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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Soni et al. World Journal of Pharmacy and Pharmaceutical Sciences


SJIF Impact Factor 5.210

Volume 4, Issue 10, 2234-2249 Research Article ISSN 2278 – 4357

BROCCOLI SULFORAPHANE: AN INSIGHT INTO THE


ANALYTICAL ASPECT AND ULTRAVIOLET SPECTROSCOPIC
METHOD DEVELOPMENT AND VALIDATION

Kriti Soni 1 and Kanchan Kohli2

Department of Pharmaceutics, Jamia Hamdard, Hamdard University-110062

ABSTRACT
Article Received on
20 Aug 2015, Sulforaphane is an isothiocyanate present in broccoli which is cleaved

Revised on 12 Sep 2015, out of glucoraphanin as a result of activity of enzyme myrosinase. It is


Accepted on 01 Oct 2015
insoluble in water but shows good solubility in organic solvents. In
addition to this the drug is highly prone to degradation subjected to

*Correspondence for thermal and basic stresses. Inspite of these it has been found to be
Author highly active as an anticancer molecule which works at a very low
Dr. Kanchan Kohli dose and shows almost no toxic effect. The present paper highlights the
PhD, Associate Professor
extraction of sulforaphane from broccoli, its structural elucidation by
Department of
simulation studies and development and validation of its ultraviolet
Pharmaceutics, Jamia
Hamdard,Hamdard spectroscopic method of analysis. The paper also highlights various
University-110062. important analytical methods developed by other researchers such as
HPLC, GC-MS, LC-MS, FTIR, NMR and Mass spectroscopic
analysis. These methods have been discussed here in so as to provide readers with an insight
into the analytical methodologies of these compound. The development of these methods by
authors will be discussed in subsequent papers.

KEYWORDS: Sulforaphane, Glucoraphanin, HPLC, LC-MS, Ultraviolet spectroscopy,


Validation.

1. INTRODUCTION
Consumers are increasingly aware of the health benefits associated with the phytochemicals
or secondary metabolites found in fruits and vegetables. The effects of these phytochemicals
on human health and nutrition depend upon numerous factors including the chemical
structure, matrix, and quantity consumed.[1] Vegetables are good sources of natural

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antioxidants and biologically active components and play an important role in human
nutrition in supplying certain constituents that are deficient in other foods.[2] Epidemiological
studies indicate that a diet rich in cruciferous vegetables, such as broccoli, cabbage, Brussels
sprouts, cauliflowers, kale, kai choi and watercress, can reduce the risk from a number of
cancers,[3-7] Brassicaceous vegetables (e.g. broccoli and red cabbage) have been widely
approved for their beneficial effects on human health and contain high concentrations of
vitamins, minerals and, in particular, a special group of phytochemicals sulfur-containing
glucosides named glucosinolates.[8] These compounds, upon hydrolysis by the endogenous
enzyme myrosinase (thioglucoside glucohydrolase), yield a variety of bioactive products,
including isothiocyanates, thiocyanates, nitriles, oxazolidine-2-thiones, sulfate and D-
glucose, depending on the chemical structure of parent glucosinolates and the conditions,
such as temperature and pH during the enzymic cleavage.[9-11] Among these, sulforaphane [l-
isothiocyanato-4-(methylsulfinyl)-butane], which has been identified in broccoli (a member
of the Brassicaceae) as a product of enzymatic- or acid hydrolysis of the corresponding co-
(methylsulfinyl)-alkyl-glucosinolate (glucoraphanin), has been a great deal of interest [12, 13].
Myrosinase, an enzyme present in fresh broccoli and its sprouts, hydrolyzes glucoraphanin
(GRA), the inert glucosinolate precursor of sulforaphane (SFN), into the biologically active
isothiocyanate. The process starts as soon as the fresh vegetable is chewed or otherwise any
causing process could damage the cells. Consequently, there is partial conversion even before
the compound reaches the stomach. Myrosinase is also present in the microbial flora of the
lower intestine of animals and humans; hence, a significant fraction of GRA is expected to be
hydrolyzed and become bioavailable as SFN by the time it fully passes the gastrointestinal
system.[14]

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Figure 1. Fate of glucoraphanin

(Source: Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-
glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells
Available at http://carcin.oxfordjournals.org/content/23/8/1399/F5.expansion)

Sulforaphane (SFN) (4-methylsulfinylbutyl isothiocyanate) is a naturally occurring


isothiocyanate, which was first identified in broccoli extracts as the principal inducer of the
quinone reductase and glutathione S-transferases activity, which protect against carcinogens
and other toxic electrophiles.[12,15,16] Sulforaphane exhibits anti-cancer, antidiabetic, and
antimicrobial properties in experimental models. D, L-sulforaphane is a synthetic racemic
analogue of sulforaphane.[17] In many studies, sulforaphane can reduce the incidence of
a number of forms of tumor. [18-21] Sulforaphane has attracted researchers’ attention as
a promising cancer chemopreventive agent.[22]

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Even though broccoli is in general consumed raw because of its higher healing effects, its
consumption after cooking is also very common. Traditional cooking methods such as
conventional boiling in water, microwaving, steaming, or oven cooking may affect both the
texture and the nutritional values of the vegetables. The nutrient losses occur due to plant
tissue damage and subsequent loss of glucosinolates that may differ depending on the
cooking treatments,[23-26] The disruption of the vegetable tissue brings glucosinolates into
contact with myrosinase from within the intra- and inter-cellular vacuoles to initiate their
hydrolysis. However, cooking methods may partially or completely denature myrosinase,[27]
Therefore, the acidity and temperature,[28] of the medium, activity and cofactors of
myrosinase and concentrations of residual glucosinolates may affect the nature and
proportion of metabolites of glucosinolates produced during cooking and ingestion of cooked
vegetables. Epidemiological studies indicate that brassicaceous vegetables might have
preventing effects on cancers, particularly against colon, lung, breast, and prostate
cancers,[29,33] The chemoprotective effects of brassicaceous vegetables have been found to be
correlated with their glucosinolate content.[8] Therefore, the determination of the
glucosinolate profile (especially GRA and SFN contents) of a given brassicaceous vegetable
happens to be a necessary step in the study of its chemopreventive activity. Numerous
[34-36]
analytical methods including high-performance liquid chromatography (HPLC), gas
chromatography (GC),[37,38] GC with a mass detector,[39] micellar electrokinetic capillary
chromatography(MECC),[40-42] and hydrophilic interaction liquid chromatography(HILIC).[43]
have been previously used for determination of the glucosinolate profile of fresh broccoli, [44]
broccoli seeds,[45] and broccoli sprouts,[46] as well as many other HPLC based analytical
methods.[12, 47, 48]
This article highlights the various analytical assesments of sulforaphane
obtained from broccoli which provides excellent cancer remedy at a very low dose. One great
problem presented by this molecule is its less stability and high thermal degradation. The
research papers highlighting the various spectras such as Fourier transmission infrared
spectroscopy, Nuclear magnetic resonance and mass spectroscopy as reported by Hadas
Ganin et al in Sulforaphane and erucin, natural isothiocyanates from broccoli, inhibit
bacterial quorum sensing has been presented . This article brings to light the ultraviolet
spectroscopic analysis and band structure of sulforaphane as created by simulation software
Quntumwise ATK.

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2. Few Reported analytical methods and assesments


HPLC method as reported by Ana M et al in Optimized Formation, Extraction, and
Determinationof Sulforaphane in Broccoli by Liquid Chromatography with Diode Array
Detection, Food Anal. Methods (2014) 7:730–740, DOI 10.1007/s12161-013-9766-6 states
that a new method has been developed to determine sulforaphane in broccoli florets, stems,
and leaves using liquid chromatography coupled to diode array detection. The hydrolysis
reaction, required to convert glucoraphanin to its breakdown product, sulforaphane, and the
extraction procedure, based on solvent extraction with methyl t-butyl ether followed by solid
phase extraction with silica cartridges, were optimized for the three broccoli matrices; the
resulting recovery values ranged from 92 to 102 % in all cases. A fast separation was
performed on a C18 analytical column with a mobile phase composed of ammonium formate
(20 mM) in water and acetonitrile (55:45, v/v) in isocratic elution mode. This method was
fully validated and was found to be selective, linear from 2.5 to 800 mg/kg, and precise
(percent relative standard deviation values below 5 %). Moreover, the limits of detection and
quantification were 0.8 and 2.5 mg/kg, respectively. Finally, the proposed method has been
applied to the analysis of sulforaphane in broccoli samples of two different cultivars
(Parthenon and Marathon).

Gas Chromatography/Mass Spectrometry method as reported by William C. K. Chiang et al


in Gas Chromatography/Mass Spectrometry Method for the Determination of Sulforaphane
and Sulforaphane Nitrile in Broccoli, J. Agric. Food Chem. 1998, 46, 1018-1021 analyses
were performed using a gas chromatograph with pressure control connected to an
autosampler and mass selective detector. 5% phenyl methyl siloxane stationary phase was
used. Injector and detector temperatures were maintained at 250 and 300 °C respectively.
Column oven temperature was initially set at 40 °C for 2 min, then increased to 270 °C and
held for 5 min. The desired carrier gas flow rates were monitored for the various
experimental conditions. Mass spectra were obtained by electron ionization (EI) over a range
of 50 550 atomic mass units.

LC–MS–MS instruments and conditions as reported by HuWang et al in Development and


Validation of an LC–MS–MS Method for the Simultaneous Determination of Sulforaphane
and its Metabolites in Rat Plasma and its Application in Pharmacokinetic Studies, Journal of
Chromatographic Science, Vol. 49, November/December 2011 states that a highly sensitive
and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS–

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MS) assay is developed and validated for the quantification of sulforaphane and its
metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione
(SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from
rat plasma by methanol–formic acid (100:0.1, v/v) and analyzed using a reversed-phase
gradient elution on a Develosil 3 μm RP-Aqueous C30 140Å column. A 15-min linear
gradient with acetonitrile–water (5:95, v/v), containing 10 mM ammonium acetate and 0.2%
formic acid, as mobile phase A, and acetonitrile–water (95:5, v/v), containing 10 mM
ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its
metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits
of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The
calibration curves are linear over the concentration range of 25 -20,000 ng/mL of plasma for
each analyte. This novel LC–MS–MS method shows satisfactory accuracy and precision and
is sufficiently sensitive for the performance of pharmacokinetic studies in rats.

Figure 2: FTIR as reported by Parvane Abassi et al in Study on the interaction of


sulforaphane with human and bovine serum albumins,
doi:10.1016/j.jphotobiol.2013.02.001

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Figure 3: NMR Spectra of sulforaphane as reported by Hadas Ganin et al in


Sulforaphane and erucin, natural isothiocyanates from broccoli, Royal Society of
Chemistry, 2012 (Bruker Avance DPX400 ,400 MHz)

Figure 4: Mass Spectra of Sulforaphane as reported by Hadas Ganin et al in


Sulforaphane and erucin, natural isothiocyanates from broccoli, Royal Society of
Chemistry, 2012.

3. MATERIALS
Field-grown broccoli was bought from local market.The broccoli extracts were made from
fresh broccoli or from a frozen broccoli. Standard Sulforaphane 95% was obtained by sigma

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chemicals. Methylene chloride, Acetonitrile (HPLC grade), Methanol (HPLC grade), Ethanol
and water of HPLC grade were purchased from SD fine Chemicals, India. All other
chemicals used in the process were of analytical grade.

4. EXPERIMENTAL METHODS
4.1 Extraction
The fresh broccoli (1 kg) and 1000mL of warm distilled water were homogenized at medium
speed for 25 mins using a mixer. After homogenization, the homogenate was left for
complete hydrolysis after adjusting pH to 3. Then the homogenate was separated in a
separating funnel using methylene chloride or centrifuged at 8000 rpm for 5 min and then
filter the supernatant using 0.45 micrometer filter paper. 100 mL of methylene chloride was
added to the filterate and vortexed for 5 mins and then it was centrifuged at 4000 rpm for 10
mins to form 2 phases. The lower layer i.e. the methylene chloride layer was collected and
the aqueous layer was extracted again by using 50 mL of methylene chloride. The methylene
chloride layer was dried over the sodium sulfate and filtered afterwards using 0.45
micrometer filter paper. The filtrate was concerntrated using a high vaccum concentrator.

4.2 Simulations for constructing the chemical structure and band structure
Simulations were performed and resultant structure was obtained using simulation software
Quntumwise ATK and band structure thus produced was obtained.

4.3 Ultraviolet spectroscopy


4.3.1 Preparation of Dilutions
The standard stock solution was prepared by dissolving sulforaphane in ethanol, distilled
water, phosphate buffer 6.8 and phosphate buffer 7.4 to make final concentration of 2ppm,
4ppm, 8ppm, 10ppm, 20ppm and 50 ppm. Different aliquots were taken and λ max was found
by UV (Perkin Elmer). It was found to be 245nm, 240nm, 213nm and 215nm respectively.
Absorbance was measured. The calibration curve was prepared by plotting absorbance versus
concentration.

4.3.2 Validation of UV spectrophotometric method


4.3.2.1 Linearity and range
The linearity was determined by analyzing 6 independent levels of calibration curve in the
range of 2-50 ppm. Absorbance of each solution against methanol was recorded at curve of

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absorbance vs. concentration was plotted and correlation co-efficient and regression line
equation for sulforaphane were determined.

4.3.2.2 Precision
Intra-day precision was determined by analyzing sulforaphane (2-50 ppm) at three different
time points of the same day and determined by analyzing sulforaphane and inter-day
precision was determined by analyzing sulforaphane at three different time points on different
days and %RSD was calculated.

4.3.2.3 Accuracy
Accuracy was determined by performing recovery studies by spiking different concentrations
of pure drug within the analytical concentration range of the proposed method at three
different set at level of 80%, 100% and 120%. The amount of sulforaphane was calculated at
each level and % recoveries were computed.

4.3.2.4 Limit of Detection (LOD) and Limit of Quantitation (LOQ)


The LOD and LOQ were estimated from the set of 5 calibration curves used to determine
method linearity. LOD= 3.3*σ/S and LOQ= 10*σ/S
Where, σ = the standard deviation of y-intercepts of regression lines S = the slope of the
calibration curve

5. RESULTS
5.1 Structural simulation

Figure 5: Chemical structure of Sulforaphane as created by simulation software


Quntumwise ATK

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Figure 6: Band structure of Sulforaphane as created by simulation software


Quntumwise ATK

5.2 Ultraviolet spectroscopy


Calibration plots

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5.3 validation of UV methods of analysis


5.3.1 Linearity Linearity results have been summarized in table 1.
Table 1: Linearity data of Sulforaphane 5.3.2 Precision
S.No Dilution medium λ max(nm) Equation of line R2
1 Distilled water 240 Y=0.0618x-0.0186 0.9901
2 Ethanol 245 Y=0.038x+0.0802 0.9865
3 Phosphate buffer 6.8 213 Y=0.0977x-0.0951 0.9915
4 Phosphate buffer 7.4 215 Y=0.0594x+0.0423 0.9951

5.3.2.1 Interday precision % RSD of dilutions in distilled water, ethanol, phosphate


buffer 6.8 and phosphate buffer 7.4 have been listed in table 2.

Table 2: Interday Precision data of Sulforaphane


S.No Dilution medium %RSD
1 Distilled water 0.25-1.23
2 Ethanol 0.32-1.55
3 Phosphate buffer 6.8 0.28-1.30
4 Phosphate buffer 7.4 0.39-1.34

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5.3.2.2 Intraday precision


% RSD of dilutions in distilled water, ethanol, phosphate buffer 6.8 and phosphate buffer 7.4
have been listed in table 3.

Table 3: Intraday Precision data of Sulforaphane


S.No Dilution medium %RSD
1 Distilled water 0.19-0.98
2 Ethanol 0.29-1.32
3 Phosphate buffer 6.8 0.36-1.27
4 Phosphate buffer 7.4 0.31-1.12

5.3.3 Accuracy
Accuracy of the method was confirmed by recovery study from marketed formulation at three
level of standard addition. % Recovery for sulforaphane was found to be 99.11-101.18.

5.3.4 Limit of Detection and Limit of Quantitation


LOD and LOQ have been given in the table 4.

Table 4: LOD and LOQ data of Sulforaphane


S.No Dilution medium LOD(ppm) LOQ(ppm)
1 Distilled water 0.62 1.25
2 Ethanol 0.28 0.98
3 Phosphate buffer 6.8 0.43 1.02
4 Phosphate buffer 7.4 0.46 1.09

6. CONCLUSION
Thus, it can be concluded that most of the important analytical parameters of the
sulforaphane extracted from broccoli have been developed and validated.Sulforaphane offers
huge potential to be chemically conjugated with other agents such as gold nanoparticles,
silver nanoparticles or antibodies and help in anticancer drug delivery, this is the reason for
simulating the chemical structure and band structural changes after conjugation will help to
study the differences. The drug is highly labile to thermal and basic stresses which was
analysed by developed HPLC method and will be reported as a separate paper. The
developed ultraviolet spectroscopic method was validated and was done in different dilution
media such as distilled water, ethanol, phosphate buffer pH 6.8 and phosphate buffer pH 7.4
which will aid in further formulation development and in vitro and in vivo studies. The article
briefs not only about major analytical methods available for sulforaphane, which will be an
accumulated source of information for researchers but also brings about a part of UV method

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development and simulation studies carried out which will help in further research. The
linearity, precision, accuracy, LOD and LOQ data shows that the method was well validated
and good for estimation of sulforaphane.

7. ACKNOWLEDGEMENT
I would like to thank Department of Science and Technology for providing fellowship in the
form of INSPIRE award. I would also like to express my gratitude to Dr. Shatendra Sharma
from Jawahar Lal Nehru University, New Delhi for his help in simulation studies of
Sulforaphane.

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