What is sulforaphane

Sulforaphane also known as 1-isothiocyanato-4-(methylsulfinyl)-butane, is a phytochemical extract derived from cruciferous vegetables such as broccoli, cabbage and cauliflower and radish ¹. Glucoraphanin in broccoli is enzymatically hydrolyzed by myrosinase (thioglucoside glucohydrolase), an enzyme that is released when the plant is chewed or processed (Figure 1) ². Heating broccoli partially denatures and inactivates myrosinase, leaving the glucoraphanin at least partially intact. In the gut of healthy individuals any intact glucoraphanin is then metabolized by myrosinase-producing bacteria ³. Because broccoli sprout or seed extracts taken orally contain no myrosinase to hydrolyze the glucoraphanin, transformation to sulforaphane must be carried out by the gut microflora ⁴. In individuals with compromised intestinal flora and low myrosinase activity, it is unclear if glucoraphanin exerts the same systemic effects as observed in individuals with normal intestinal flora ⁵. Sulforaphane is unstable in aqueous medium and at high temperature; thus, the stability of sulforaphane during storage is the focus of its biological activity research. Studies have shown that −20°C and 4°C are the best storage temperatures for sulforaphane ⁶.

Sulforaphane is often used as an anticancer and/or anti-inflammatory drug in traditional Chinese herbal medicine ⁷. Sulforaphane is known to upregulate genes that fight oxidative stress, inflammation and DNA damage, all of which are believed to be involved in autism spectrum disorder symptoms that lead to stimming. Sulforaphane has reported anti-cancer effects through cell cycle arrest and apoptosis in various cancer cells, such as prostate, lung, breast, and colon cancers ⁸. The chemoprevention properties of sulforaphane against cancer are through both “blocking” and “suppressing” effects ⁹. The “blocking” function of sulforaphane is achieved through inhibiting Phase 1 metabolism enzymes that convert procarcinogens to carcinogens and inducing Phase 2 metabolism enzymes that promote excretion of carcinogens ⁹. Subsequent studies revealed the “suppressing” effects of sulforaphane in modulating diverse cellular activities to inhibit the growth of transformed cells ¹⁰. The ability of sulforaphane to induce apoptosis and cell cycle arrest is associated with regulation of many molecules including Bcl-2 family proteins, caspases, p21, cyclins and cdks ¹⁰. Sulforaphane was also shown to suppress angiogenesis and metastasis by down-regulating VEGF, HIF-1α, MMP-2 and MMP-9 ¹⁰.

Currently, it is well known that evaluation of the bioavailability of natural compounds is one challenge in the design of clinical trials for studying their biological activity. Recently, Fahey et al. ¹¹ identified that changes of inflammatory-related genes in peripheral blood mononuclear cells have significant influence on the sulforaphane bioavailability in 20 healthy participants. Similarly, another research has been carried out to evaluate the bioavailability of sulforaphane in 14 women and found that repeated dosing of sulforaphane could not result in the accumulation of toxic metabolites in urine over time ¹². Moreover, sulforaphane-loaded nanostructured lipid carriers were developed and optimized to effectively improve its bioavailability and cytotoxicity efficacy against cancers ¹³. These findings provide valuable recommendation to better design the clinical trials to study the sulforaphane functionality in the future. To date, a preliminary randomized controlled trial was performed to demonstrate that pretreatment with broccoli sprout extract could improve the bioavailability and chemopreventive activity of sulforaphane, together with downregulation of several prostate cancer development-associated genes in the biopsy from 98 men ¹⁴.

Figure 1. Glucoraphanin metabolism into sulforaphane

[Source ¹⁵ ]

Sulforaphane health benefits

Sulforaphane is one of the most frequently studied plant-derived isothiocyanate organosulfur compounds ¹⁶. Sulforaphane has been reported to exhibit a wide range of biological effects including antioxidant ¹⁷, antibacterial ¹⁸, anticancer ¹⁹, anti-inflammatory ²⁰, anti-aging ²¹, neuroprotective ²² and antidiabetic ²³.

An additional benefit of sulforaphane is that it plays a major role in energy metabolism and can mitigate obesity by increasing energy expenditure ²⁴. In an animal model, sulforaphane or its precursor (glucoraphanin) enhanced reduction of food intake, adiposity, and weight gain caused by intraperitoneal injection of leptin ²⁵.

Table 1. Summary of clinical trials using broccoli and sulforaphane for disease indications.

Agent	Disease/ condition	Participants, agent, dose and schedule	outcome	Reference
Broccoli/ sulforaphane	Cancer – breast	54 breast biopsy candidates; Broccoli seed extract; 514 μmol glucoraphanin rich/ day; 56 days)	Lower Ki67, lower HDAC3 in benign tissue, lower HDAC in peripheral blood mononuclear cells compared to placebo	26
	Cancer – lung	30 healthy, young smokers; steam-cooked broccoli; 250 g per day; 10 days	Increased DNA repair activity in peripheral blood mononuclear cells compared to control diet	27
		291 healthy participants; broccoli sprout beverages; glucoraphanin (600 μmol) and sulforaphane (40 μmol); 84 days	Rapid and sustained increases in urinary excretion of benzene (61%) and acrolein (23%)	28
		50 healthy participants; glucoraphanin- or sulforaphane-rich broccoli sprout beverage; 7 days	20-50% increase in excretion levels of glutathione conjugates of acrolein, crotonaldehyde and benzene in glucoraphanin, sulforaphane or both compared to baseline	29
	Cancer – gastrointestinal tract	40 H. pylori-infected subjects; broccoli sprouts; 70 g/ day/ 8 weeks	Reduced urease, inflammation and bacterial colonization in broccoli intervention group compared to alfalfa control group	30
	Cancer – prostate	90 men with biochemical recurrence after radial prostatectomy; 60 mg of prostaphane daily; 6 months	Significantly lower log prostate-specific antigen (PSA) slope compared to placebo	31
	Diabetes	103 Scandinavian type 2 diabetes patients; broccoli sprout extract; 150 μmol sulforaphane per dose; 12 weeks	Improved fasting glucose and hemoglobin A1C (HbA1C) in obese participants	32
		81 type 2 diabetes patients; broccoli sprout powder (22.5 μmol /g sulforaphane) ; 5 g or 10 g per day; 4 weeks	Reduced fasting glucose, reduced inflammatory markers and serum insulin compared to placebo	33
	Skin disorders	5 subjects with Epidermolysis bullosa simplex; topical application of broccoli sprout extract (500 nmol sulforaphane/ ml); 1 week	Increase in K17 expression, variable but induced expression on K6 and K16	34
		6 volunteers; topical application with broccoli sprout extract (200 or 400 nmol sulforaphane); 3 doses every 24 hours	Reduced erythema (mean = 37.7%) caused by UV radiation	35
	Heart and vascular disease	37 subjects with high cardiovascular disease risk; standard or high glucoraphanin rich broccoli; 400 g per week; 12 weeks	Reduced plasma LDL-C (low density lipoprotein or “bad” cholesterol) in high glucoraphanin rich group	36
		77 type 2 diabetes patients with a positive H.pylori stool antigen test; broccoli sprout powder or in combination with standard therapy; 6 g/ day; 28 days	Improvement in systolic and diastolic blood pressure in the combination group	37
		14 adults with sickle cell disease; broccoli sprout homogenate; 50-150 μmol dose escalation for 21 days	Increase in whole blood mRNA levels of heme oxygenase 1 and trend for same with subunit of fetal hemoglobin	38
	Developmental/ behavioral disorders	27 young males with moderate to severe autism spectrum disorder; sulforaphane derived from lyophilized broccoli sprout; 50-150 μmol sulforaphane; 18 weeks	Improvement in social interaction, abnormal behavior and verbal communication	39
		10 schizophrenia patients; broccoli seed extract; 69 μmol glucoraphanin rich (3 tablets, daily); 54 days	Improvement in cognitive function tests	40
	Respiratory conditions	29 subjects inoculated with FluMist live attenuated influenza virus; broccoli sprout homogenate; 100 μmol sulforaphane; 21 days	Increase in peripheral blood NK cell expression and reduction in circulating influenza RNA	41
		16 young, healthy smokers; broccoli sprout homogenate; 200 g per day; 4 days	Reduction in virus-induced inflammation; reduction in influenza sequences in nasal lavage fluid from smokers	42
		45 moderate asthmatics; sulforaphane; 100 μmol daily; 14 days	Reduction in bronchoconstrictor effects of methacholine; reduction in airway resistance	43
		29 healthy subjects who tested positive for cat allergens; sulforaphane-rich broccoli sprout extract; 100 μmol per day; 4 days	54% reduction in diesel exhaust particle-induced nasal white blood cell counts	44

Sulforaphane and cancer

Sulforaphane exerts anti-cancer activity against multiple cancer types ¹⁶. Epidemiological studies have also shown that sulforaphane consumption is associated with reduced risk of various human cancers, including lung, colon, breast, stomach, and prostate ⁴⁵. The chemopreventive activity of sulforaphane involves inhibition of phase 1 enzymes (thereby preventing the conversion of procarcinogens to carcinogens), activation of phase 2 enzymes (leading carcinogens detoxification and excretion from the body), and suppression of pro-inflammatory responses ⁴⁶. Sulforaphane also exerts direct effects on cancer cells by inhibition of growth, induction of cell cycle arrest, and activation of apoptosis in various types of cancer cells ⁴⁷. Also, sulforaphane modulates epigenetic changes that regulate various molecular targets involved in cell proliferation, differentiation, apoptosis, or cell cycle, and viability of cancer stem cells ⁴⁸. The chemopreventive and therapeutic effects of sulforaphane have been comprehensively investigated in various cancers in vitro and in vivo ⁴⁶. Therefore, sulforaphane is a rational candidate for development as a therapeutic agent for endometrial cancer ⁴⁹. The safety of sulforaphane and sulforaphane-rich broccoli sprout extract has been tested clinically ⁵⁰ and multiple phase I and phase II trials have been completed evaluating the efficacy of sulforaphane amongst patients with prostate cancer and breast cancer. These promising activities suggest that sulforaphane may also have value for use in endometrial cancer treatment regimens. However, further additional investigation, mainly well-designed clinical trials, are required to establish correlations and allow to further verify the efficacy, safety, and possible adverse reactions of sulforaphane products.

Table 2. Mechanism of action of sulforaphene in human tumors

Tumors	Action	Outcome	Model Used	Reference
Breast cancer	Akt–mTOR–S6K kinase pathway↓	Reversal multidrug resistance, apoptosis↑	SKBR-3, BT-474	51
Triple-negative breast cancer	Hedgehog↓, MMP-2↓, MMP-9↓	Migration and invasion↓, apoptosis↑, proliferation↓	MCF7, T47D, MCF10A, MCF10AT1, MCF10CA1a, SUM159	52
Triple-negative breast cancer	EGR1 ↑, cyclinB1↓, Cdc2↓	Apoptosis↑, cell cycle G2/M phase arrest	MDA-MB-231, MDA-MB-453, MDA-MB-436, MDA-MB-468	53
Hepatocellular carcinoma	caspases -3/7 and -9↑, caspase-8↓	Apoptosis↑, cell cycle G0/G1 phase arrest	MFC-7, HT-29	54
Hepatocellular carcinoma	ROS↑, microtubule polymerization↑	Apoptosis↑, radiation-induced cell death↑	HB-8065	55
Hepatocellular carcinoma	NF-κB↓	Apoptosis↑, proliferation↓	HepG2, Hep3B	56
Lung cancer	PI3K-Akt↓, PTEN↓	Apoptosis↑, migration and invasion↓, proliferation↑	A549, H460, H446, HCC827, H1975, H1299	57
Non-small cell lung carcinoma	ROS↑, Bcl-2↓, Bax↓, cytochrome C↑, caspase 9/3↑	Apoptosis↑, proliferation↓	A549	58
Cervical cancer	Caspase 3↑, caspase 9↑, EGFR↑	Apoptosis↑, proliferation↓	HeLa	59
Ovarian cancer	ROS↑, mitochondrial membrane depolarization	Apoptosis↑, proliferation↓	SKOV 3, SNU 8	60
Colon cancer	p38, CDK1, CDC25B	Apoptosis↑, cell cycle G2/M phase arrest	HCT116, HT-29, DLD1, KM12	61
Gastric cancer	ROS↑, cytochrome c↑, Casp-3↑, Casp-8↑, PARP-1↑	Apoptosis↑, migration and invasion↓	AGS	62
Lymphoma	CRM1, p62↑, AMPK↑	Apoptosis↑	U937, HUT78, Raji, JeKo-1, U2932	63
Thyroid cancer	Ras↑, MEK↑, ERK↑, B-Raf↑	Apoptosis↑, proliferation↓	FRO	64

Abbreviations: MMP = matrix metalloproteinases; EGR1 = early growth response 1; Cdc2 = cell division cycle gene 2; ROS = reactive oxygen species; PTEN = phosphatase and tensin homolog; Bcl-2 = B-cell lymphoma 2; CDK1 = cyclin dependent kinase 1; CDC25B = cell division cycle 25 B; CRM1 = chromosome-region-maintenance-1; AMPK = AMP-activated protein kinase; MEK = mitogen-activated protein kinase; ERK = extracellular signal-regulated kinase. ↑ = activation/up regulation; ↓ = suppression/down regulation.

Sulforaphane in breast cancer

Breast cancer is one of the most common malignant tumors in the world, especially in women ⁶⁵. According to statistics, among women younger than 45, breast cancer is undoubtedly the leading cause of cancer-related death ⁶⁶. At present, the treatment of breast cancer mainly includes surgical resection, radiotherapy, and chemotherapy. Previous research found that pretreatment with sulforaphane, at as low concentration as 5 μM, inhibited cell clonogenicity by nearly 70% in breast cancer cells, when compared to untreated cells. Human epidermal growth factor receptor 2 (HER-2) is known to be involved in the proliferation and division of breast cancer cells ⁶⁷, specifically through the Akt–mTOR–S6K kinase pathway ⁵¹. The anti-HER2-targeted drug lapatinib is often used in breast cancer patients with HER2 overexpression ⁶⁸. Studies have found that the combination of sulforaphane (2.5 μM) and lapatinib (100 nM) could effectively induce cell apoptosis and decrease cell viability mainly by inhibiting the Akt–mTOR–S6K pathway in breast cancer cells, thus improving the therapeutic effect of lapatinib ⁶⁹. Triple-negative breast cancer is a common subtype of breast cancer lacking estrogen receptor, progesterone receptor, and HER2 gene overexpression ⁷⁰. sulforaphane also has the significant therapeutic potential against triple-negative breast cancer. In recent years, the Hedgehog (Hh) pathway has been identified as a key signaling pathway that drives tumorigenesis in triple-negative breast cancer ⁵². Downregulation of the Hedgehog (Hh) signaling pathway by inhibitors can reduce cell migration and invasion ⁷¹. Sulforaphane can significantly inhibit the Hedgehog (Hh) pathway, thereby reducing the activity of the downstream signal modulators matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and inhibiting the invasion of human triple-negative breast cancer cells ⁷². Early growth response 1 (EGR1) is an immediate early gene induced by estrogen, growth factor, or stress signal that can exert both cancer-suppressive and -promoter activities ⁷³. At the same time, EGR1 was successfully verified as a uniformly activated marker after sulforaphane treatment in triple-negative breast cancer cell lines MDA-MB453 and MDA-MB-436. The data indicated that sulforaphane could inhibit the expression of cyclin B1 and phosphorylated Cdc2 by mediating tumor suppressor EGR1, thus inducing G2/M phase arrest of triple-negative breast cancer cells ⁵⁴.

Sulforaphane in hepatocellular carcinoma

Hepatocellular carcinoma (liver cancer) is one of the deadliest and most common cancers in humans. The treatment of liver cancer mostly involves surgical resection, transplantation, and ablation, but the therapeutic effect is not good ⁵⁵. Some researchers have found that sulforaphane can promote apoptosis of hepatocellular carcinoma cells, which is morphologically manifested as cell contraction, blistering, chromatin condensation, and nuclear fragmentation. They also found that sulforaphane was most toxic in HepG2 cells. sulforaphane exhibited an IC50 value of 33.8 μM when incubated with HepG2 cells for 72 hours. An annexin V assay found that the same treatment increased caspases 3/7 and 9 activities, while caspase 8 activity decreased ⁷⁴. Oxidative reactive oxygen species (ROS), which are responsible for killing cancer cells, also affect secondary signaling networks ⁵⁶. Sulforaphane can induce the generation of intracellular ROS and inhibit the polymerization of microtubules, leading to the apoptosis and necrosis of hepatocellular carcinoma cells ⁷⁵. The transcription factor nuclear factor-κB (NF-κB) is a key transcriptional regulator in the inflammatory response. The NF-κB pathway is one of the important pathways activated during liver injury and inflammation and has been widely studied in the development of liver cancer ⁷⁶. Sulforaphane can inhibit NF-κB activity and downstream gene expression of the NF-κB pathway in hepatocellular carcinoma cells. sulforaphane can increase the radiation sensitivity of hepatocellular carcinoma by blocking the NF-κB pathway ⁷⁷.

Sulforaphane in lung cancer

Lung cancer is the leading cause of cancer death in the world ⁷⁸. Non-small cell lung cancer (NSCLC), the most frequent subtype of lung cancer, has increased in both incidence and mortality ⁷⁹. At present, research advancement in the field has revealed the tumor promotion roles of PI3K–Akt overactivation in non-small cell lung cancer ⁵⁷. The PI3K–Akt pathway promotes proliferation, migration, invasion, and resistance to treatment by activating a variety of mechanisms, including the loss of the negative regulator phosphatase and tensin homolog (PTEN) and/or Akt1 itself ⁵⁸. Sulforaphane-treated non-small cell lung cancer cells have significant inhibitory effects on the PI3K–Akt signaling pathway, including inhibition of PTEN expression and inhibition of Akt phosphorylation ⁸⁰. Sulforaphane (7.5 μM) combined with the chemotherapy drug carboplatin (20 μM) can significantly induce mitochondrial membrane potential and intracellular ROS depolarization. By activating caspases, destroying matrix metalloproteinases and arresting the cell cycle, combination treatment with sulforaphane and carboplatin synergistically promotes the apoptosis and antiproliferative effects of human non-small cell lung cancer cells A549d and enhances the tumor toxicity effect of conventional therapy alone ⁸¹.

Sulforaphane in cervical cancer

Cervical cancer remains the third most common cancer in developing countries, despite a wide range of screening procedures ⁵⁹. The therapeutic effects of photodynamic therapy in cervical intraepithelial neoplasia (CIN) and cervical cancer have been extensively studied ⁸². Effects of photodynamic therapy with a very low dose of sulforaphane (2.0 μg/ml) and radachlorin (0.5 μg/ml) at a fluence of 27 J/cm² (30 mW/cm², λmax ∼ 670 ± 3 nm) on human cervical cancer cells HeLa has shown a synergistic effect in inducing cell apoptosis. This combination therapy activates the mitochondrial apoptotic pathway primarily through upregulating the levels of caspase 3 and caspase 9. This therapeutic strategy also activates the caspase 8-dependent death receptor pathway and inhibits cell proliferation by downregulating epidermal growth factor receptor (EGFR) ⁶⁰.

Sulforaphane in gastrointestinal cancer

In the Netherlands Cohort Study ⁸³, it was observed that consumption of Brassica vegetables was inversely associated with development of gastric cardia adenocarcinoma. In an ecologic study where colorectal cancer rates between Maori and non-Maori people in New Zealand were compared, the authors attributed the low prevalence of cancer amongst Maori people to the consumption of vegetables like watercress ⁸⁴, in spite of their high red meat consumption. Interestingly, another study showed that consumption of cruciferous vegetables increased the urinary excretion of PhIP, a DNA adduct forming carcinogen present in cooked meat ⁸⁵.

Reduced urease and inflammation was seen in Helicobacter pylori-infected humans fed a broccoli sprout-rich diet indicating that isothiocyanates could likely be used to block stomach carcinogenesis in high-risk individuals ³⁰. Similar encouraging results have been shown in smaller trials with a lower number of participants ⁸⁶. However, in another trial using 250 mg standardized broccoli sprout yielding 1000 μg sulforaphane twice daily for four weeks, no significant changes were observed in urea breath test and gastric juice ammonia concentrations but modest changes were seen in lipid peroxidation in the gastric mucosa compared to placebo ⁸⁷.

Sulforaphane in prostate cancer

The efficacy of sulforaphane against prostate cancer has been tested in a few small clinical trials. Treating 20 prostate cancer patients with sulforaphane-rich extracts (200 μmol/ day) for 20 weeks did not result in a significant decline (≥50%) in prostate-specific antigen (PSA) levels ⁸⁸. However, in another study, men with biochemical recurrence after radical prostatectomy showed promising results after daily ingestion of 60 mg (~340 μmol) of stabilized free sulforaphane in a commercial dietary supplement for 6 months ⁸⁹.

Sulforaphane in other tumors

Sulforaphane obviously also has cytotoxic effects on other human malignant tumor models. For example, cisplatin is a first-line chemotherapy drug for a variety of cancers, including ovarian cancer ⁶¹. Sulforaphane can sensitize cisplatin by enhancing ROS and mitochondrial membrane depolarization and can activate multiple apoptotic pathways to synergistically inhibit the proliferation of ovarian cancer SKOV3 and SNU8 cells and induce apoptosis. Therefore, sulforaphane could be used as a promising chemotherapy sensitizer to improve the efficacy of cisplatin in ovarian cancer ⁶³. Sulforaphane can also reduce the viability of gastric cancer cells and induce apoptosis ⁶⁴. In addition, sulforaphane can induce cell cycle arrest and apoptosis in the G2/M phase of colon cancer cells, accompanied by the phosphorylation of CDK1 and CDC25B inhibitory sites and the upregulation of the p38 and JNK pathways ⁹⁰. Surprisingly, sulforaphane can selectively clear lymphoma cells via CRM1-mediated SQSTM1/p62 overexpression and AMPK activation. At the same time, sulforaphane protects normal lymphocytes by inducing cophage and apoptosis ⁹¹. Sulforaphane and photosensitive fiber-mediated photodynamic therapy can induce the apoptosis of thyroid cancer cells via significantly upregulating Ras, mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and B-Raf protein expression levels. After combined treatment, their proapoptosis and antiproliferative effects were both significantly enhanced to a much higher level than the single dose ⁹².

Sulforaphane and autism

Sulforaphane treatment (50-150 μmol daily, for 18 weeks followed by 4 weeks without treatment) improved autism spectrum disorder-related outcomes (largely based on the Social Responsiveness Scale) in young, male patients ³⁹. This finding was particularly important because sulforaphane potentially addressed the pathophysiological hallmarks of autism spectrum disorder (oxidative stress and antioxidant deficiency) instead of treating symptoms of autism spectrum disorder as done by standard therapy ⁹³. Such results are in alignment with the findings from a recent report that showed that sulforaphane was able to reduce damage caused to mouse cortical cultures by chemicals that mimicked the action in several brain disorders, including autism spectrum disorder ⁹⁴. There are multiple clinical trials in the pipeline evaluating the utility of sulforaphane/ broccoli preparations in autism spectrum disorder and findings from these studies would add valuable insight to incorporating these compounds into autism spectrum disorder treatment methods. Dietary broccoli sprout extract also improved outcomes related to schizophrenia in patients ⁹⁵, suggesting that isothiocyanates may collectively have a very important role to play in treating neurological and developmental conditions.

Sulforaphane and diabetes

In a randomized, double-blind, placebo-controlled study, 103 Scandinavian type 2 diabetes patients were given daily oral broccoli sprout extract (containing 150 μmol sulforaphane per dose) for 12 weeks, and improved fasting glucose and hemoglobin A1C (HbA1C) was observed with the most robust improvement occurring in dysregulated, obese participants ³². Most of these patients were concurrently on metformin treatment, therefore the therapeutic effect of sulforaphane alone in type 2 diabetes is unknown from this study. Previous studies in Iran showed that broccoli sprout powder consumption (112 or 225 μmol/day of sulforaphane equivalents given as a commercial glucoraphanin-rich dietary supplement) for 4 weeks, reduced serum insulin concentration in type 2 diabetes patients ⁹⁶. The same intervention resulted in favorable lipid profiles in type 2 diabetes patients suggesting that isothiocyanates have utility in reducing diabetes-related complications too ³³. Whether or not sulforaphane and other isothiocyanates could replace or complement current diabetes medications such as metformin requires further investigation.

Sulforaphane and skin disorders

Topical application of broccoli sprout extract for keratin-based disorders was tested by Kerns and colleagues in a small study ⁹⁷. Here, five subjects with epidermolysis bullosa simplex (caused by mutations in keratin 14 or 5) applied the extract (500 nmol sulforaphane/ mL) daily. Variable but induced expression of keratins 16 and 6 were observed after application indicating the potential of broccoli sprout extract to be used in similar keratin-associated disorders. sulforaphane-rich broccoli sprout extract application was shown to protect skin of volunteers against erythema caused by ultraviolet radiation ⁹⁸. However, more extensive studies with larger sample sizes are needed to understand the broad spectrum of possible uses of isothiocyanates in skin disease.

Sulforaphane and cardiovascular disease

In 2015, a study showed that a broccoli diet containing high levels of glucoraphanin reduced plasma LDL cholesterol (low-density lipoprotein or “bad” cholesterol) levels significantly ⁹⁹. Cardiovascular disease risk in Helicobacter pylori-infected type 2 diabetes patients was shown to be reduced after administration of broccoli sprouts powder ¹⁰⁰. However, in 40 hypertension patients consuming 10 g of dried broccoli sprouts for 4 weeks, no changes in blood pressure or flow mediated dilation were detected ¹⁰¹. The exact mechanism by which sulforaphane/ broccoli is able to protect against heart and vascular disease is currently unknown but is likely related to redox changes associated with NRF2 signaling ¹⁰². This is an active area of research that warrants more clinical studies. In a phase 1 study that used sulforaphane-containing broccoli sprout homogenate (50-150 μmol dose escalation for 14 days) in adults with sickle cell disease, increases in whole blood mRNA levels of heme oxygenase1 and subunit of fetal hemoglobin were observed ¹⁰³.

Sulforaphane and respiratory conditions

A broccoli sprout homogenate was shown to reduce influenza-related outcomes in human volunteers ¹⁰⁴. Influenza virus-induced markers of inflammation were significantly lower in smokers after consumption of broccoli sprout homogenates ¹⁰⁵. In another study, daily 100 μmol sulforaphane for 14 days was shown to improve the bronchoprotective response in asthmatics ¹⁰⁶. Broccoli sprout extract (dose equivalent to consumption of 100 – 200 g broccoli) was also shown to reduce the nasal allergic response to diesel exhaust particles in human subjects ¹⁰⁷. When 25 or 150 μmol sulforaphane was orally administered to smokers with chronic obstructive pulmonary disorder (COPD) for four weeks, no significant changes in inflammatory markers were observed compared to placebo ¹⁰⁸. This outcome may be related to the fact that baseline oxidative stress and inflammation is already very high in such a patient population and cannot be reversed by a dietary agent.

Sulforaphane foods

Foods high in sulforaphane include cruciferous vegetables, such as broccoli, watercress, kale, cabbage, collard greens, brussels sprouts, bok choy, mustard greens, and cauliflower ¹⁰⁹. In cruciferous vegetables, sulforaphane is present as its precursor glucosinolate glucoraphanin ¹¹⁰. When the tissues of cruciferous plants are processed by cutting, cooking, freezing, or mastication, glucoraphanine and an enzyme called myrosinase in the cruciferous vegetables are exposed to each other, which removes the glucose moiety on glucoraphanine and hydrolyzes glucoraphanine to sulforaphane ¹¹¹. Beta-thioglucosidases occurring in the human gastrointestinal microbiome also convert precursor glucoraphanine into bioactive sulforaphane ¹¹².

Critical to the formation of sulforaphane is the plant enzyme myrosinase (thioglucoside glucohydrolase) and beta-thioglucosidases occurring in the human gastrointestinal microbiome ¹¹³, which convert precursor glucosinolate glucoraphanin into bioactive sulforaphane. A human homolog of myrosinase has not been described. Directly administered sulforaphane has much higher bioavailability than glucosinolate glucoraphanin ¹¹⁴, reflecting their intrinsic lipophilicity, but more importantly reflecting the need for ingested glucosinolates to first be converted to isothiocyanates prior to absorption and further metabolism. Measurement of urinary excretion of sulforaphane and sulforaphane metabolites has been used routinely to determine oral bioavailability in various trials. In a randomized, cross-over clinical trial where participants were administered either sulforaphane- or glucoraphanin-rich beverages, it was observed that participants who received sulforaphane-rich beverage had substantially higher rates of urinary excretion of sulforaphane metabolites than those who received glucoraphanin-rich beverage ¹¹⁵. Only 5% of the administered glucoraphanin-rich was recovered as sulforaphane metabolites as compared to 70% when sulforaphane was administered. Egner and colleagues ¹¹⁶ also showed in this cohort of healthy Chinese adults that there was rapid clearance of urinary metabolites of sulforaphane, a finding that had been reported by others previously. Four healthy volunteers who were fed an extract of 3-day old broccoli sprouts that had been hydrolyzed with myrosinase (mean dose of isothiocyanates ~201 μmol) showed rapid excretion rates of isothiocyanates (mean cumulative 8 hour excretion ~117 μmol; about 58% of the administered dose) ¹¹⁷.

Others have shown that consumption of fresh broccoli sprouts resulted in significantly higher plasma and urinary concentrations of sulforaphane than achieved following consumption of a commercial dietary supplement claiming to be rich in glucoraphanin, but lacking myrosinase ¹¹⁸. In another small clinical study, a different commercial supplement rich in glucoraphanin but without myrosinase, provided identical crude pharmacokinetics as a laboratory-prepared, freeze-dried broccoli sprout extract ¹¹⁹, indicating inter-individual variability observed in glucoraphanin delivery. Similarly, it was shown that high myrosinase activity corresponded with higher bioavailability of sulforaphane and somewhat shorter excretion half lives (2.2 hours for high activity compared with 3.1 hours for low activity) of urinary sulforaphane conjugates compared to heat-applied broccoli florets with low myrosinase activity ¹²⁰.

The finding that myrosinase-containing preparations of broccoli have higher bioavailability than those that have no myrosinase has been echoed by other studies ¹¹⁹. A comparison of fresh broccoli sprouts, glucosinolate-rich broccoli powder lacking myrosinase and a combination of both, yielded the highest urinary sulforaphane recovery from the sprouts followed by the combination and the sulforaphane supplement powder, respectively ¹²¹. In this study, appearance of sulforaphane metabolites in urine and plasma was delayed after consumption of the sulforaphane supplement powders compared to that from consumption of broccoli sprouts, likely due to the lack of active and readily available, ingested myrosinase and a dependence on the gut microbiome to supply it. Not surprisingly, methods of cooking broccoli and other cruciferous vegetables can have a significant impact on the formation and content of isothiocyanates. Fresh broccoli yields approximately 3 times higher levels of isothiocyanates compared to cooked broccoli ¹²², leading to the clear suggestion that retention of endogenous myrosinase activity by avoiding heat is an important consideration for sulforaphane delivery from cruciferous vegetables. These concerns are supported by epidemiologic data reviewed by Tang and colleagues ¹²³.

Sulforaphane supplement

In the United States, both broccoli and watercress are available in the form of dietary supplement capsules. Many of these products have not been tested for efficacy in randomized clinical trials or monitored for bioactive content, and therefore present areas of research or manufacturing that require more robust work. While most manufacturers don’t directly make health or disease treatment claims on the labels of these products, some do so overtly. The Food and Drug Administration (FDA) recently issued a series of warnings to companies that produce supplements that claim cancer preventive and therapeutic properties ¹²⁴. Amongst the common statements found on broccoli and watercress supplements are claims to support detoxification – a finding that has been essentially extrapolated from the larger body of research on these plants and their bioactives and not necessarily by direct testing the respective products. Hence, it is important not only to conduct rigorous scientific experimentation on these products but also to raise public awareness so that people know what to consume, how and when.

Advances have been made to circumvent some of the challenges associated with sulforaphane delivery. To account for the variability – or lack – in myrosinase activity in preparations or individuals that could lead to lower or higher sulforaphane concentrations, tablets containing both the glucoraphanin as well as active myrosinase have been manufactured and are sold in the US and internationally. Additionally, cruciferous crops that produce higher content of glucoraphanin can been selected-for and emerging technologies in the field are evaluating how bacteria can be engineered to bind specific cells and secrete myrosinase in a targeted chemoprevention approach ¹²⁵. A recent study compared the delivery efficiency of an alpha-cyclodextrin inclusion sulforaphane preparation with a commercial sulforaphane-rich nutritional supplement ¹²⁶.

Sulforaphane dosage

Based on available research, typical dosage for broccoli sprout and seed extracts is 50-100 mg sulforaphane glucosinolate (glucoraphanin) daily in divided doses ¹⁵.

Sulforaphane side effects

Because of sulforaphane’s toxicity to certain cells, it is of great significance to evaluate the clinical safety of sulforaphane ¹²⁷. Some researchers have tested sulforaphane in acute toxicity analyses. After fasting overnight, 48 mice were given five different doses of sulforaphane at 400, 300, 225, 168.8, and 126.6 mg/kg (8 in each group), and any serious effects or mortality were carefully observed after administration. After 14 days, all eight mice treated with 126.6 mg/kg sulforaphane survived during treatment. However, eight, seven, four, or two animals treated with 400, 300, 225, or 168.8 mg/kg sulforaphane died within 24 hours of dosing ¹²⁸. In addition, one mouse treated with 225 or 168.8 mg/kg sulforaphane died within 48 hours. For the 126.6 mg/kg sulforaphane group, no physical or abnormal changes were observed in sleep patterns, behavior patterns, fur, skin, eyes, mucous membranes, tremors, or salivation ⁸⁰. In another study, scientists implanted lymphoma cells in nude mouse xenografts and administered sulforaphane to them twice a week, 100 mg/kg each time. After 10 days, there was no significant change in body weight compared with the control group, indicating that sulforaphane is less toxic ⁹¹. Thus, the dose-associated superiority of sulforaphane in reducing adverse reactions is obvious in current preclinic research. In addition, the findings from Li et al. ¹²⁹ have shown that sulforaphane could be able to evidently restrain the pathological process of diseases in C57BL/6J mice associated with increased intestinal inflammatory factors. They demonstrated no apparent toxicity to animals induced by sulforaphane administration.

Several studies have been conducted to assess the safety of sulforaphane in humans. A randomized, placebo-controlled, double-blind study showed broccoli sprout extracts were without significant side effects at doses of 25 and 100 μmol glucoraphanin for seven days ¹⁵. Another randomized, placebo-controlled study involving 200 healthy adults consuming broccoli sprout infusions daily for two weeks (400 μmol or approximately 175 mg glucoraphanin) showed no adverse effects ¹³⁰. In a dose escalation safety study, broccoli sprout extracts containing sulforaphane doses as high as 340 nmol were topically applied three consecutive times to forearm skin. Researchers reported significant induction of phase II enzyme activity in biopsied tissue without any adverse reactions ¹⁵.

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