Cancer is a disease in which a group of cell undergoes uncontrolled growth, invasion, and sometimes metastasis. These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, and do not invade or metastasize. Most cancers form a tumor but some like leukemia. Cancer can be cause by the genetic makeup, cancer causing substance, problem of thee immune system, day to day environment, and the viruses. This disease is still not having any vaccine and it cannot be predict. Scientists are still researching in the field and try to find out the way to control this disease [WHO, 2010]
Start from late 19th century and early of 20th century; the research of cancer was start to growth and focus in the medical field. The first congresses on cancer had been held in Europe, the further advanced research mostly were done in America, start by Dr Gaylord and Dr Tyzzer who are the founder of the American Association Cancer Research (AACR). Afterward, discover of the x-ray by Roentgen and radium, isolated by Curies, proved immensely useful to the medical practitioner and soon become recognized as valuable tools to the cancer investigator [Bernard W. et al, 2008]
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The body is made up of many types of cells. These cells grow and divide in a controlled way to produce more cells as they are needed to keep the body healthy. When cells become old or damaged, they die and are replaced with new cells.
However, sometimes this orderly process goes wrong. The genetic material (DNA) of a cell can become damaged or changed, producing mutations that affect normal cell growth and division. When this happens, cells do not die when they should and new cells form when the body does not need them. The extra cells may form a mass of tissue called a tumor. Benign tumors aren't cancerous. They can often be removed, and, in most cases, they do not come back. Cells in benign tumors do not spread to other parts of the body. Malignant tumors are cancerous. Cells in these tumors can invade nearby tissues and spread to other parts of the body. The spread of cancer from one part of the body to another is called metastasis
Cancer affects people at all ages with the risk for most types increasing with ages. Based on the statistic of cancer patient in Malaysia, there are total of 21,773 cancer cases were diagnosed among Malaysians in Peninsular Malaysia in the year 2006. It comprises of 9,974 males and 11,799 females. This means 128.6 per 100,000 male population and 135.7 per 100,000 female populations were facing cancer incidence. Cancer seems to be predominant among Chinese as compare to Malay and Indian. The most common cancer among population of Peninsular Malaysia in 2006 is breast, colorectal, lung, cervix and nasopharynx. This is one of the serious issues in the country, so the control of the cancer growth is required for Malaysian's healthy. [National Cancer Registry, Ministry of Health Malaysia, 2006]
Table 1: Cancer incidence per 100,000 populations and Age-standardized incidence, by sex, Peninsular Malaysia 2006
Table 2: Cancer Incidence per 100,000 population and Age-standardized incidence, by ethnicity and sex, Peninsular Malaysia 2006
[National Cancer Registry, Ministry of Health Malaysia, 2006]
From the studies of carcinogenesis, there is many ways to reduce the risk of cancer. One of the ways to reduce the risk of cancer growth is from the diet of the subject. The anti-carcinogenic or phytochemical substance contained in the food can help to reduce the cancer cell in the body and prevent the cell mutation in the body. When subject intake the food contained anti-cancer substance or phytochemical, this chemical can help to reduce the risk of cancer possibly due to the dietary fibers polyphenol antioxidant and anti-inflammation effect. Broccoli was chose as the target of this project because contain the highest concentration of sulforaphane which is one of the phytochemical substance in all the vegetables, but the presence of the sulforaphane is not been determined and proved the concentration of the sulforaphane in broccoli is the highest among the vegetables. To determined and proved the statement, some instrument such as gas chromatography/ mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) had been use to find out the presence of sulforaphane in the broccoli and the concentration of the sulforaphane in broccoli.
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Brassica oleraces, also known as broccoli is a plant of kale family or mustard family. Broccoli's name was came from the Italian words which is "brocco," means the "sprout" or "shoot," which comes from the Latin, "brachium," meaning "arm" or "branch." Leaves and flowers can be eaten but mostly flowers is more common to consumers. It is common to the Mediterranean and was originally eaten for its stems, and later bred to have large, delicious flowers. Cauliflower was a development of broccoli during this period of improvement [Buck P.A., 2008]. From the beginning, broccoli is the wild vegetable in the eastern Mediterranean. Broccoli traveled from Italy to France, on to Britain, then United State. Broccoli was first introduced to the United States by immigrants but did not become widely known until the 1920s. By the 1930's it was a important vegetable of the American diet. The demand for broccoli is higher than cauliflower after the Second World War, when Americans, returning from Europe and the European demanded the vegetable at home.
The two major types of broccoli are: those which are grown for heads of flowers, and those which are grown for heads of flowers and their leaves. It was out of the first type that cauliflower was bred: the broccoli flower was made larger and white. Broccoli was a popular vegetable in Greece and Italy, but did not become popular in the rest of Europe until the king of France, Louis XIV favored the vegetable (Buck P.A., 2008).
Broccoli contain high amount of vitamin C, K and A, low amount of calorie and protein. Broccoli offers 0.73 mg of calcium per 100 gram, as much calcium as 4 oz. of milk. Besides broccoli also contain a sufficient iron requirement, the absorption of iron also help by the vitamin C to the body. Broccoli contains about 35% more beta-carotene compare with frozen broccoli and fresh broccoli because the frozen packages consist mainly of the florets. Most of the beta carotene is stored in the florets. It is a very good source of enzyme-activating manganese; muscular-system-supporting potassium, and magnesium. Besides that, broccoli also contains energy-producing such as vitamin B2, vitamin B6, and phosphorus; and anti-inflammatory omega-3 fatty acids. The vitamin B1, vitamin B3, vitamin B5, and iron; bone-healthy calcium; and immune-supportive zinc and vitamin E are also contained in the broccoli (USDA national nutrient database, 2010).
Because of its impressive nutritional profile that includes beta-carotene, calcium, fiber, and phytochemicals, specifically indoles and aromatic isothiocynates, broccoli and it skin may be responsible for boosting certain enzymes that help to detoxify the body. These enzymes help to prevent cancer, diabetes, heart disease, osteoporosis, and high blood pressure
Though definitive proof is not yet published, the National Cancer Institute suggests that broccoli, along with its cruciferous family members, may be important in the prevention of some types of cancer. Recently in 2006, the first evidence shows that the sulforaphane contained in broccoli able to induce the histone acetylation and suppress the tumor formation in rats. This proved that broccoli can help in prevention of the cancer disease (Melinda C., 2006).
Figure 1: molecular structure of sulforaphane (H. Liang
The 1-Isothiocyanato-4-methylsulfinylbutane (C6H11NOS2), also known as sulforaphane, is one of the organosulfur substances that showing anticancer, antidiabetic and antimicrobial properties. Sulforaphane is also classified as an isothiocyanate. Its molecular weight is 177.29 daltons and it is insoluble in water. Salforaphane can be detected under the evaporative light-scattering detector (ELSD) but not in ultra violet light. It highly obtained from the cruciferous plant such as Brassica vegetables. Besides broccoli, it also found in brussel sprouts, Chinese kele, mustard, cabbage, and watercress. The content of sulforaphane is 30-50 times more than other example vegetable compare with the matured broccoli. Sulforaphane is not a nature compound in the broccoli. It is from as a sugar molecule in the plant called sulforaphane glucosinolate. Sulforaphane glucosinolate undergoes hydrolysis and breakdown to sugars and sulforaphane when in the body. Sulforaphane glucosinolate (glucoraphanin) is also known as 4-methylsufinylbutyl glucosinolate, it works as a catalyst and boosts Phase 2 enzymes that exert antioxidant activity; broad spectrum activity eliminates many types of free radicals, cycling over and over again; creates an "army" of antioxidants, ready to neutralize free radicals over a period of time. These sugars will breakdown to glucose and sulforaphane and release to body and only this the sulforaphane formed. Sulforaphane does not cause any effect to human body whether is overdose or insufficient because sulforaphane is not an essential chemical in body. Although is not harm to human body, people are advised to get consult from the doctor before taking the sulforaphane [Sciencelab.com, Inc, 2008].
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Properties of sulforaphane.
The discovery of the phase 2 enzyme inductions by sulforaphane, many other mechanisms of chemoprotection had been exanimate. Recent studies also suggest that slforaphane offer protection against tumor development during the "post-initiation" phase and mechanism for "suppression" effect of sulforaphane.
Sulforaphane inhibits can carcinogenesis with the inhibition of Phase 1 enzymes and then the induction of Phase 2 enzymes at the initiation stage of cancer. A summary of the "blocking" targets are shown in Table 3.
Sulforaphane play an indispensible in cellular resistance to the carcinogenic attack. In the post initiation, sulforaphane can interrupt the cancer development through various molecular targets which is involved in controlling cell such as proliferation, differentiation, apoptosis, or cell cycle. Sulforaphane can modulate these targets and lead their activities towards apoptosis or cell cycle arrest, thereby eliminating initiated cells from the cell population (John D. et al, 2008).
Table 3: Effect of sulforaphane in "blocking "mechanism
Figure 2: Metabolism of sulforaphane involving (A) hydrolysis of glucosinolate to its isothiocyanate via myrosinase enzyme activity and (B) metabolism of sulforaphane via the mercapturic acid pathway.
GST= Glutathione-S-Transferase; GTP= Î³-Glutamyltranspeptidase; CGase= Cysteinylglycinase; HAT= histone acetyltransferase
2.2.1 Metabolism and bioavailability of sulforaphane
The metabolism of sulforaphane is summarized in Figure 2. The initial of reaction is involves the enzymatic hydrolysis of glucoraphanin, which can form the sulforaphane in the further. This reaction catalyzed by myrosinase, which the found in plant to protect plant against herbivour. In this reaction, breakdown of the glycone from the glucosinolate forming glucose, a hydrogen sulfate and one of many different aglycones. This reaction is depending on the reaction pH in the environment, glucosinolate, and availability of ions (Bones.AM, 1996). Most of the glucosinolate hydrolysis products are stable isothiocyanates in a neutral pH. After absorption, sulforaphane is predominantly metabolized through the mercapturic acid pathway. The electrophilic central carbon of the -N=C=S group in sulforaphane reacts with the sulfhydryl group of glutathione to form a dithiocarbamate glutahione conjugate in these reactions.
Interestingly, sulforaphane is also can instigate its own metabolism via induction of GSTs. The last step in sulforaphane metabolism is the formation of sulforaphane-Cys and finally sulforaphane-N-acetylcysteine (Kassahun K, 1997). There are some factors that affect the absorption and bioavailability of sulforaphane. The first factor is involves the hydrolysis of sulforaphane from glucoraphanin via myrosinase activity. This initial step is important because only the isothiocynates form is thought to be biologically active and promote the desired anticancer properties. Importantly, mammalian cells do not occurred endogenous myrosinase activity. The myrosinase enzyme is separated from the the plant cell wall in the cruciferous plant, but upon physical interruption such as chopping, cutting, or chewing. The enzyme is released and the isothiocynate is released too (Kassahun K, 1997). Another source of intestinal microbial flora is another source of the myrosinase activity. Evidence from experiments done with isolated human fecal bacteria (Conaway CC, 2000) and others using rats dosed with glucoraphanin, indicates that sulforaphane can be converted by glucoraphanin from colonic microbial flora and that enterohepatic circulation is requisite for efficient metabolism.
However, the bioavailability of sulforaphane is six times less when metabolism of the glucosinolate to the isothiocynate had not occurred prior to ingestion (Sapiro TA, 2001), revealing a strong reliance on plant myrosinase activity, as opposed to the intestinal gut flora. Nonetheless, there is an important factor proved the inter-individual sulforaphane bioavailability is variability in the gut microbial flora when the nonhydrolyzed glucosinolate is taken. The last factor that affects sulforapahene bioavailability is related to polymorphisms in phase 2 sulforaphane metabolizing genes, such as GSTs. GSTs play a important role in determining the detoxifying ability in human body. In general, GST enzymes catalyze the conjugation of GSH to electrophiles such as sulforaphane. There are 6 different classes of GST isoenzymes, which is: Î±, Î¼, Ï€, Î¸, Ïƒ, and Îº, and each functional unit is composed of two subunits. In general, the substrate specificity for the different isoenzymes overlaps but, specifically, each class has different degrees of reactivity for different substrates. GST null genotypes are quite prevalent in the population, with up to 50% of people being GSTM1 null and 47% Glutathione S-transferase theta-1(GSTT1) null. The effect that GST genotype has on tumor development and chemoprevention is complex. In the context of high cruciferous vegetable intake, evidence is mounting in favor of a GST null genotype providing a protective effect against lung, colon, and breast cancers (Seow A, 2005; Lampe JW, 2002)
Since GST activity plays an important role in the sulforaphane metabolism and subsequent excretion, lower GST activity in individuals with GST polymorphisms could result in slower elimination and longer disclose to isothiocyanates after cruciferous vegetable consumption. For example, a study of investigating a population in Singapore reported has higher isothiocynate discharge among GSTT1 positive individuals in comparison to GSTT1 null (Seow A, 1998), implying shorter exposure times to the potential beneficial metabolites of sulforaphane for GSTT1 positive individuals. This study in Singapore indicating that GST null genotype coupled with high cruciferous vegetable intake and reduced the risk of colorectal cancer (Seow A, 2002). But, other studies by Gasper have shown that the GSTM1 null genotype produced a greater number in the area under time-concentration curve (AUC) for metabolite concentrations in plasma, a sulforaphane metabolite excretion with a significantly higher rate, and a higher percentage of sulforapahne excreted 24 hours after ingestion, indicating shorter retention times of sulforaphane and its metabolites (Gasper AV, 2005). In addition, in other studies, a GSTM1 null genotype had no increase in prostate cancer risk, while men in the high vegetable consuming group that have GSTM1 present exhibited the greatest reduction in cancer risk (Joseph MA, 2004). These inconsistencies may be explained by differences in the predominant ITC consumed (3-butenyl-isothiocynate and4-pentenyl-isothiocynate versus sulforaphane), the class of GST null genotype present (GSTT1 versus GSTM1), or the cancer of interest. Notwithstanding these conflicting results, the impact of polymorphism on nutrient bioavailability is an important area of research that will aid in our understanding of response variability to sulforaphane in human populations.
Application of sulforaphane
The induction of phase 2 enzyme to protect against the cancer development by the sulforaphane, a lot of experiments had been done by many scientists to prove the sulforaphane can be use as the anti-carcinogens chemical.
Effective at inhibiting Helicobacter pylori growth
Helicobacter pylori, is one of the bacteria can cause the stomach cancer in human body, H. pylori is a Gram-negative, bacilliform, motile, microaerophilic bacterium that colonizes the gastric mucosa in humans. It can cause a chronic low-level inflammation of the stomach lining and is strongly linked to the development of duodenal and gastric ulcers or stomach cancer. In one of the experiment done by the Jed W. Fahey and Paul Talalay in 2002, the sulforaphane can inhibits the helicobacter pylori and prevent the benzo[a]pyrine to induce the stomach tumor cells (Jed W. et al, 2002)
In the experiment, the result shows that sulforaphane is lethal to both extracellular and intracellular forms of Helicobacter pylori, by mechanisms that are not yet understood. The experiment also show that sulforaphane protects the mouse against the neoplastic effects of benzo[a]pyrene, and that this effect depends on induction of phase 2 enzymes because it is abolished in mice deficient in the nrf2 gene, which controls these inductions. Thus, the dual properties of sulforaphane as an antibiotic and anticancer agent provide a two-tiered, and possibly synergistic, approach to eliminating Helicobacter pylori and reducing the incidence of gastric disease.
In the experiment, the sulforaphane is isolated from broccoli and add into horse blood agar with contain 3 types of helicobacter pylori and observe the bacteria activity by using time-to-kill assay. The result will be determined by the minimal inhibitory concentration (MIC). MIC is use to diagnose laboratories to confirm resistance of microorganisms to an antimicrobial agent.
The result show the high concentration of the sulforaphane in the Helicobacter pylori culture is decrease the activity of the bacteria. The lower concentration of the sulforaphane inhibits and decrease the activity of the Helicobacter pylori bacteria but the inhibition is not very successful. This means the sulforaphane does affect the activity of the bacteria. When the high concentration of sulforapahen is applied, the activities of bacteria are drop to the lowest and control the bacteria (Jed W. et al, 2002)
Figure 4: Bactericidal potency of sulforaphane (SF) on two strains of H. pylori (A, LBN201, clinical isolate; B, 26695, reference strain) MIC of SF at pH 5.8. (Data points are the means of duplicate determinations; the asterisk indicates a point below the limits of detection.)
Sulforaphane can protect skin against skin cancer
Over expose to the ultraviolet radiation (UVR) such as the sun can cause to the skin cancer. UVR can emit 3 type of band: UVA, UVB and UVCbands. This 3 light bands can damagethe collagen fiber and accelerate the aging of the skit. UVB and UVC can direct damage the DNA in the cell but not UVA. UVA cann produce highly reactive chemical intermediate, for example hydroxyl and oxygen radicals, which turn can damage DNA (Torma, 1988)
Dr Paul Talalay done an experiment in 2007 and founds that apply the sulforaphane topically in human and animal skin, the sulforaphane can protect against a carcinogen in human is catalytic can long lasting. In the experiment, there are 6 subjects (male and felame, 28-53 years old) were ask to refrain from consuming the crusiferous plants and coffee, then sulforaphane applied on the allowing the skin to equilibrate under the weight of the chrometer.
In the result of the experiment, the mean reduction of the enrythema by broccoli extract in all subjectand highly significant. The protection of the sulforaphane is highly significant in 3 subjects. The reason of other three subject do not have the same positive result is because differences in protective responses among individuals related to the genetic polymorphisms among the gene encoding for phase 2 enzyme
Figure 5: Effect of treatment with sulforaphane (broccoli sprout extract) on the erythema induced by UV radiation
126.96.36.199 sulforaphane act as histone deacetylase (HDAC) inhibitors
Histone deacetylase is an enzyme that can remove the acetyl groups from a Îµ-N-acetyl lysine amino acid from histone. The function of the histone deacetylase is to remove the acetyl groups from histone , increasing the positive charge of histone tails and supporting high-affinity binding between the histones and DNA backbone. This effect will prevent the transcription in the DNA structure. With this condition, the mutation of the cell will occur because of the cell cycle is not lead the cell to apoptosis stage.
Histone deacetylase inhibitor (HDI) is the enzymes that inhibits the HDAC and induces the differentiation and/or apoptosis of transformed and inhibit the tumor cell to growth. HDI found in the cells, which can disrupt the cell cycle in G2, acquiescing cells to prematurely enter the M phase, as well as interfering directly with the mitotic spindle checkpoint. One experiment had been done by Roderick H. Dashwooda, he done the experiment by in vitro with human body and in mice body.
From the experiment, Dr. Roderick's experiment is to find out can or not the inhibition of HDAC activity in nuclear from human colon cancer cells can be treated with sulforaphane. So, the experiments start with the mice to find out the hypothesis. First, the mutated human colon cell will be implanted into the nude mice and cell growth characteristic had been observed. The necessary amount of sulforaphane injected to the mice and the result will be observed for 3 weeks. The quantity of sulforaphane is not over then 15 Î¼M. This is to avoid the possible complications of oxidative stress and apoptosis, which occurs at higher doses of sulforaphane in mice body.
There is a significant result show that the tumor growth in the mice body were decreased and controlled after sulforaphane is induced into mice body. This means the sulforaphane can inhibits the histone deacetylase and lead the cell to apoptosis from mutated. To make sure the sulforaphane is effects in the nude mice, blood samples and various mouse tissues also were examined; there was significant inhibition of HDAC activity in the prostate and peripheral blood mononuclear cells.
Then, the experiment was preceded to human body to further experiment. The experiment is almost similar to mice, the subjects will consume certain amount of broccoli which rich in sulforaphane and the result will be observed in 48 hours. The volunteer's ages is range 18 to 55, healthy, no history of non-nutritional supplement use, and did not consume any cruciferous vegetable for 48 hours. After the volunteer consumed broccoli, the volunteer's blood had drawn at 0, 3, 6, 24, and 48 hours. From the result, HDAC activity was inhibited by the sulforaphane as 3 h after broccoli sprout intake, and returned to normal by 24 hours. This is the first evidence that human consumed broccoli, which contained high concentration of sulforaphane, showed a marked effect on HDAC activity. There was a significant induction of histone and aceylation which function as the HDI in first 3 hours and 6 hours, HDAC acivity back to normal in 24 hours, and histone hyperacetylation was evident for at least 48 hours. This is the evidence that the sulforaphane from broccoli were affecting the activity of HDAC in blood circulation in 48 ours, this is proving that the sulforaphane can act as HDAC inhibitor in human body and can help to prevent or reduce the growth of tumor cell in human body (Roderick H, 2007).
Figure 6: HDAC inhibition by SFN in human colon and prostate cells, and suppression of xenograft growth in mice.
Figure 7: HDAC inhibition by SFN in mouse colon, and suppression of intestinal polyps
Synthesis of sulforaphane.
The sulforaphane is very useful in the protection against the tumor growth, the synthesis of the sulforaphane had been studied by scientist to invent the medicine or drugs to control the tumor growth. The compound the synthesized is not similar to the sulforapane but having the same function as sulforaphane. There is experiment of the cancer chemopreventive activity by the sulforamate derivatives.
This is the pathway of the synthesis of sulforaphane by the N-(4-Bromobutyl)phthalimide. Sodium thiomethoxide and methanol added to N-(4-Bromobutyl)phthalimide, stir the mixture in room temperature for overnight and then heat to reflux for 2 hours. The mixture will undergo hot filtrate and dissolve in diethyl ether and water. The mixture then filtrate, the tan solid which is N-(4-Methlysulfanylbutyl)phthalimide collected.
Next, 3-chloroperbenzioc acid which dissolve in dichloromethane was added to N-(4-Methlysulfanylbutyl)phthalimide in -5oC and stir for 2 hours to let the N-(4-Methlysulfanylbutyl)phthalimide undergo oxidation to form N-(4-Methlysulfinylbutyl)phthalimide. Then, methanol and N-(4-Methlysulfinylbutyl)phthalimide were mixed. Hydrazine monohydrate was added and the mixture heated to reflux for another 2 hour, and settle overnight. The 4-methanesulfinylbutylamine will obtain after the purification. Then, the compound was later treated with triethylamine, carbon disulfide and then iodomethane to yield sulforaphane derivative compound in good yields.
This synthesis of sulforaphane method is not usually use to produce the commercial sulforaphane product. This is because the process for all the synthesis is very complicated and it takes a long time for the synthesis. The complete synthesis will need more than 24 hours and this is not a better procedure for produce. Besides, the potassium pthalimide is not easy to obtain or purchase and it take a lot of cost to do the synthesis and the yield percentage is very low. So, most of the sulforaphane product in market is extract from the original broccoli plant.
Figure 8: Synthesis of sulforaphane by N-(4-Methlysulfanylbutyl)phthalimide
Bioavailability and Kinetics of Sulforaphane in Humans after Consumption of Cooked versus Raw Broccoli
When human consume the broccoli, glucoraphaninwill under chewing and enzymatically hydrolyzed by myrosinase breakdown to isothiocyanate, sulforaphane, and other breakdown products. Most of the time, the broccoli is cooked before ate and the raw broccoli is seldom consume by human. In this case study, the thing that focus is the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, from raw and cooked broccoli because we does not know about the concentration of the sulforaphane in raw broccoli is more or less compare to the cooked broccoli. In the experiment carry out by Martin Vermeulen in 2008, eight healthy men consumed broccoli in a cross-over design study. The sulforaphane in raw and glucoraphanin in cooked broccoli were determined, and metabolites were measured in urine and blood. In the urine the sulforaphane can detect by sulforaphane mercapturic acid and in blood, sulforaphane can be detected by the sulforaphane conjugates.
Volunteers will consumed certain amount of crushed broccoli, raw or microwave cooked, together with a warm meal on two separate days, in the morning, with 1 day of wash out between in a randomized, freeliving, open cross-over trial. Fasted is not necessary for the volunteer before taking the meals. The warm meal consisted of a fixed amount of a meat burger and mashed potatoes and volunteers were refrained from eating crucifers on the days before treatment.
Glucosinolates were determined by a modified method proposed by Vermeulen, M. (Vermeulen, M., 2006). In briefly, glucosinolates were extracted and trapped then desulfated in the solid phase extraction columns. Water were used for the desulfoglucosinolates, measured using highperformance liquid chromatography (HPLC) coupled to a diode array detector (DAD), and using the relative response factors to determine result. Isothiocyanates were conjugated with 2-mercaptoethanol and determined by HPLC-DAD.
Figure 9: Urinary Sulforaphane Mercapturic Acid Pharmacokinetic Secondary Parameter Summary after a Single Dose of 200g of Broccoli, either Raw or Cooked, Was Consumed
There are few methods can be use to determine the sulforaphane in broccoli. The first experiment of determination of sulforaphane in broccoli done by Dr. Talalay in the Johns Hopkins University is by using the IR and the HPLC. The most popular instrument uses to determine the sulforaphane are high-performance liquid chromatography (HPLC), chromatography (GC) and GC/mass spectrometry (GC/MS).
There is difference between the GC/MS and HPLC machine. Liquid chromatography is using a "column" which is made from bare or bonded silica; the tested compound will be separate by polarity of the compound. This can be run in a mixture compound to separate the contain or find out the presence of chemical that desired
Gas chromatography also using a column, but it is a capillary column that not using a liquid to carry the experiment mixture. the column is filled with gas and sample is separated uses a carrier gas, for example: nitrogen. You can vary the temperatures in both LC and GC to aid better resolution.
Gas chromatography is used for more volatile compounds and liquid chromatography is used for the less volatile compounds. HPLC machine mostly refers to reversed phase which means the polarity sample is opposite to the column, normal phase is where the column is in silica which is very polar.
From the case study, there is two instrumental had been use to determine the presence of sulforaphane in broccoli. The GC/MS instrument will used to determine the presence of the sulfaraphane in the broccoli. The HPLC will used to determine the concentration of the sulforapahane in different part and determine the concentration of the sulforaphane in broccoli is higher than other vegetables.
For the GC/MS method, the procedures are similar to the HPLC method. The broccoli will be autolyse in room temperature for 30 minutes. One gram of the broccoli was homogenized in warm (50oC) distilled water at medium speed for 5 minutes using a mixer. The homogenate was centrifuge at 8000g for 5 minutes, and the supernatant was filtered through a 0.45Î¼m filter. 25mL of the methylene chloride was added to the filtered supernatant and vortexed the mixture for 1 minute. After that, the mixture was centrifuge at 4000g for 5 minutes to form two phase. The lower methylene chloride layer was collected by pipette. The aqueous was extracted for the second times with methylene chloride as describe above. Sodium sulfate was used to dry the organic liquid in the mixture and remove the sodium sulfate by the 0.45Î¼m filter. The filtrate then will be concentrated under reduced pressure at 35oC to <1mL using a rotary evaporator. The concentrated extract will then diluted to 1.0mL with methylene chloride prior to inject into the GC/MS.
Analyses were performed on a Hewlett-Packard (HP) 5890 Series II Plus gas chromatograph with electronic pressure control (EPC) connected to an HP 7673 auto sampler and an HP 5972 mass selective detector. The split/splitless injector was operated in splitless mode using a 4 mm inside diameter (i.d.) injection liner (Hewlett-Packard, Palo Alto, CA). An HP-5MS fused silica capillary column (Hewlett-Packard, 30 m, 0.25 mm i.d., 0.25 Î¼m film thicknesses, cross-linked to 5% phenyl methyl siloxane stationary phase) was used. The entire system was controlled by MS ChemStation software (Hewlett-Packard, version B.02.04). Injector and detector temperatures were 250 and 300 Â°C, respectively. Column oven temperature was initially set at 40 Â°C for 2 min, then increased to 270 Â°C (ramp, 10 Â°C/min), and held for 5 min. The EPC was used to provide the desired carrier gas flow rates for the various experimental conditions. For the constant flow conditions, the electronic pressure controller was programmed to maintain a flow rate of 1.0 mL/min throughout the chromatographic separation. For fast initial injection flow conditions, the carrier gas flow rate was programmed for an initial pressure of 25.0 psi (3.0 mL/ min). After 1 min, the pressure was reduced at a rate of 20.0 psi/min to a pressure of 7.1 psi (1.0 mL/min). To maintain a constant 1.0 mL/min flow rate throughout the chromatographic separation, the pressure was increased at a rate of 0.47 psi/ min to a final pressure of 20.3 psi, which was then held for additional 5 min. Mass spectra were obtained by electron ionization (EI) over a range of 50-550 atomic mass units. Ion source temperature was 177 Â°C, and the electron multiplier voltage was 1753 eV.
The broccoli was purchase from a farm and let it autoclave in the room temperature for 30 minutes. After the autolyzing, the sample was extracted two times with methylene chloride, which was combined and salted with anhydrous sodium sulfate. The methylene chloride fraction was died at 30oC under a rotary evaporator. The residue was dissolved in acetonitrile and was then filtered through a 0.22Î¼m membrane filter prior to inject into HPLC.
The Hitachi HPLC apparatus equipped with Hitachi model L-7100 pumps, L7420 tunable absorbance detector, and reversed-phase C18 column (250 x 4.6mm, 5Î¼m, DiamodsilTM) were used in the analysis of the sulforaphane. The solvent system consisted 20% of acetonitrile in water was changed linearly over 10 minutes to 60% acetonitrile, and the acetonitrile maintained in 100% for 2 minutes to purge the column. The temperature of the column was set at 30oC, flow rate was 1mL/min, and 10Î¼L portions were injected into the column. The sulforaphane will injected and the result will be recorded (H. Liang, 2005).
Figure 10: Mass spectra of sample: (A) sulforaphane, (B) sulforaphane nitrile, and (C) 3-butenyl isothiocyanate.
Figure 11: GC/MS chromatogram of sulforapahne standard using the (A) split/splitless injector with 4.0mm splitless inlet liner and constant flow and (B) on-column injection with programmed temperature vaporization (PVT).
4.2 HPLC METHOD
Figure 12: Chromatograms of the HPLC elution profile of sulforaphane from broccoli (A) and cabbage (B) sample
Table 4: Sulforaphane content (mg/g, FW) in the edible tissues of 18 varieties of broccoli and cabbage.
Table 5: Sulforaphane content (mg/g, FW) in various tissues of broccoli
*FW = fresh weight
5.1 GC/MS Method
From topic 4.1, the spectra result of the sulforapahene, sulforaphane nitrile, and, 3-butenyl isothiocyanate. The first spectra result was showing the MS (EI) of sulforaphane, m/z (%): 72 (100), 39 (12), 45 (12), 55 (33), 60 (5), 64 (17), 85 (57), 114 (8), 119 (3), 160 (68), 177 (M+, 1). In the spectra, the molecular size of sulforaphane (177.3mol/g) does not appear in the spectra. The highest intensity of the spectra is the m/z 72, which is the functional group of the isothiocyanate(NCSCH2), and the second highest is the m/z 160, which is C6H10NS2 and the m/z 85 (C2H3NCS) and m/z 64(CH4SO). This fragment is the small fragment of the sulforaphane. The actual size of the sulforaphane is not found in the spectra is because the structure of the sulforaphane is already destroy when in the GC/MS due to the high temperature.
Sulforaphane is a stable compoung but the structure will break under high temperature in the instrument. The second spectra is the spectra of the sulforaphane nitre, one of the stable compound form when the hydrolysis by the glucafinin. the MS(EI) of sulforaphane nitrile,m/z (%): 55 (100)(C4H7), 39 (19),41 (29), 45 (11), 63 (21)(CH3SO), 64 (57), 78 (10), 82 (33)(NC5H8), 145(M+, 10). The complete structure was found in the spectra because the structure of sulforapahen nitrile is a stable structure. The molecular weight of sulforaphane nitrile is 145g/mol, (C6H11NOS). Smaller structure such as m/z 55, which is butane, and m/z 63 which is methane sulfermonoxide was found in the spectra.
The structure can be found in the spectra were The trird spectra is the 3-butenyl isothiocyanate with the MS(EI) of 3-butenyl isothiocyanate, m/z (%): 72 (100), 53 (8), 55 (27), 85 (8), 113 (M+, 44). The molecular weight of the 3-butenyl isothiocyanate is 113, (C4H7NCS). The functional group of isothiocyanate was determined in the spectra by the evidence of m/z 72 (NCSCH2). The isothicyanate which contain in the broccoli is the highest intensity in the spectra. As all the 3 spectra, the presence of the thiocyanate and the sulfoxide was proved that the presence of sulforaphane in the broccoli that tested in the experiment.
In this experiment, The split/splitless mode of operation of the GC/MS, employing a 4.0 mm i.d. splitless inlet liner with a constant carrier gas flow rate controlled by the EPC, is typically satisfactory for the analysis of small, volatile, and thermostable molecules. Preliminary experiments demonstrated that these conditions were not acceptable for the analysis of the relatively thermolabile sulforaphane molecule. Degradation of sulforaphane nitrile was not observed. Mass spectral detection of a sulforaphane standard solution indicated that â‰ˆ80% of the sulforaphane was degraded to 3-butenyl isothiocyanate. The identification of 3-butenyl isothiocyanate as a primary thermal degradation product of sulforaphane is reported here for the first time. Confirmation that the formation of 3-butenyl isothiocyanate was the result of the thermal degradation of sulforaphane was obtained by comparison with the technique of on-column injection with programmed temperature vaporization (PTV). By minimizing exposure of the sample to heat, essentially 100% of the standard was detected as sulforaphane.
To conclude this case study, Broccoli is of great interest in the field of human nutrition and disease prevention. Sulforaphane and sulforaphane nitrile, two compounds produced by the enzymatic hydrolysis of the primary glucosinolate found in broccoli, are of particular interest. GC/MS method for the analysis of sulforaphane and sulforaphane nitrile is sensitive, rapid, reproducible, and suitable for routine analysis. This method is now being used to screen >70 different varieties of broccoli and other Brassica vegetables to identify those that are high in glucoraphanin and which yield high levels of sulforaphane and low levels of sulforaphane nitrile upon hydrolysis.
5.2 HPLC method
The HPLC method was used to quantitate sulforaphane in a sample of fresh broccoli and cabbage. The fresh weight (FW) was used because it gives a better idea of the content really ingested. It is reported that, even at dietary doses, sulforaphane can modulate the xenobiotic-metabolisming enzyme systems, shifting the balance of carcinogen metabolism toward deactivation (Yoxall et al., 2005). Thus, it is more important to analyse the content of sulforaphane in the edible tissues of broccoli and cabbage. Table Shows the sulforaphane content (triplicate analyses) of 18 different varieties of broccoli and cabbage. The highest content of sulforaphane in broccoli was found in the accession ZhQ no. 26 (32.9 mg/g, FW), while the lowest content was found in ZhQ no. 172 (1.4 mg/g, FW). Differences in contents of sulforaphane among the cabbage accessions were relatively small. In contrast, it was shown that there was a significantly higher level of sulforaphane in broccoli than that in cabbage such that the mean sulforaphane content in broccoli was nearly five-fold higher than that in cabbage. In this study, the content of sulforaphane was detected in different tissue types of broccoli (Table 2). Data showed significant differences in the content of sulforaphane among the various broccoli parts. The highest content of sulforaphane was found in the florets, while the lowest content was found in the leaves. The mean content of sulforaphane in florets was nearly two-fold higher than that in stalks and nearly 10-fold higher than that in leaves. It is interesting to note that the highest sulforaphane content is found in the edible tissues of broccoli.
In the research of preventing and decrease the tumor growth activity in human body, researcher found that the broccoli is one of the effective vegetable to consume. The phytochemical that contain in broccoli can help to induce the phase two enzyme and promoting the inhibition of the tumor growth in human body. Besides this the phytochemical, sulforaphane, giving the other benefits to human body, such as providing a good nutrient to human body and prevent of the heart from inflammation that can lead to atherosclerosis.
Sulforaphane, one of the phytochemical compound found in cruciferous vegetable having effect against the cancer cell. the broccoli is the vegetable having the highest concentration of sulforaphane in the vegetable family. Sulforaphane is the organic compound and it is still a new chemical for researcher because the effect of the sulforaphane was discovered in few decades. To study the sulforaphane, the synthesis of the sulforaphane also been discovered by researcher to analyze the structure and usage of the sulforaphane. But this theory of synthesis mostly is used in the academic research and not in the commercial product because the process is very complicated and the cost is high for the process. The yield percentage of the synthesis also is not in a convenient amount or range.
In the analytical of sulforaphane in broccoli, many methods had been tested and there is two methods is the most popular, which is using the gas chromatography/mass spectrometry (GC/MS) and the high performance liquid chromatography machine (HPLC). This two methods is very successful to determine the presence of the sulforaphane in broccoli because the described method was found to be rapid, linear, reproducible, specific, accurate and capable of analysing a large number of samples without elaborate sample preparation.
As a conclusion, broccoli is a vegetable which contained the high amount of the sulforaphane is benefits to human health. Inside the broccoli contained the various nutrients and the phytochemical, for example, sulforaphane, 3,3'-Diindolylmethane, and the selenium. This phytochemical can help to against the tumor activity and prevent mutation of the cell in the cell cycle. The raw broccoli was founds to have more high sulforaphane concentration compares to the cooked broccoli. Although there is a different concentertion of sulforaphane in raw and cooked broccoli, the different is just very small and there is no any extra benefit to human body. So, increase the consumption amount of broccoli can significantly improve one's health benefits and decrease the tumor activity in human body.