rays, radioactive elements present in the soil, man-made medical radiation such as x-rays, food irradiation, airport security scanners, and exposure as a result of above-ground nuclear detonation from testing."

In the modern world, gamma radiation exposure, even at low levels, is inestimably higher than it was before WWII - it's a fact of life. So are irradiated processed foods, and consumers have expressed widespread concern about the likelihood of irradiation destroying food's nutritional value. But when researchers irradiated Tualang honey with gamma radiation, they were astonished to find that the antioxidant, free-radical scavenging properties of the honey increased up to 111 percent - a most unexpected outcome. Irradiation made the honey's benefits more potent.

A Google search will return Tualang honey sellers - many on Ebay and Etsy. Caveat emptor (buyer beware) - look for sellers with good ratings and reviews. Multiple sellers are on Amazon, and a search will return both Tualang and Manuka honeys. Reading product descriptions is an education, as Tualang comes in black, yellow, and red grades. But prepare for sticker shock; the hand-harvested honey can cost up to $21 an ounce.

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A Gift from the Malaysian Jungle: Tualang Honey | Gaia

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Research has established that exposure to the sun, with its damaging ultraviolet wavelengths, is responsible for most skin . Skin cancer development due to exposure to destructive light wavelengths is called "photocarcinogenesis." Ultraviolet B (UVB) rays damage the skin's surface, while Ultraviolet A (UVA) penetrates more deeply into the dermis.

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Boswellia serrata is an ancient herb used in traditional medicine for arthritis and other inflammatory diseases. Its ability to relieve inflammation-related pain and symptoms is well-documented in medical studies. Read on to learn how to take Boswellia serrata, the dosages for different medical conditions, and the necessary precautions to keep in mind.

Different types of Boswellia serrata supplement

When we talk about "how to take boswellia", it all depends on which boswellia supplement you are taking.

There are 3 main types of Boswellia supplements:

1. Supplements with dried Boswellia gum resin

2. Boswellia extracts, standardized to a high percentage of boswellic acids

3. Boswellia extracts that are high in AKBA, the most important boswellic acid

The main difference between these supplements is how potent there are.

Supplements with dried Boswellia gum resin are the least potent as they contain little amounts of the active ingredients in Boswellia.

Next in potency are standardized Boswellia extracts. These extracts contain a certain percentage of boswellic acids, which are the active ingredients in Boswellia resin. Typically, the extracts have somewhere between 30% and 65% of Boswellic acids. The higher the percentage, the more potent is the supplement.

Finally, there are special, patented formulations that are high in AKBA - which is the most important and beneficial type of boswellic acid.
>>> Read more on how to choose the right Boswellia supplement.
Boswellia serrata dosage

General dosage recommendations for Boswellia

There are no general guidelines for Boswellia dosage. The dosages that we represent below are taken from manufacturers' suggested use and studies made on Boswellia and various medical conditions.

Dosage for supplement that contain Boswellia gum resin (so-called whole herb supplements)

Typical dosage is 800 mg, taken twice or three times per day.

Dosage for standardized Boswellia serrata extracts

Standardized supplements usually contain 60-65% standardized boswellic acids. For these, the recommended dosage ranges from 300-500mg taken thrice a day. They are typically used for alleviating inflammatory symptoms and pain.

Dosage for AKBA-enriched solutions

These are patented technologies that are generally more powerful due to higher concentration of AKBA. They contain 10-30% of AKBA as opposed to 1-3% in normal standardized extracts.

Supplements with 20 to 30% AKBA can be taken in much lower dosage - 100mg to 300 mg taken once per day.

For products with 10% AKBA, the suggested dosage is up to 500mg taken once or twice daily.

Boswellia serrata dosages for specific medical conditions

For osteoarthritis

For best results, go for special Boswellia serrata formulations with 20-30% AKBA. You'll only need 100-300mg a day.

If you're taking regular standardized extracts with 60-65% boswellic acids, raise the dosage to 300-400mg three times daily.

For rheumatoid arthritis

AKBA-concentrated formulations are believed to be more effective than normal standardized extracts at just 100-300mg per day dosage.

On the other hand, standardized Boswellia serrata has been successfully taken at a total of 1,200-3,600mg a day for improved joint swelling in rheumatoid arthritis ( 1) ( 2).

For bronchial asthma

German study has shown that taking 3 capsules of Boswellia resin daily (each with 300mg of gum resin) eased the symptoms of bronchial asthma ( 3).

For ulcerative colitis

Two studies ( 4), ( 5) examined the effects of Boswellia resin on ulcerative colitis. One used 350mg and the other 300mg of resin; taken three times daily in both studies.

The results have shown that Boswellia is more successful than the drug sulfasalazine in treating ulcerative colitis and triggers remission of the disease in larger percentage of patients.

For Crohn's disease

400mg of standardized Boswellia serrata extracts taken thrice daily is clinically proven effective and well-tolerated in Crohn's disease patients ( 6).

For diabetes

One study shows that a total of 900mg Boswellia serrata gum resin taken daily for 6 weeks increased levels of blood HDL, lowered cholesterol, LDL, fructosamine, and other blood parameters in type 2 diabetes patients ( 7).

For cancer

Studies on cerebral edema and glioma have used a total daily intake of 3,600-4,200mg of standardized Boswellia serrata extracts.

Clinical trials on Boswellia treatment for cancer

Patients receiving irradiation of the brain for primary brain tumors or brain metastases of solid tumors were administered 4,200mg a day of Boswellia serrata extracts during radiotherapy. 60% of patients had more than 75% decrease in the edema by the end of the treatment (8) (9).

A study on glioma patients who took 3,600mg a day for 7 days prior to surgery ended up with greatly reduced perifocal edema plus improvement in clinical symptoms. Those who took 2,400mg per day had a lower degree of perifocal edema reduction (10) (11).

In a smaller trial with 12 patients with brain tumors and progressive cerebral edema, 3 of the 7 people with glioblastoma and tumor progression plus 3 of the 5 patients with radiochemotherapy-related leukoencephalopathy had reduced edema after taking 3,600mg daily for 12 weeks. All leukoencephalopathy patients also reported a clinical improvement (12).

A 39-year-old woman with symptomatic multiple brain metastases of a mammacarcinoma took 2,400mg per day of standardized Boswellia serrata for 10 weeks. At the same time, she began receiving brain radiation therapy and capecitabine chemotherapy. Her brain metastases were no longer visible in the CT scan by the treatment's conclusion (13).

When and how to take Boswellia serrata supplement

Most Boswellia extracts are packaged in capsule or softgel form. Take your supplement with a glass of water after having a high fat meal. Boswellia is fat-soluble so it will be better absorbed this way ( 14).

Boswellia serrata for colon conditions

If using Boswellia serrata for inflammatory bowel disease (ulcerative colitis or Crohn's disease), it is thought to be best to take the supplement before meals.

Recommended time of day to take Boswellia serrata supplement

AKBA-rich Boswellia serrata supplements like ApresFlex and 5-LOXIN can be taken during the first meal of the day.

For the less potent standardized extracts, take one capsule every 8 hours. This is how long the substance stays in the body after taken orally ( 15).

Boswellia side effects and safety

Boswellia serrata is regarded as generally safe and well-tolerated ( 16), ( 17), ( 18). On rare occasions, it may induce side effects such as:

Nausea

Diarrhea

Acid reflux

Skin rash

Allergic reactions-if you experience symptoms like chest pain, difficulty in breathing, swelling in different areas of the body, stop taking Boswellia serrata. Consult your doctor to make sure you don't have allergic reactions to the herb.

There has been one case of a woman who ended up with a bezoar, a solid mass of indigestible material that builds up in the digestive tract ( 19). However, it has been speculated that her celiac disease might have been the culprit behind this complication and not Boswellia serrata.

How can you minimize the side effects of Boswellia serrata?

There is no definite way to avoid side effects. However, it has been suggested that if Boswellia serrata causes you to suffer from heartburn, taking esomeprazole (Nexium) alongside it might help.

Boswellia serrata and long-term supplementation

Majority of studies on Boswellia lasted somewhere between 2-12 weeks. However, there have been a few studies that lasted 6, 9, and even 12 months.

Boswellia serrata's has been well tolerated with few side effects; mostly gastrointestinal distress. The dosages of Boswellia used in these studies ranged between 1,200mg and 3,600mg daily ( 20) ( 21).

People who shouldn't take Boswellia supplements

Pregnant and breastfeeding women

Boswellia serrata can induce menstruation, birth-related problems and even miscarriage. If you are pregnant, lactating or planning to have a baby, stay away from Boswellia serrata.

Patients with liver failure

Those who are suffering from liver failure are at greater risk of the side effects because the damaged liver will not be able to process Boswellia serrata properly. This might result in higher levels of Boswellia in blood and increased side effects.

Patients with stomach ulcer or GERD

Steer clear of Boswellia serrata if you have stomach ulcer or GERD as it might worsen your symptoms.

Patients with degenerative lung disease

Boswellia serrata can potentially cause more harm to your lung tissues. Further investigation is needed to confirm this.

Children

Boswellia serrata's safety in children is not well-researched. It is best if you don't give this herb and its extracts to children.

Boswellia serrata: interactions with food, drugs or medications

Blood sugar medications

Boswellia serrata has been found to stabilize blood sugar levels. If you are taking medications for blood glucose regulation, don't take Boswellia serrata extracts as your blood sugar might fall to low.

Other pain relievers (ibuprofen, aspirin, or naproxen)

Refrain from taking Boswellia supplements with non-steroidal anti-inflammatory drugs (NSAIDs) of similar purpose. The combination could cause damage to the liver.

Warfarin

Taking Boswellia serrata with warfarin, a medication that inhibits blood clotting, can trigger excessive bleeding ( 22).

Boswellia serrata may also increase the effects of:

Anti-cancer medications

Asthma medications like Singulair

Glucosamine or chondroitin (supplements for joint health)

Garlic and red yeast (allergic reaction)

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Different types of Boswellia serrata supplementWhen we talk about "how to take boswellia", it all depends on which boswellia supplement you are taking.There are 3 main types of Boswellia supplements:Supplements with dried Boswellia gum resinBoswellia extracts, standardized to a high percentage of boswellic acidsBoswellia extracts that are high in AKBA, the most important boswellic acidThe main difference between these supplements is how potent there are.

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How I make Apricot Milk for My Friend with Stage 4 Pancreatic Cancer

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Known to some as the "king of oils," frankincense is derived from sap found in trees of the genus. It's most often found in the Boswellia sacra tree. These trees are commonly found in Middle Eastern countries, such as Oman and Yemen, and African countries, such as Somalia and Ethiopia.

This sap is thought to have a number of medicinal properties, making it a staple in many herbal and alternative therapies. Some research has suggested that frankincense oil may be a natural way to treat certain kinds of cancer. More research is necessary to determine whether this is a viable treatment option, as well as whether there are short- and long-term effects associated with its use.

People throughout history have used frankincense it to improve personal satisfaction and combat various ailments. The oil's aromatic properties are said to promote feelings of relaxation, peace, and overall wellness.

It's also thought that frankincense can help support cellular function, so it's often used to soothe skin and reduce the appearance of blemishes. Researchers have found boswellic acid, the active component found in frankincense, to have anti-inflammatory properties.

In recent years, researchers have looked at the possible effects of frankincense or its extract, boswellia, on certain cancers.

Frankincense oil has been linked to treatments for ovarian, breast, and skin cancers. Studies are generally done in vitro, or on cells in a laboratory. No studies have been conducted on people living with cancer.

The findings of one 2015 study suggest that breast cancer cells may stop growing and die off when exposed to frankincense oil. The researchers concluded that their approach is cost-effective and less time consuming than other methods.

Researchers in a looked exclusively at frankincense oil derived from the Boswellia carteri species and assessed its anti-tumor activity on bladder cancer. Researchers concluded that, when administered, the oil appears to differentiate between healthy and cancerous cells. The oil can also suppress cancer cell viability.

Similar results were found in a assessing the effects of oil from B. sacra on breast cancer cells.

More research is necessary to determine whether the oil or its extract can be consistently and effectively used to treat people who have these cancers.

If you're interested in adding frankincense to your treatment, consult your doctor. They can help determine whether this is the best option for you and advise you on potential next steps.

Although some may recommend ingesting a small amount of frankincense with food, this isn't a medically sound approach. You shouldn't ingest any essential oil.

Instead, dilute one to two drops of frankincense oil with 1 to 2 drops of a carrier oil, such as coconut or jojoba oil. Carrier oils help reduce the potency of essential oils to prevent your skin from having an adverse reaction.

You can also diffuse to the oil into the air to get potential aromatic benefits. If you have a diffuser, add 3 to 4 drops and allow it to disperse. If you don't have a diffuser, you can add 3 to 4 drops to a pot of boiling water. The steam will allow the scent to disperse into the air.
Check out: Skin cancer alternative treatments "
Applying undiluted frankincense oil to your skin may cause inflammation, irritation, or burning.

Ingesting frankincense oil or its extract, boswellia, isn't advisable. It isn't yet clear how ingesting either substance can affect you in the short- or long-term.

Ingestion has risks. If you're taking P-glycoprotein drugs, the extract may affect how you absorb and metabolize these drugs. If you're taking anticoagulants or antiplatelet drugs, boswellia may increase your risk of bleeding.

Cancer is usually treated through a plan that you work out with your oncologist and other doctors. Depending on the stage and grade of your cancer, these treatments may include:

surgery to remove any cancerous tissue and prevent the cancer from spreading

chemotherapy drugs to kill the rapidly dividing cancer cells

radiation therapy to kill cancer cells in targeted areas

Talk with your doctor before adding frankincense to your treatment regimen. They can help ensure that the essential oil or its extract won't interfere with any other medications or therapies you may be using. They can also help you find a dosage that will work for you.

The short- and long-term effects of using frankincense are still unclear. Although some researchers have suggested that frankincense may be an effective treatment, more research is necessary to fully assess the potential benefits and risks.
Keep reading: Breast cancer alternative treatments "

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Known to some as the "king of oils," frankincense is derived from sap found in trees of the genus. It's most often found in the Boswellia sacra tree. These trees are commonly found in Middle Eastern countries, such as Oman and Yemen, and African countries, such as Somalia and Ethiopia.

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Asthma, Bowel, Cancer: The Boswellia ABCs presentation by Cheryl Myers - 11/17/2016

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Is Frankincense Oil for Cancer a Good Natural Alternative   Dr  Josh Axe

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The Fastest Technique to Get Rid of Shingles Pain

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An immunotherapy drug that could save some cancer patients from the ordeal of extreme chemotherapy may also help them live longer, researchers say.

In a trial, pembrolizumab kept head and neck cancers at bay for an average of two years - five times longer than under chemotherapy.

The patients also suffered far fewer side-effects.

Cases of head and neck cancers are rising in the UK and most are diagnosed late, when they are harder to treat.

'I can get on with my life'

Derek Kitcherside, 70, from Leicestershire, believes he wouldn't be here without pembrolizumab.

He was diagnosed with cancer of the voicebox in 2011. When he started coughing up blood three years later, after receiving standard treatment, he was told the cancer had spread to his lungs and was probably incurable.

Derek asked to go on a drug trial and travelled to London every three weeks for two years for treatment with pembrolizumab.

"My tumour was shrinking all the time and I felt a bit better every time I went," he said. "It made a huge difference to my life."

Regular scans show the disease is now stable and the tumour is still getting smaller.

"I'm very pleased I can get on with my life," Derek said.

"I don't think I'd be here without it."

What is immunotherapy?

It is a treatment that does not kill cancer cells itself but instead stimulates the body's immune system to attack them.

Pembrolizumab is already being used to treat a wide range of advanced cancers, including melanoma - a type of skin cancer that spreads easily.

Experts believe the drug has the potential to treat many more.

When is it used?

Normally, immunotherapy is used after standard treatments like chemotherapy have failed but this trial, in 882 patients from 37 countries, suggests it should be used earlier - and for some people it should be the go-to option.

The drug is given to patients regularly through a drip when their cancer has returned or spread, and is considered incurable.

Why is it better than current treatment?

It is kinder, safer and can keep patients alive for longer, the study in The Lancet found.

But it may not work for everyone.

In people with advanced head and neck cancer who responded to the drug - one in four - their cancer shrank or stabilised for an average of 23 months.

By comparison, although more patients (36%) responded positively to standard chemotherapy treatment, the improvements lasted on average for only four and a half months.

Those with larger, more aggressive tumours were given the drug in combination with chemotherapy to help slow progress of the disease - which was kept in check for an average of seven months.

So who could benefit?

The trick is identifying people with tumours that will respond, says Prof Kevin Harrington, oncologist at The Royal Marsden NHS Founation Trust and professor of biological cancer therapies at the Institute of Cancer Research, who led the study,

A test for the presence of immune marker PD-L1 in the tumour means doctors can work this out.

Prof Harrington says approximately 85% of people with advanced or relapsed head and neck cancer would be eligible for pembrolizumab - around 1,300 patients a year.

Why is it kinder?

At the moment, the recommended treatment is a toxic combination of chemotherapy and antibody therapy, which often makes people feel very sick.

The extreme treatment can also cause damage to the kidneys, hands and feet.

Patients given the immunotherapy drug experienced far fewer side-effects.

"It's more sensible and less toxic - patients live longer and feel better," says Prof Harrington.

What does this mean for patients?

"This study is very exciting", says Prof Paul Workman, from the Institute of Cancer Research.

"Firstly because it shows that immunotherapy can have dramatic benefits for some patients with head and neck cancer when used as a first-line treatment, and secondly because the researchers have devised a test for picking out who is most likely to benefit."

He said all new drugs coming on to the market should be accompanied by a test to target their uses as precisely as possible.

In the US and the EU, pembrolizumab has been approved for use on its own and with chemotherapy for advanced head and neck cancer, but not yet in the UK.

The National Institute for Heath Care and Excellence is currently appraising pembrolizumab and it could be approved for use on the NHS by next summer.

Facts on head and neck cancer

Around 12,000 people in the UK are diagnosed with head and neck cancer each year

Half are diagnosed at stage III or IV when it is hard to treat

Most cases are linked to smoking and alcohol but the recent rise in cases seems to be linked to HPV (human papillomavirus)

This virus infects the skin and cells lining the inside of the body and can be spread through close skin-to-skin contact

Boys and girls are now being vaccinated against HPV at school, but it will be several decades before HPV stops being a risk factor

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Cancer immunotherapy drug 'prolongs life'

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Abstract

Keywords: Apricot kernel, Peach kernel, Amygdalin, Colon cancer, S-phase block

Background

The National Cancer Registry of South Africa has recorded that colorectal cancer (CRC) is one of the top ten most common cancers in both men and women [ 1]. Although advances in radiotherapy, chemotherapy and surgery has improved survival rates, approximately 1 in 17 people over the age of 50 years will develop colon cancer in their lifetime, with more than 50% of these individuals eventually dying from the disease [ 1]. Colon cancer is nutrition dependent [ 2] with alcohol consumption accounting for the greatest risk factor [ 3, 4], followed by a high-fat diet, poor dietary fibre intake, obesity, smoking and lack of exercise [ 2]. Colon cancer may be treated by the resection of the colon, chemotherapy, targeted therapy, hormonal therapy and immunotherapy [ 5], with pharmaceutical hormonal drugs resulting in undesirable adverse effects [ 6]. The efficacy of 5- Fluorouracil (5-FU), the mainstay of colon cancer treatment [ 7] do not have a consistent drug response in patients [ 8].

Ruan et al. (2006) [ 9] have shown that apricot and peach kernels dispensed in its raw form, induce apoptosis and differentiation of tumour cells. Human DU145 and LNCaP prostate cancer cells showed morphological characteristics associated with apoptosis as well as increased Bax expression and caspase-3 enzyme activity after treatment with apricot kernel extracts [ 9]. The apricot and peach kernels are composed of glycosides including amygdalin, fixed oils including oleic acid and linoleic acid, volatile oils such as benzaldehyde [ ﻿10﻿]. In addition, the peach and apricot kernel has polyphenols such as flavanoids [ 10﻿] and gallic acid respectively [ 11]. Epidemiological studies support evidence that colon cancer is preventable by adjusting the diet [ 12] and a protective effect is attributable to polyphenols and foods such as fruits and vegetables [ ﻿13﻿]. Wu et al. (2011) had found that high content of phenolic compounds (4.1593 mg GAE/g) contribute to the antioxidant activity of the kernels oil [ ﻿14﻿]. The presence of oleic and linoleic acids in the peach kernel enhances its nutritional and medicinal value [ 15]. Both amygdalin and its patented form, Laetrile®, have been promoted and sold as "vitamin B-17", although neither compound is a vitamin.

Cyanogenic glycosides are considered non-toxic until cyanide is released. This usually occurs as a result of enzymatic hydrolysis by β-glucosidases following grinding of plant tissue which activates intracellular β-glucosidases, or by the gut micro-flora. This reaction can also result from chewing. Amygdalin is a cyanogenic glycoside plant compound found in kernels of the Rosacea family (Persica Semen and Armeniacea Semen) that release cyanide upon enzymatic metabolism. Cyanide acts through the inhibition of cytochrome-c oxidase in the respiratory electron transport chain of the mitochondria, impairing both oxidative metabolism and the associated process of oxidative phosphorylation, thereby causing death through energy deprivation and oxygen uptake [ 16]. The rationale for using amygdalin / laetrile rests on the fact that non-malignant cells have higher levels of a liver mitochondrial enzyme, rhodanese, a sulphur transferase, which catalyzes the detoxification of cyanide by sulphuration in a double displacement mechanistic reaction [ ﻿17﻿]. In animal tissue rhodanese is multifunctional, but the physiological role is to catalyze the formation of thiocyanate that is excreted by the kidneys [ 1﻿8﻿]. The apricot and peach kernel's role in nutrition and as a substance with medicinal properties makes it a target for anti-cancer drugs.

In South Africa, practitioners of Traditional Chinese Medicine prescribe apricot and peach kernels imported from the China as part of herbal formulations to their patients. It would therefore be of value to both the practitioner and the patient to know whether the kernels from South Africa could have similar effects.

The aim of this research was to investigate the potential anti-proliferative properties and possible cytotoxic effects of apricot and peach kernel extracts from South Africa and China on HT-29 colon cancer cells. The study included the evaluation of cell cycle progression after exposure to the various kernel extracts.

Methods

Cell culture

HT-29 colon cancer cells (ATTC®HTB-38™, Middlesex TW11 OLY, United Kingdom), a gift from Prof WCA Gelderblom (CPUT, Bellville, Cape Town), were cultured in Dulbecco's Minimum Essential Medium F12 (Thermo Fisher Scientific, South Africa) supplemented with 15% heat inactivated Fetal Bovine Serum (FBS) (GIBCO), penicillin (100 μg/mL) and streptomycin (100 μg/mL) (GIBCO). The cells were grown and maintained in sterile 25cm ² tissue culture flasks at a 5% CO ₂, 95% humidified air atmosphere at 37 °C. Tissue culture flasks, serological pipettes and filters were obtained from BIOCOM/Biotech.

Kernel extracts preparation

Raw Chinese apricot and peach kernels were obtained from China through a private Traditional Chinese Medicine (TCM) practitioner, Dr. Li Chunlan, Newlands, Cape Town, South Africa. The South African kernels were obtained from De White Industries (Pty) Ltd. (P.O Box 400, Montagu, Western Cape, South Africa) producers and exporters. All kernels were grounded using a household coffee blender. The Chinese apricot (CAK), Chinese peach (CPK), South African apricot (SAK) and South African peach (SPK) organic extractions, namely total (tot), lipophilic (lipo) and hydrophilic (hydro) extractions, were obtained using 80% ethanol. Organic extractions were achieved by evaporating ethanol extracted kernels using a desktop rotatovapour (Buchi rotavapor R-114) at 55 °C to obtain a viscous oily liquid and stored at − 20 °C. To make up a stock solution of the respective CAK, CPK, SAK and SPK kernel extractions, 1 g of the respective oils were added to 9 mL of DMSO to make up a final volume of 10 mL.

Dimethyl-Sulfoxide (DMSO) (9 mL) and 1 g of the respective oils was used to constitute the respective concentrations of 100, 500 and 1000 μg/mL for the organic extractions. The kernel extractions hereafter are referred to as the treatment. Routinely the vehicle concentration (Ethanol/DMSO) was limited to 0,1% in the low concentration of extracts used and 0,3% in the high concentrations of extracts used in the experiments.

Cell proliferation

HT-29 cells were seeded in 96 well plates at 5000 cells/well and incubated for 24 h (h). The cells were exposed to the organic extracts at concentrations of 100, 500 and 1000 μg/mL respectively for 24 h, 48 h and 72 h. A crystal violet assay was used to determine cell proliferation [ 19] and the absorbance of the samples was analysed at 570 nm using a Glomax plate reader.

Morphological studies

Statistics

Statistical analysis of the quantitative data was done by GraphPad Prism (GraphPad Prism version 6.04 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com). Cell viability studies were repeated thrice, with sample size (n) - 12 in each experiment and analyzed by means of two-way ANOVA followed by Tukey's multiple comparisons test. A P-value of less than 0.05 was accepted as significant. Means of quantitative experiments are presented in bar charts, with T-bars referring to the standard error of the mean (SEM). All experiments included a set of appropriate controls. Changes in cellular kinetics and morphology were carried out to illustrate possible explanations for the changes in cellular proliferation and not statistically analysed. The software used for acquisition of cell cycle progression data, was Cellquest Pro version 5.2.1. Analysis and results obtained of acquired data was done with Modfit version 3.3 software and not presented as mean ± SD.

Results

Cell proliferation

Cell numbers of the HT-29 colon cancer cells were affected differently by the kernels of different origin. Cell numbers decreased in a time and dose-dependent manner when exposed to the Chinese apricot kernel extracts for 24 h and 48 h (Fig. a and b). After 72 h however, CAK-hydro (100 μg/mL) and CAK-hydro and CAK-lipo (1000 μg/mL) stimulated cell proliferation. All the extracts of the South African apricot kernels (SAK) had a dose related bi-phasic proliferative effect on the HT-29 cells (Fig. ). The lowest and highest concentrations of the SAK extracts had an outspoken stimulatory effect on cell proliferation after 24 h while at 500 μg/mL, it significantly inhibited cell proliferation (Fig. a). In contrast, after 72 h, the lowest and highest concentrations (100 & 1000 μg/mL) of all SAK extracts significantly ( p ≤ 0.001) inhibited cell proliferation while the 500 μg/mL extracts now stimulated ( p ≤ 0.001) cell proliferation (Fig. c). For the 48 h exposure period, the SAK extracts followed the same trend as observed in the 24 h exposure samples, however the effect was not significant (Fig. b).

For the peach kernels (Fig. ), the South African kernel extracts (100 μg/mL) at 24 and 48 h, the 1000 μg/mL (24 h) and the 500 μg/mL (72 h) induced an outspoken increase in cell proliferation. These results are similar to that observed with the South African apricot kernel extracts. The most outspoken inhibition of cell proliferation by the CPK organic extracts was induced after 24 h and 48 h by the 500 and 1000 μg/mL concentrations (Fig. a and b). The inhibitory effects induced by the total and lipophilic extracts is possibly due to the presence of glycosides, fixed and volatile oils as well as the high nutritional value due to the high lipophilic extract percentage.

In contrast to the inhibitory effect seen after 24 h and 48 h, the cells exposed to CPK-H after 72 h showed an increase in cell proliferation (Fig. c).

Morphological effects

Colony forming HT-29 colon cancer cells show characteristic round to elliptical shapes (Fig. a). In response to the treatment with the 100 μg/mL CPK-H (24 h) (Fig. b) cells displayed cell membrane blebbing (arrow) possibly due to changes in membrane permeability; to 100 μg/mL CAK-T extract (48 h) (Fig. c) irregularly shaped cells and 1000 μg/mL SPK-Tot and 500 μg/mL SAK-H (72 h) (Fig. d and e) cellular shrinking. These changes could possibly be associated with cells undergoing apoptosis. After 72 h however, the cells seem to resume their initial growth appearance and increase in colony size.

To verify the possible presence of apoptosis, treated cells were stained with Hoechst33342. Only a few apoptotic cells were observed, negating the hypothesis that the decrease in cell numbers in the SAK and SPK treated cells was due to apoptosis induction (Fig. a-d).

Cell cycle kinetics

Compared to the control in Table the SPK extracts showed a consistent increase in the number of cells in the S-phase. After 24 h exposure, the SAK hydrophilic extract at 100 μg/mL (S = 86.4%), and 65.57% (500 μg/mL) and 74.47% (1000 μg/mL) showed the most significant accumulation of cells in the S-phase (Table ). Similar increases in the S-phase cell fraction after SAK and SPK kernel treatments were observed after the 48 h exposure period with the largest build-ups caused by SPK-H (100 μg/mL) at 76.08%, 500 μg/mL SAK-L at 70.74% and 1000 μg/mL SPK-T at 77.62% (Table ). Cells treated with the CPK organic fractions resulted in an increase in the number of cells in the S-phase (S = 56.73%) when exposed to 500 μg/mL CPK organic extractions after 72 h exposure (Table ). The percentage values of SAK and SPK extract exposed cells in the G ₁ and S-phases vary greatly between each other indicating a higher variance as compared to the CAK and CPK exposed cells.

Discussion

Colon cancer treatment entails surgical removal of the tumor that is followed by chemotherapy with or without radiation. However, the notorious effect of such treatments has in many cases exacerbated the overall diminishing health and quality of life of the recipients. Although early detection has improved survival, the chemotherapeutic agents used in the treatment of colon cancer have not proven to be efficient in all patients. Studies strongly correlate the relationship between dietary constituents and the risk of specific cancers [ 10, 13, 14]. With colon cancer, a relation commonly found in epidemiological studies is an increase in risk associated with a high fat and low fibre dietary pattern [ ﻿14﻿]. Certain dietary fatty acids (FA) can irritate the colon by increasing pro-inflammatory responses. The presence of glycosides such as amygdalin, oleic and linoleic acid in the kernels render them as possible chemotherapeutic agents. Replicating cancer cells have an increased requirement for lipids for membrane formation, hence dietary interventions with fatty acids possessing anti-carcinogenic properties represent a novel, practical and relatively safe approach to reduce proliferation of colorectal cancer [ 21]. Inhibition of cell proliferation and the induction of apoptosis could be an important approach in chemotherapeutic treatment. Understanding how dietary components regulate proliferation and cell survival could play a critical role in development of new agents that can prevent and treat cancer with reduced risk of toxicity. Conventional chemotherapy lacks aqueous solubility, selectivity and due to its nonspecific targeting of cancer cells, damages rapidly proliferating normal cells [ 6]. Epidemiological studies have revealed that colon cancer is preventable by adjusting the diet leading to a reduction in the incidence. Thus identification of chemopreventive compounds in commonly consumed natural dietary sources such as fruits, vegetables and teas is important in that it provides a means of everyday cancer prevention. Laboratory studies have indicated a positive association between cancer reduction and intake of dietary plant foods which is attributable to the presence of phytochemicals [ 22]. One group of phytochemicals that has shown potential in chemoprevention studies are plant polyphenols [ 23].

Plant glycosides, which are flavonoids and polyphenolic, have a bio-action, are larger and chemically more complex than in mammals, are therapeutic in small doses but toxic in larger doses [ ﻿24﻿]. They thus have a narrower therapeutic index. This is reflected in the time-dose-dependent manner as well as the type of extract preparation and its effects on the HT-29 colon cancer exposed cells. Reflecting the anti-proliferative effect at low doses at 24 h and 48 h exposure and a recovery after 72 h exposure to the kernel extractions.

This investigation demonstrated that organic extracts of apricot and peach kernels of Chinese and South African origin may significantly reduce cell viability, inhibit cell proliferation, induce morphological changes and affect cell cycle progression of the HT-29 human colon cancer cell line. These findings agree with reports showing growth inhibiting, anti-proliferative and pro-apoptotic effects of amygdalin on promyelocytic leukaemia [ 25], colon cancer [ 26], cervical cancer [ ﻿27﻿], and bladder cancer cells [ 28]. As opposed to previous research where the isolated cyanogenic compound was used, in the present research the whole kernel was used. This may have contributed to the slight variation in the results. The rationale for using the whole kernel was based on the principle that the whole is better than the parts, whose constituents act synergistically as a whole. The method of preparation (in the form of an herbal medicine as administered by a Chinese medicine practitioner) and consumed as a food supplement were other factors considered. Since the Chinese apricot and peach kernels were bought from a Chinese medicine practitioner it was expected that there would be less fixed and volatile oils as a result of the preparation process of the kernels. The aim of the present study was to examine the influence of CAK/SAK as well as CPK/SPK on colon cancer cell proliferation to determine whether these compounds affects cell morphology and cellular kinetics, thus mechanisms influencing cell growth.

Although the organic extracts of both the apricot and peach kernels affected cell proliferation, it was the Chinese apricot and peach total and lipophilic kernel extracts that had the most outspoken inhibition on cell viability after 24 h. This may be due to the specific ratio of amygdalin and the presence of volatile oils, fixed oils and glycosides in the kernels originating from China. The glycosides in the kernels causes polarisation of water insoluble compounds that could possibly also contribute to the growth inhibitory effect [ ﻿24﻿]. The antioxidant potential of polyphenols depends on the chemical structure particularly the positions of the hydroxyl groups [ 29]. Thus the fixed and volatile oils may be greatly reduced in hydrophilic extracts possibly resulting in a reduced inhibitory effect by the hydrophilic extracts compared to the total and lipophilic extracts. The HT-29 cancer cell growth inhibitory effect of the CAK and CPK treatments was abrogated after 72 h exposure with an increase in cell proliferation observed in most of the treated samples. In contrast with the anti-proliferative effects observed after treatment with CAK and CPK, the South African kernel extracts stimulated cell proliferation in the 24 and 48 h exposure periods. The bi-phasic stimulation/inhibition by these extracts (Figs. and a, b) would certainly impact on the use of these kernels in patients as the dose effect could potentially affect the treatment. The aggressive stimulation by low and high doses SAK/SPK (24 h) (Figs. a and a) in contrast to the absence of the stimulatory effects of the 500 μg/mL can possibly be related to the biphasic nature of the respective components in these kernels. Several forms of cell death may typically be induced within the same tissue although apoptosis is the fastest, while other forms, like necrosis or autophagy, only become visible when apoptosis is inhibited. Treatment of the cells with the various extracts caused limited morphological changes, and it was mainly the Chinese kernel extracts that caused changes that could be associated with apoptosis. Changing of intracellular ATP levels can affect the form of cell death. High ATP levels lead to apoptosis, while a low ATP level leads to necrosis confirming that an intracellular ATP depletion switches the energy-dependent apoptotic cell death to necrosis. As cyanide acts through the inhibition of cytochrome-c oxidase in the respiratory electron transport chain of the mitochondria, impairing both oxidative metabolism and the associated process of oxidative phosphorylation causing death through energy deprivation, it is possible that ATP levels decreased in HT-29 cells influencing the type of cell death observed after the kernel treatment switching the energy-dependent apoptotic cell death to necrosis.

Regulation of the cell cycle is important in the growth and development of cancer. Cell cycle progression revealed a significant S-phase peak in the cells treated with SAK and SPK. The intra-S phase checkpoint activated by genotoxic insults causes only temporary, reversible delay in cell cycle progression, mainly by inhibition of new replicons initiation and thereby slowing down DNA replication, but not permanently arresting DNA replication [ 30].

Bladder cancer cells [ ﻿27﻿] exposed to amygdalin only, showed a significant delay in cell cycle progression and a G ₀/G ₁ arrest whereas the HT-29 cells showed the induction of an intra-S phase block which is shown by an increase in the number of cells in the S phase and a decrease in the number of cells in the G ₁ and G ₂ phases. The accumulation of cells in the S-phase started at 24 and continued throughout the different time and doses indicating that the South African kernels affect the cell cycle progression possibly by activating the intra-S-phase checkpoint. Activation of the intra-S-phase block slows down the processes of DNA synthesis allowing time for DNA repair thus preventing genomic instability. A long term intra-S-phase block (48 h) could limit the amount of sister chromatids and therefore reduce available templates for efficient repair by homologous recombination [ 30, 31]. Complete inhibition of cell cycle regulating enzymes and prolonged intra-S phase arrest may cause regaining of replication competence of already fired origins, making the recovery process prone to over-replication of at least parts of the genome. It is therefore possible that the insult caused by SAK and SPK treatments could act by inducing this block that could later progress to cell death [ ﻿31﻿].

The organic extractions of the Chinese apricot kernels showed an increase in the number of cells in the G ₀/G ₁ and no relative increase in cell numbers in the S-phase as compared to the Chinese peach kernels which showed a slight increase in the number of cells in the S-phase.

The inhibitory effect of the Chinese kernels lasted for 48 h only. Therefore, repetitive treatment of the cells every 24 h or 48 h would greatly reduce and maintain the reduction of colon cancer growth. It can therefore be deduced that the inhibition of proliferative activity was not due to a permanent cytotoxic effect of the kernels, but rather a modulation of cell cycle progression and the induction of a temporary S-phase block. Mitosis is therefore influenced in a different manner depending on the type of kernel extraction, dose and time of exposure. Future studies will include determination and quantification of the specific type of cell death induced by the different extractions. The presence of S-phase block will also be verified by studying changes in cyclin A levels.

Conclusion

The growth inhibition effect of the CAK and CPK extracts may be due to increased necrosis and possibly autophagy as an alternative to decreased proliferation in the cells. This possibility should be evaluated in further studies. Low ATP levels, caused by the presence of amygdalin in the kernels, can also switch to pycnosis or necrosis. Willis and Rhind 2009 [ 32] suggest that the intra S-phase checkpoint may be more concerned with tolerating and accommodating damage during replication rather than repairing it, thus the HT-29 cells most probably proceed to necrosis in the presence of outspoken DNA damage by specially the high concentrations CAK/CPK (24 h and 48 h) and SAK after 72 h.

Cancer cells are thought to have more β-glucosidase and less of the liver enzyme rhodanese which converts cyanide to relatively harmless compound thiocyanate. Thus the cancer cells are more susceptible to the effect of amygdalin than the healthy cells. Daily intake due to the narrow therapeutic index and reduced risk of toxicity based on the form of administration and preparation may make these kernels a viable chemopreventive agent.

Acknowledgments

1. Prof WCA Gelderblom (CPUT, Bellville, Cape Town) for the HT-29 human colon cancer cells

2. Dr. Li Chunlan a Traditional Chinese Medicine (TCM) practitioner (Cape Town) and De White industries (Pty) Ltd. (South Africa) for the Chinese and South African apricot and peach kernels respectively.

3. Mr. Ronnie Dreyer (Department of Immunology, University of Cape Town) for assistance with the flow cytometric analysis of the specimens.

Abbreviations

Authors' contributions

MDK and WC put together the concept and design of this study, analysed and interpreted the data, and wrote and revised the manuscript. MDK and WC conducted the experiments. Both authors contributed to final editing and approval of the manuscript.

Notes

Ethics approval and consent to participate

N/A

Consent for publication

Not applicable.

Competing interests

The author's declare that there have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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The anti-proliferative effect of apricot and peach kernel extracts on human colon cancer cells in vitro

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Colorectal malignant neoplasms is one of the leading causes of death in both men and women in the developed world and the incidence has recently increased markedly in South Africa. Studies have highlighted the beneficial effects of Amygdalin, a cyanogenic ...

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