Herbs: Challenges in Chemistry and Biology - American Chemical

Chapter 12

Thioglucosidase-Catalyzed Hydrolysis of the Major Glucosinolate of Maca (Lepidium meyenii) to Benzyl Isothiocyanate Mini-Review and Simple Quantitative HPLC Method 1,2

Downloaded by CORNELL UNIV on July 7, 2012 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch012

Matthew W. Bernart 1

2

Analytical Laboratory, Herb Pharm, Williams, O R 97544 Current address: Science Department, Rogue Community College, 3345 Redwood Highway, Grants Pass, O R 97527

This H P L C method is for the quantification of benzyl isothiocyanate (BITC, 1) released by the action of the thioglucosidase enzyme on the substrate glucotropaeolin (2), the predominant glucosinolate of maca hypocotyls. Maca, a native Peruvian member of the cabbage family, is a popular herbal product. Details of the H P L C method for BITC, enzymatically produced from maca powder extracts, as well as highlights of BITC's relevance to human health, are provided.

Introduction Maca root, or hypocotyl, (Lepidium meyenii Walpers), also known as L peruvianum, is now receiving nearly worldwide popularity as a nutritional supplement, following centuries of obscurity (1). The glucosinolate content of maca, a potential indicator of herbal quality, has been recently shown to consist predominantly of glucotropaeolin (2, 2-4). Best known in, but by no means restricted to the Brassicaceae, or cabbage family, glucosinolates constitute a structure class containing over 120 different molecules, all of which are © 2006 American Chemical Society

In Herbs: Challenges in Chemistry and Biology; Wang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

157

158


substrates for the enzyme β-thioglucosidase (E.C.3.2.3.1), also called myrosinase (3,5). Depending on conditions but especially near p H 7, this waterdependent hydrolysis often yields volatile and bioactive isothiocyanates (ITC) in addition to other compounds (5-S). Considering the literature, we deduced that benzyl isothiocyanate (BITC, 1) enzymatically produced from glucotropaeolin contained in maca (Figure 1), is a biologically relevant marker compound and so endeavored to devise a simple, rapid analytical high performance liquid chromatographic (HPLC) method that could be used to quantify BITC directly from hydroethanolic solutions containing thioglucosidase and maca extractives.

Figure 1. Thioglucosidase enzyme reaction using glucotropaeolin (2) substrate.

Rationale The biological importance of BITC to human health hinges on the thioglucosidase activity resident in the intestinal flora (5,7-9), thus giving an organism consuming dietary glucotropaeolin at least the capability of releasing BITC into the digestive tract. Among the variety of ITCs released from glucosinolates by the enzyme, BITC possesses some unique biological activities. Over three decades ago, BITC from papaya fruit was reported as fungistatic to sporangia of Phytophthora parasitica (10). Papaya seed extract also contains BITC, which was shown to be anthelmintic to the nematode Caenorhabditis elegans (11). In vitro work has demonstrated that, among all ITCs and nitriles tested, BITC was the most potent antiproliferative against human erythroleukemic K562 cells (12). The mechanism of BITC's chemopreventive action may be exerted through the inactivation of a cytochrome P450 enzyme that is involved in the carcinogenesis process (13). Although it appears that most ITCs inhibit cancer when administered before or during carcinogen exposure, B I T C inhibited mammary tumor formation in rats when administered subsequent to treatment with the carcinogen 7,12-dimethylbenz[ûf]anthracene (14). Taken together, these studies indicate significant bioactivity, and hence, a need for a


159 simple analysis to quantify enzymatically produced BITC in foodstuffs and herbal products.


Methods of B I T C and Glucosinolate Analysis Historically, BITC was quantified by a gas chromatographic (GC) method that, in the case of papaya, required extensive sample cleanup (10). This methodology also required endogenous thioglucosidase activity in the biomass itself to produce the analyte, and so would not be applicable to dehydrated or otherwise processed samples, nor to hydroethanolic extracts or tinctures. A n isocratic H P L C method, requiring a derivitization step, has also been reported (75). Isocratic elutions are often unsuitable for complex herbal matrices; hence, gradient elution on H P L C is preferred (16,17). H P L C methods for the nonvolatile glucosinolates have appeared in the literature (2,18-20); however, these methods may require extensive sample cleanup and ion-pairing conditions; moreover, the glucosinolate standards, i f available, are quite expensive. As reported in the literature, major glucosinolates of maca are depicted in Figure 2:

R- Group:

Name: Glucoallyssin

-H C 2

HO -H

Glucobrassicanapin

-H C 2

OH

Glucosinalbin

-H C 2

Glucotropaeolin

OMe

Methoxybenzylglucosinolate

Figure 2: Major glucosinolates of maca: substrates for thioglucosidase hydrolysis to corresponding isothiocyanates.



160 HPLC-amenable glucosinolate derivatives from maca have been produced via a sulfatase enzyme reaction to produce desulfoglucosinolates (4); however, the availability of these compounds as standards is also in question. The same thioglucosidase enzyme used in the present work was recently applied to the quantification of maca glucosinolates by Dini et al. (3). In their work, this enzyme was demonstrated to exhibit hydrolytic activity in the presence an of organic solvent, dichloromethane. Furthermore, the glucotropaeolin content was estimated from the glucose liberated from the hydrolysis, as measured by G C M S , not by direct detection of BITC. Dini's thioglucosidase-coupled technique requires extensive sample cleanup to ensure that other glucose pools in the plant material do not interfere with quantitative analysis. A H P L C method for BITC and its metabolites formed by microsomal cytochrome P450 2B1 has been reported (21). A reversed-phase method on a C 18 column, it employs a linear gradient of acetonitrile in an aqueous buffer, monitored by liquid scintillation counting. Under those conditions, the retention time of BITC was 39.9 minutes. Other technologies that have seen utility in investigations of this structure class have included *H N M R (22), as well as micellar electrokinetic capillary chromatography of the parent glucosinolates (23). Because BITC is so reactive, especially with methanol or any substance capable of generating nucleophilic species in solution (10,24), we wanted to achieve reproducible chromatography using pure water with no modifiers in the aqueous phase, and to eliminate the use of methanol in any of the extraction and chromatographic procedures.

Experimental

Safety A lab coat and safety glasses should be worn while performing all bench work, especially the heating of sealed EtOH-containing vials and the subsequent syringe filtering of the solutions contained therein. Good ventilation is necessary as the use of acetonitrile is required. General considerations for the use and storage of flammable solvents apply.

Materials A l l organic solvents were hplc grade except for 95% EtOH, which was food grade. Distilled water (Sparkletts, Albertsons) was filtered through a 0.45 μηι nylon membrane prior to use. Thioglucosidase, isolated from Sinapis alba, was


161 purchased from Sigma. One enzyme unit is defined as producing 1.0 μπιοΐβ glucose per min at p H 6,25° C. The number of enzyme units per mg of enzyme preparation may vary from lot to lot. Glucotropaeolin (2) and BITC (1) were obtained from Chromadex. Maca dried-hypocotyl (maca powder) was obtained from commercial sources and from the Herb Pharm quality assurance department retain samples. Hydroethanolic maca extract was also provided from Herb Pharm stock.


Instrumentation and Equipment A Dionex Summit H P L C system, consisting of a Gina 50T autosampler, a P580A L P G pump, and a U V D 170S U V - V i s detector, all controlled by Chromeleon™ software, was employed. A n Eppendorf TC-30 column heater and TC-50 temperature controller was used to control the column temperature. The column was a Y M C Pro™ C18, 5μ, 120 À, 4.6 χ 150 mm, protected by a guard column of the same stationary phase, 4 χ 20 mm, and an Upchurch A-430 2μπι P E E K inline filter. Samples were sonicated and heated in a Branson 2210 sonicator / heater. Volumetric flasks and pipets were class A . Fluid transfers of one ml or less were handled with a Gilson P-1000 Pipetman.

Chromatographic Conditions The column temperature is maintained at 40° C, the mobile phase flow rate set to 1.0 ml / min, and the detector set to 246 nm (1 nm bandwidth). Five to 30 μΐ injections, typically made for expected BITC concentrations ranging from 0.02 - 0.2 mg / ml, fell within the linear range of the detector. The gradient elution profile is outlined in Table I below:

Table I. Linear Gradient Elution of BITC on Y M C Pro™ C18 Column Time (minutes) -10 0 5 9 23 25

%

H0 97 97 92 52 38 1 2

% Acetonitrile 3 3 8 48 62 99


162 Standard and Sample Preparation BITC (2, oily liquid), kept refrigerated until use, was diluted with acetonitrile using volumetric glassware to 0.02 - 0.2 mg / ml, then kept in a freezer for as long as one week with no evidence of decomposition. Maca powder (100 mg) was sonicated for 1 hr at 50° C in 10 ml of 47.5% E t O H / H 0 (v/v). Separately, 10 enzyme units are dissolved in 10 ml H 0 . Using a volumetric pipet, transfer at least one enzyme unit, in its aqueous solution, into a volumetric flask, calculating that at least one enzyme unit is needed for every four ml of hydroethanolic maca extract. Bring the flask to volume with the maca extract, recording the dilution of the extract obtained, and mix well. Transfer an aliquot of extract-enzyme mixture to a scintillation vial or other suitable gas-tight container and incubate in a water bath at 45° C for 30 minutes, then filter through a 0.45 urn PTFE syringe-filter prior to injection on H P L C . 2


2

Results and Discussion

The chromatogram of an acetonitrile solution of the BITC standard is seen in Figure 3 below:

698-

030904

UVV1S 1 VWL246nm|

BITC

.nAU

BfTC 400-

200-

•52-0.0

1

Γ

Γ"" 25

5.0

7.5

' I » 10.0

' I ' 12.5

' 1 15.0

1

'·

»

175

200

22.5

25.0

Figure 3. HPLC chromatogram at 246 nm of BITC standard in acetonitrile.

The typical chromatographic efficiency was calculated by the Chromeleon™ software to be 85,000 to 100,000 theoretical plates, according to the European Pharmacopoieal method. Using a standard least-squares linear regression including a y-intercept, a standard curve was generated for the quantification of each day's analyses. For example, a six-point standard curve, plotting peak area at 246 nm versus concentration, was generated for a set of low BITC concentration standards. The equation takes the form y = mx + b, where m is the


163 slope and b is the y-intercept, giving the linear relationship depicted in Figure 4 below:


Q.DOQO

Q.Q10D

0.0200

0.0300

0.0400

0.0500

0.0600

00700

O.QBQO

2

Figure 4. BITC standard curve with slope 257, y-intercept 0.4, and r 0.9999.

When a BITC solution in acetonitrile was kept in the freezer, it gave three standard curves with the same slope for assays conducted over three successive days.

Stability Indication One of the important parameters to be addressed in a single laboratory method development project is that of stability indication. A valid analytical method should be able to distinguish between an analyte and its degradation products. Due to its inherent reactivity, it was simple to produce dégradants of BITC with heat, dégradants that were fortuitously separated using this hplc method. When BITC was heated at 80°C for one hour on a water bath in 50% E t O H / H 0 , the chromatogram (Figure 5) seen below resulted: 2

031204 nAU

UV VIST WVL246 nm BITC

A

I

L

min -00

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

Figure 5.Chromatogr am ofhydroethanolic BITC solution following heating.


164 In addition to the prominent BITC peak eluting at 21.6 minutes, two peaks attributed to BITC dégradants are observed in the 18-19 minute range. B y this method, BITC was determined to be more heat-stable in acetonitrile than in the alcohols M e O H , EtOH, or iPrOH.

Development of the Method Though thioglucosidase from Sinapis alba was active in organic solvent (5), we chose an aqueous environment for these reasons:


1. 2. 3.

It was easier to obtain a homogeneous enzyme preparation using only distilled water for dissolution. Maca is very sugary (2), with even more sugar produced in the hydrolysis. A commercially available maca product was a hydroethanolic extract.

We found BITC levels in maca extracts made with 50/50 (vol/vol) aqueous mixtures of tetrahydrofuran, acetone, dioxane, or acetonitrile to be one half those seen in the hydroethanolic extract, all other variables held constant (data not shown). Given that the water / w-octanol partition coefficient of BITC has been determined at 0.006, the most lipophilic ITC tested (25), we needed a relatively high percentage of organic solvent in the sample prep. In a series of experiments where the ethanol /water ratio was varied, the optimum for production of BITC from maca extracts was determined to be 47.5% EtOH/water (vol/vol). Figure 6 below shows a chromatogram obtained from the thioglucosidase reaction with a maca hydroethanolic extract as per sample prep in the Experimental Section:

Figure 6. HPLC chromatogram obtainedfrom a maca extract incubation with thioglucosidase enzyme preparation.



165 A n earlier version of our hplc method did not incorporate the high-aqueous flush at the beginning of the gradient. We found flushing (Table 1, minus 10 to 5 minutes) to be a crucial component of the method. Without it, filters and precolumns became irreversibly clogged after a few samples. This presumably occurred when sugars in the samples precipitated in the H P L C system upon encountering a strong organic (40% acetonitrile) mobile phase. With the current modification to the gradient, which accommodates high sample throughput before any significant systemic pressure spikes, it appears that sugars are eluted from the column without precipitating. Further experiments showed that given the conditions in the experimental section, the biomass can be exhaustively extracted at 10 mg biomass per ml extraction solvent. A n Italian group (2) has reported the endogenous BITC content of dry maca tuber by G C / M S , without enzyme treatment, to be 32 ng/g. Such a small amount may be well below the limits of detection and quantification of the proposed method. In fact, when a maca extract was injected on H P L C without previous enzymatic treatment, we obtained the chromatogram seen in Figure 7 below:

962

lot 212550

UY VIS 1 WVL246 nrr

nÂÏT

50.0

Herbs: Challenges in Chemistry and Biology - American Chemical

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