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August 1, 2005 | Naegel, Ludwig C.A.; Alvarez, Jesus I. Murillo

Biological and chemical properties of thesecretion from the hypobranchial gland of thepurple snail Plicopurpura pansa (Gould, 1853).

ABSTRACT The hypobranchial gland of the muricid Plicopurpura pansa (Gould,

1853) is so active that the snails can be stimulated periodically without harming them

to expulse the secretion. This property is a great advantage in the study about its

biologic and chemical characteristics. No statistical difference could be determined

between the incidence of expulsion and the sex of the animals. Also the test on

whether the size of the animals had an influence on the frequency of expulsion

showed no relation. The incidence of expulsions is the same between the different size

classes. From between September 2003 and February 2005 collected snails (total

number 3,577) 1,724 (48.2%) expulsed secretion. The proportion of snails that

expulsed or not varied from month to month, however no clear seasonal trend could

be observed. We determined in the laboratory the amount of the total organic

compounds in the "milk" and found great variations (from 34.2 mg/100 animals to

337.8 mg/100 animals). We determined from 11 samples collected during different

months an average of 148.9 mg organic compounds/100 animals. The "milk"

expulsed from the hypobranchial gland of P. pansa contains 6.15% ([+ or -] 1.07 SD

n = 3) total solids, 21.3 mg/ml ([+ or -] 17.8 SD n = 38) soluble proteins, and 6.01

mg/ml ([+ or -] 3;2 SD n = 38) carbohydrates. In organic extracts from the secretion

of the hypobranchial gland we determined in a microwell assay a 50% lethal dose

([LD.sub.50]) of 81.72 [micro]g/mL (SD 35.78 n = 5) against Anemia nanplii. In

assays to determine possible antibacterial activities in organic extracts we found two

inhibition zones against Staphylococcus aureus. To quantify the microbial activity we

determined a lowest inhibitory concentration of 125 [micro]g/disk. By thin layer- and

column chromatography, as well as by IR spectroscopy, we could preliminary identify

some of the organic compounds in the "milk" and in organic extracts. By comparing

previously reported front reference values (Rf-values) we could identify the dye

precursor tyrindolinone, tyriverdin and bromoisatin. By column chromatography the

extract was separated with different mixtures of organic solvents. In a first step

gradient fractionation we obtained three fractions, which were unstable in light and

Journal of Shellfish Research



turned immediately purple, and one yellow, light stable fraction. Two light stable

brownish-colored fractions turned purple after acid hydrolysis. They were united and

subjected to further fractionation, where four fractions and the green insoluble

tyriverdin were obtained. By IR spectrophotometry and comparison with reported

spectra it could be shown that one fractionated compound was a salt of probably 6-

bromo-2-methylsulfonylindoxylsulfate and the other 6-bromoisatin. In organic

extracts of the secretion free radical scavenging activities were determined by the 2,2-

dipbenyl-l-picrylhydrazyl radical method (DPPH). We observed two yellow patches

above a purple background. By IR spectroscopy of the organic extract used we could

determine the chromogen IV, probably 6-bromo-2 methylsulfonylindoxylsulfate, as a

substance responsible for the free radical scavenging activity.

KEY WORDS: purple snail, Plicopurpura pansa, hypobranchial gland, biological and

chemical properties

INTRODUCTION

The majority, if not all, of the marine snails from the family Muricidae produce in the

hypobranchial (mucous) gland a colorless secretion, which turns on exposure to air

and light to "Tyrian purple" (Fretter & Graham 1994).

In pre-Roman and Roman times "Tyrian purple" from the Mediterranean muricids

Murex trunculus, M. brandaris and Purpura haemastoma was a most expensive

luxury article, however with the Arab conquest of Palestine in 638 A.D. and finally

with the fall of Constantinople in 1453 A.D. the use of "Tyrian purple" became, with a

few exceptions, extinct in the Old World and the details about the dyeing methods

were forgotten.

For the scientific world it was therefore a big surprise when in 1685 William Cole

reported that the contents of the hypobranchial gland of the muricid Nucella lapillus

could directly be applied to linen and after a series of chemical reactions in the

presence of light and oxygen "Tyrian purple" is formed (Cole 1685). After his finding

numerous scientists tried to understand the chemical processes involved in the

production of "Tyrian purple". Bizio (cited in Ghiretti 1994) showed in 1835 that color

differences in the pigment from M. brandaris and M. trunculus are species-specific

and not related to ecology, as considered before, and that the dye had the chemical

properties of indigoid pigments. By processing the hypobranchial glands of 12,000 M.

brandaris snails Friedlander (1909) obtained 1.4 g of the pure pigment and by

elemental analysis he showed that the pigment contained bromine and that it was

6,6'-dibromoindigo. Syntheses confirmed his conclusion. Using advanced analytical

methods, Fouquet (1970), Baker and Duke (1973), Michel et al. (1992), Koren (1994),

Withnall et al. (2003) among others, have confirmed that the major pigment from

muricids is 6,6'-dibromoindigo, with dibromoindirubin and monobromoindigo as



minor components. The exception is M. trunculus where the secretion contains both

brominated and nonbrominated dye precursors and because of cross coupling of

different indoxyl or indoleninone chromogens leading to a mixture of a high

concentration of 6-bromoindigo with indigo, indirubin and dibromoindigo (Wouters

1992, Koren 1994, Cooksey 2001). However, 6,6'-dibromoingigo and the other minor

indigoid pigments as such do not occur in the live animal, but are formed from

colorless dye precursors during a sequence of chemical reactions, requiring light,

oxygen and specific enzymes. First a yellow color is immediately formed, followed by a

greenish shade, and under the influence of oxygen and light changing to bluish,

which in turn changes finally into the purple dye, while liberating garlic-like smelling

volatile products, which have been determined by gas chromatography-mass

spectroscopy as methylmercaptan and dimethyl-disulfide (Shiomi et al. 1983).

After the development of new analytical methods the exact determination and

description of the different precursors leading to "Tyrian Purple" was only recently

possible. Cooksey (2001) described the different precursors and summarized the

entire process of the purple generation from excretions from the hypobranchial gland

from M. brandaris and M. trunculus.

The first steps in the chemical reactions towards purple are probably the degradation

of the essential amino acid tryptophan to indole and the hydroxylation into the

colorless indoxyl (Verhecken 1989). Indoxyl sulfate is formed through the sulfation of

indoxyl, which undergoes bio-bromination in the presence of hydrogen peroxide and

bromide by the membrane bound enzyme hromoperoxidase, leading to the colorless

tyrindoxyl sulfate.

Gribble (1998) described the reactions leading from the natural bromide to

organobromine compounds (bio-bromination) in marine organisms. Jannun and Coe

(1987) determined in homogenates of the hypohranchial gland of M. trunculus

bromoperoxidase for the probably peroxide-induced bromination reaction.

The required enzyme for the hydrolysis of the sulfate group in tyrindoxyl sulfate

leading to the yellow tyrindoxyl has been determined as the cytoplasmatic

arylsulfatase by histochemical (Erspamer 1946) and enzymatical methods (Erspamer

1946, Fouquet 1970). In the presence of oxygen the red tyrinindoleninone and the

yellow tyrindolinone are formed. Those indoxyls, which have substituents in the 2-

position, are oxidized to indoleninones that dimerise to give the green photolabile

tyriverdin. Photolysis of tyriverdin gives dibromoindigo, the main component of

Tyrian purple, and the odorous dimethyl disulfide (Cooksey 2001).

After the main chemical routes leading to Tyrian purple were described the question

about the biologic role of the hypobranchial secretions still remains open. Fretter and

Graham (1994) consider the main function of the hypobranchial gland to be a



secretor of mucus for trapping and cementing particulate matter sucked into the

mantle cavity with the respiratory water current, prior to its expulsion. In the

hypobranchial gland the purple precursors and the enzymes that induce the

transformation of the precursors into pigments are kept separate so that no reaction

occurs. The final dye "Tyrian purple" as such does not occur in live animals.

The pharmacologic action by extracts of the hypohranchial gland was discovered by

Dubois (1909), and he described for the first time their toxic effects limiting the

movement and finally paralyzing actions on the central nerve system in both warm-

and cold-blooded animals. More recently toxins and narcotizing agents have been

described from the hypobranchial gland such as serotonin (5-hydroxytryptamine),

murexine (urocanylcholine), choline ester and biogenic amines (Erspamer 1946, 1952;

Erspamer & Benati 1953, Whittaker 1960, Malaszkiewicz 1967, Huang & Mir 1971,

Andrews et al. 1991, Roseghini et al. 1996, Shiomi et al. 1998).

All the earlier described determinations and descriptions of the chemical composition

of the precursors and the final pigments, the enzymatic reactions, and biologic

functions of the hypobranchial gland had to be done with dead animals, because the

earlier described muricids do not expel from the hypobranchial gland secretions in

sufficient quantity for analysis. In this respect the muricid Plicopurpura pansa

(Gould, 1853) is an exception. The hypobranchial gland of this intertidal gastropod is

so active that after mechanical stimulation the secretion of the hypobranchial gland

can be obtained periodically by "milking" the animals without harming them. P.

pansa offers therefore the enormous advantage to study the biologic and chemical

properties of the secretion of live animals. Astonishingly, until now only very limited

information exists about the biologic functions and the chemical properties of the

hypobranchial secretion of P. pansa.

Information exists about the chemical composition of Tyrian purple obtained from P.

pansa. Schunck (1880) isolated from a sample of cotton dyed with the secretion from

P. pansa the pigment that he called punicin. Thirty years later, it was shown by

Friedlander in 1909 that Schunck punicin was 6,6'-dibromoindigo (Friedlander

1909). Using up-to-date analytical methods, like UV/ VIS spectrophotometry, high

performance liquid chromatography (HPLC) and mass spectrometry Withnall et al.

(2003) showed that Tyrian purple from P. pansa contains 90% 6,6'-dibromoindigo,

9% 6-monobromoindigo and 1% 6,6'-dibromoindirubin, and that under high light

intensity 6-bromoisatin formed in a minor reaction pathway, leading to a low

concentration of 6,6'-dibromoindirubin in the purple dye.

The carnivorous, gonochoristic marine purple snail P. pansa inhabits intertidal rocky

shores exposed to the high impact waves of the open sea. The range of distribution of

P. pansa extends from the northwest coast of Mexico (Baja California Sur) (Clench

1947, Keen 1971) to northern Peru (Pena 1970, Paredes et al. 1999). The snail is not



too small (shell length: average about 30 mm, but can reach a total shell length of 90

mm), and at low tides it is relatively easily gathered.

In view of the growing interest in marine, naturally occurring compounds with

pharmacologic properties an investigation about the biologic and chemical properties

of the secretion of the hypobranchial gland of the marine muricid P. pansa (Gould,

1853) seemed to be justified. The objective of this study is (1) to describe the relation

between "milk expulsions", sex and total shell length of P. pansa at Playa Cerritos; (2)

to identify preliminarily by thin layer- and column chromatography, as well as by IR

spectroscopy, organic compounds in the "milk" and in organic extracts; (3) to

determine the concentration of soluble proteins, total carbohydrates and solids from

the secretion of the hypobranchial gland expelled by live animals and (4) the general

toxicity of organic extracts against Artemia nauplii, of antimicrobial activities and of

free radical activities.

MATERIALS AND METHODS

Relationship Between "Milk" Expulsions, Sex and Total Shell Length of P. pansa

A total of 376 adult snails were collected in September and November 2003 during

extreme low tides from exposed high intertidal rocks along the Pacific coast at Playa

Cerritos (23[degrees]19'54"N and 110[degrees]10'38"W, about 100 km southwest of

La Paz, Baja California Sur, Mexico). The total shell length of the snails was

determined with a precision of 0.01 mm and the sex by the presence or absence of a

penis, additionally the number of snails that expulsed secretion.

Collection of the "Milk" and Preparation of Organic Extracts

From May 2004 to February 2005 the secretion was collected monthly from 1,525

adult snails of different sizes. To reduce changes in the chemical composition of the

secretion by light, oxygen and enzymatic reactions, the secretion was immediately

transferred into 30 mL amber vials containing 5 mL of an aqueous solution of 20%

sodium-bisulfite and ,10 mL ethyl acetate or dichloromethane. In the laboratory the

samples were stored frozen. After thawing, the organic phase was separated and the

aqueous phase washed three times with ethyl acetate. The organic phases were

pooled and the solvent was evaporated under reduced pressure at 40[degrees]C to

42[degrees]C and diffused light. The organic extracts were dried in darkness under

nitrogen, afterwards weighed and stored frozen. The amount of organic extract

obtained from 100 snails was calculated.

Total Protein and Sugar Content in the Secretion of the Hypobranchial Gland of P.

pansa Cultured in the Laboratory

From animals cultured in the laboratory the hypobranchial secretion was obtained in



darkness under a photographic safe light for immediate analysis.

The soluble protein content was determined spectrophotometrically according

Peterson modification of the micro Lowry method with the Sigma protein assay kit

(No. P. 5656), the carbohydrate content with phenol and sulfuric acid according to

the method described by Dubois et al. (1956). The portion of the total solids in the

"milk" was determined by evaporation of the water under a nitrogen stream and in

darkness.

Thin layer chromatography combined with the method of bio-autography to

determine antibacterial properties in the "milk."

Under nitrogen stream and darkness 93.8 mg of dried "milk" from four snails was

suspended in 1 mL of a mixture of dichloromethane, ethanol (100%) and water

(6:5:1). The supernatant was dried in darkness under a nitrogen stream to give 15.5

mg of extract. The extract was reconstituted in 1 mL of the same solvent mixture.

Fifteen microliters (final concentration 232 [micro]g) were applied to thin layer

chromatography plates (TLC-plates, silica gel 60, Merck) and separated with a

mixture of toluene and methanol (9:1).

Pure strains of Staphylococus aureus (ATCC 25923), Escherichia coli (ATCC 25922)

and Pseudomonas aeruginosa (ATCC 15442) from the American Type Culture

Collection were used as target organisms. The antimicrobial properties were

determined by the bio-autographic method according to Rahalison et al. (1991).

The bacteria inoculums were prepared in Mueller-Hinton broth (Difco) and the

cellular concentration was adjusted to 12 x [10.sup.6] cells/mL. The surface of a

Mueller-Hinton medium was inoculated under axenic conditions with a cotton

applicator.

The previously developed TLC-plates were placed upside down on the medium. After

30 min of contact the TLC-plate was removed and after 24 h of incubation at

37[degrees]C the areas of antimicrobial activity could be observed and the Rf values

of the antimicrobial compounds determined.

To quantify the minimal inhibitory concentration of the extracts the method of disk

diffusion was applied. The filter paper disks (diameter 6 ram) were impregnated with

different concentrations (from 31.25 [micro]g to 1 mg) of the extract. The disks were

placed on an inoculated Mueller-Hinton medium, and after 24 h of incubation at

37[degrees]C the diameter of the inhibition zone was measured.

Toxicity of the Extract From the "Milk" of P. pansa Against Artemia Nauplii

The microwell assay using nauplii of Artemia according Solis et al. (1993) was used to



determine the toxicity of the hypobranchial secretion. "Milk-extracts" obtained as

described earlier were suspended in ethyl alcohol and added in different

concentrations to microwells containing seawater and 10-15 Artemia nauplii. After 24

h the number of live and dead animals was counted, and the 50% lethal dose

([LD.sub.50]) determined.

Free Radical Scavenging Activity in Extracts From the "Milk" of P. pansa

To determine the free radical scavenger activities in the secretion from the

hypobranchial gland the 2,2-diphenyl-1-picrylhydrazyl radical method (DPPH)

according to Cuendet et al. (1997) was used. A sample of the organic extract was

applied to a TLC-plate (silica gel 60) and eluted with toluene-methanol (9:1).

Consequently the plate was sprayed with a methanolic 0.2% DPPH solution. After 30

min the plates were examined to determine whether free radical scavenger activities

could be observed.

RESULTS

Relationships Between Sex, Total Shell Length And "Milk" Expulsion From P. pansa

In September and October 2003, 376 adult snails were collected. The sex of the

animals was determined by the presence or absence of a penis. From the 376 animals

were 223 males and 153 females (Fig. 1). More than half of the males (113 = 55.16%)

had a total shell length between 26.9 and 30.7 mm, and more than half of the females

(97 = 63.4%) a total shell length between 29.7 and 35.4 mm (Fig. 2 and Fig. 3). From

the 223 collected males 120 (= 53.8%) expulsed secretion from the hypobranchial

gland, and from the 153 collected females 76 (=49.7%) (Fig. 1). No statistical

difference could be determined between the incidence of expulsion and the sex of the

animals. The test on whether the size of the animals had an influence of the

frequency of expulsion showed no relation. The incidence of expulsions is the same

between the different size classes. Also the percentage of expulsion is nearly the same

with males and females of the different size classes (Fig. 4 and Fig. 5). From between

September 2003 and February 2005 collected snails (total number 3577) 1724

(48.2%) expulsed secretion. The proportion of snails that expulsed or not varied from

month to month, however no clear seasonal trend could be observed. The highest

proportion of expulsions (58.5%) was found middle of August, and the lowest

(36.6%) middle of November. In February 2005 where we experienced the coldest

water temperature (20[degrees]C) 52.9% of the collected animals expulsed secretion.

[FIGURES 1-5 OMITTED]

In the laboratory we determined the amount of the total organic compounds in the

"milk" and found great variations. In May 2004 the secretion from the hypobranchial

gland from 100 animals contained only 34.2 mg organic compounds. This amount



increased continuously to 232 mg until the middle of August, decreased in September

to 111.6 mg, increased in October to 337.8 mg and decreased in November to 116.38

mg. We determined from 11 samples collected during different months an average of

148.9 mg organic compounds/100 animals (Fig. 6).

[FIGURE 6 OMITTED]

Determination of Soluble Proteins, Total Carbohydrates and Solids From the

Secretion of the Hypobranchial Gland Expulsed From Live Animals

The "milk" expulsed from the hypobranchial gland of P. pansa contains 6.15% ([+ or

-] 1.07 SD n = 3) total solids, 21.3 mg/ml ([+ or -] 17.8 SD n = 38) soluble proteins

and 6.01 mg/mL ([+ or -] 3.2 SD n = 38) carbohydrates.

General Toxicity of Organic Extracts of the Hypobranchial Secretion Against Artemia

Nauplii

A microwell assay was used to determine whether organic extracts from the secretion

of the hypobranchial gland (53.7 mg from 52 animals) are toxic against Artemia

nauplii. A fifty percent lethal dose ([LD.sub.50]) of 81.72 [micro]g/mL (SD 35.78 n =

5) was determined.

Determination of Antimicrobial Activities of Organic Extracts From Hypobranchial

Secretions

In the plates incorporated with S. aureus two inhibition zones were observed (Rf. 0-

0.37 and 0.66-1.00). No antibacterial activities could be seen against E. coli and P.

aeruginosa. In addition no antimicrobial activity could be found in the stored liquid

of the mantle.

To quantify the microbial activity of the organic extract obtained from freshly

"milked" snails against S. aureus filter paper disks (6 mm) were impregnated with

different concentrations of the extract. A lowest inhibitory concentration of 125

[micro]g/disk was found.

Preliminary Identification of Organic Compounds in the "Milk" and in Organic

Extracts From the "Milk" of P. pansa by Thin Layer- and Column Chromatography as

Well as by IR Spectroscopy

From freshly expulsed "milk" and from organic extracts of the glandular secretion of

P. pansa organic compounds were separated by silica gel thin layer chromatography

using as the mobile phase a toluene: methanol mixture (9:1). Following front

reference values (Rf-values) were determined:



"Milk-1" "Milk-2" Extract-1 Extract-2

Spot 1 0 0 0 0

Spot 2 Not detect. Not detect. 0.28-0.42 0.28

Spot 3 0.45 0.44 0.28-0.42 0.46

Spot 4 0.53 Not detect. 0.57 Not detect.

Spot 5 0.67 Not detect. 0.67 Not detect.

Spot 6 0.71 0.71 0.72 0.73

Spot 7 Not detect. 0.76 Not detect. Not detect.

Spot 8 Not detect. Not detect. Not detect. 0.89

By comparing previously reported front reference values (Rf-values) for different dye-

precursors from organic extracts from the hypobranchial gland of Nucella lapillus

(Cooksey 2001, Cooksey & Withnall 2001) with the Rf values obtained from P. pansa

we could identify the dye precursor tyrindolinone with an Rf-value of 0.53 and 0.57

in the "Milk-1" spot 4 and in the "Extract-1" spot 4. The spot 3 in the "Milk-1" and in

"Milk-2" with a Rf-value of 0.45 and 0.45, spot-3 in the "Extract-1" with Rf-values

between 0.44-0.46 and spot 3 in "Extract-2" with an Rf-value of 0,46xmight be

tyriverdin because of the green coloration of the spot, which turned purple under

light and oxygen. The chemical compound in "Extract-l" spot 2 (Rf. 0.28-0.42) and

in "Extract-2" spot 2 (Rf.-0.28) might be bromoisatin because of the light stable

yellow color.

Seven organic extracts from the secretion of the hypobranchial gland collected

between May and August 2004 were pooled to give 500 mg of dry weight. The extract

was separated by column chromatography (silica gel 60, 230-400 mesh, J. T. Baker)

with different mixtures of organic solvents. We obtained in the first step gradient

fractionation (hexane, toluene and 10%, 15% and 20% methanol in toluene, followed

by a methanol wash, six fractions, which contained 67% (337 mg) of the original

extract. Three fractions were unstable in light and immediately turned purple, and

one yellow light stable fraction. Two light stable brownish-colored fractions turned

purple after acid hydrolysis. They were united (64 mg) and subjected to further

fractionation (dichloromethane, methanol, water--6:4:1) where 27 mg of the green

insoluble tyriverdin and 4 fractions were obtained. By IR spectrophotometry and

comparison with spectra reported by Fouquet (1970) it could be shown that one

fractionated compound was 6-bromoisatin and the other chromogen-4, which

Fouquet described as a salt of 6-bromo-2 methylsulfonylindoxylsulfate, or as Cooksey

(pets. comm.) suggested, as a 2-methylthio compound perhaps contaminated with a

trace of a 2-methyldithio compound.

Determination of Free Radical Scavenging Activities in Organic Extracts From

Hypobranchial Secretions and the Liquid in the Mantle



In the secretion of extracts from the hypobranchial gland two yellow patches were

observed above a purple background. One large one with a Rf-value of 0.0 and a

smaller one with a Rf-value of 0.74. Free radical scavengers are responsible for the

development of the yellow patches, the purple background is the area where no

scavengers were present. By IR spectroscopy of the organic extract used we could

determine the chromogen IV, probably 6-bromo-2 methylsulfonylindoxylsulfate as a

substance responsible for the free radical scavenging activity.

DISCUSSION

Survival strategies are essential for of intertidal snails, which have to cope with

extreme living conditions of its habitat, especially the risk of desiccation and

overheating during prolonged periods of sun and air exposure outside of the water.

During periods outside of the water the stored water in the mantle of the snail serves

as a source of oxygen, however, during high ambient temperatures the stored water

in the mantle would be an excellent medium for the growth of bacteria. For this

reason it was not astonishing to find in the secretion from the hypobranchial gland

substances with antibacterial properties which could serve as a defense against

bacterial infections. Benkendorff et al. (2000, 2001) studying the chemical

compounds and their biologic function in egg masses of the Australian muricid,

Dicathais orbita, found a range of brominated indole derivates, which were

precursors to Tyrian purple. In egg capsules Benkendorff et al. (2000, 2001)

determined that tyrindoleninone is the major antibacterial metabolite, tyriverdin

highly bacteriostatic, toxic to marine and human pathogens at a concentration of 1

mg/mL, and 6-bromoisatin showed mildly antimicrobial activities. It is commonly

assumed that many organobromine metabolites serve in a chemical protection role for

the organism (antibacterial, antifungal, antifeedant, antifouling etc), although this

function has only been established in few cases (Gribble 1999).

In our bio-autographic study the spots with a Rf-value of 0-0.37 and 0.66-1.00

showed strong antibacterial activities against Staphylococcus aureus. The bioactive

compound responsible for the antibacterial activity could have been, like in the study

by Benkendorff et al. (2000, 2001), tyrindoleninone, with a Rf-value of 0.88, and

tyriverdin with a Rf-value of 0.32.

The secretion of the hypobranchial gland contains, besides fucose-rich

mucopolysaccharides (fucomucins), mucoproteins, plasma proteins, enzymes, bit-

active substances and also small amounts of pigment precursors. In view of the single

layer, epithelium glandular cells of the hypobranchial gland, the volume secreted by

P. pansa is surprisingly large, especially since in the hypobranchial gland no organ

exists that could store the secretion. During the determination of the soluble proteins

of the hypobranchial secretion we experienced large variations in the concentration.

These variations could be explained on one side by varying contaminations with water



from the mantle during sampling, or by different secretion activities of the

hypobranchial gland. To eliminate the impact of water contaminations we tried

unsuccessfully to obtain a constant relation by relating the protein with the

carbohydrate concentration. In humans at high secretion velocities of the saliva is the

protein content relatively constant, however, at lower secretion velocities decreases the

protein concentration (Anonymous 1968). The protein concentration in the secretion

of the hypobranchial gland, however, is nearly 10 times higher than in the saliva from

humans, whereas the glucose concentration is nearly five times lower (Anonymous

1968).

Since the experiments by Dubois (1909), it is known that the secretion also contains

pharmacologically active substances with toxic and paralyzing actions, like serotonin,

murexin, choline ester and biogenic amines. During personal field observations

(unpublished) we observed that P. pansa uses the secretion to immobilize prey

(Nerita sp., Litorina sp.) in the intertidal zone, and it does not resort to drilling

through the shells of other snails.

Artemia nauplii have gained popularity as a test organism for short-term toxicity

testing, because of their rapid development and growth after hatch. For toxicity tests

however, synchronous populations of nauplii must be available. During the study

presented here it was difficult to comply with this prerequisite, because development

and growth of nauplii are not only dependent on the quality of the cysts used but also

on the duration of accumulation of nauplii and the incubation temperature (Sleet &

Brendel 1983a). Additionally, the technology as described by Sleet and Brendel

(1983b) for harvesting synchronous populations, was not available. Difficulties with

the replicability of the tests and the high variations of the results could be explained

by these problems. The in this study determined 50% lethal dose ([LD.sub.50]) of

81.72 [micro]g/mL is in the range of strychnine sulfate (77.2 [micro]g/mL), it is

double the dose for digitalin (151 [micro]g/mL), three times for ephedrine sulfate (215

[micro]g/mL) and nearly four times for caffeine (306 [micro]g/mL) (Meyer et al.

1982).

Additionally interesting to notice is the fact that during the predation no purple color

is formed on the prey, despite the presence of oxygen and intense light radiation. This

supports the finding that under normal circumstances the dye precursors aryl

sulfatase and the pharmacologically active substances are kept apart in specialized

cells in the hypobranchial gland. The large number of different cell types supports

this view (Roller et al. 1995).

The importance of the chromogens in the metabolism of P. pansa remains unclear.

The presence of the enzyme aryl-sulfatase (purpurase), which presumably occurs in

all muricids (Erspamer 1946), supports the hypothesis that the chromogens could

serve as a storage for the highly unstable indoxyls, which are formed enzymatically by



the aryl-sulfatase from sulfate esters (Fouquet 1970). Further investigation needs to

address the question of the biologic function of the indoxyls and their substituted

bromo- and methylthio-analogs. It could be possible that these bromo- and thio-

substituted indoxyls, like the iodine derivates of tyrosine could act as endogen

hormones in the metabolism of the snails. Because the chromogens, besides mucus

and bioactive substances, have their origin in specialized cells, it is feasible that the

hypobranchial gland could have additionally inner secretory activities (Fouquet

1970). The great number of different cell types and of chemical activities in the

hypobranchial gland is an indication that it has multiple biologic functions.

The formation of Tyrian purple after spontaneous expulsion of the secretion by P.

pansa is most probably accidental, because under normal conditions P. pansa does

not expulse. Although in the laboratory traces of purple were found on the wall of the

culture carboys, which had not been cleaned for a long period. It is feasible that P.

pansa continuously secretes small amounts of the precursors together with

arylsulfatase. The antibacterial properties of the secretion support this suggestion.

Indole, which is found as well in the catabolism as in the biosynthesis of tryptophan

can be considered as the primary substance for the biogenetic formation of the

indoxyles in the hypobranchial gland of muricids (Verhecken 1989). Interestingly, in

patients with various pathologies, including leukemias, indican, indigo and indirubin

can be found in the urine, derived from the metabolism of tryptophan into indole

which is absorbed and further oxidized in the liver to indoxyl. This indoxyl is then

excreted in the urine as a sulfate conjugate and is decomposed to indigo and

indirubin by bacteria (Hoessel et al. 1999).

The bromination of indoxylsulfates still needs research. Bromoperoxidase has been

isolated from extracts from the hypobranchial gland of the muricid M. trunculus

(Jannun & Coe 1987). The formation of natural organohalogens in living organisms

is well established. For many of these compounds, the mechanism for their formation

initially involves the oxidation of halide by a peroxidase enzyme and hydrogen

peroxide (Gribble 1998).

Animals that are facultative anaerobes, such as freshwater turtles, appear to deal with

the potential of oxidative stress during the anoxic-aerobic transition by maintaining

constitutively high antioxidant defenses that can readily accommodate the burst of

reactive oxygen species generation, like hydrogen peroxide, when oxygen

consumption is renewed (Storey 1996). During the enzymatic bio-bromination of

indoxylsulfate to tyrindoxylsulfate hydrogen peroxide is required in the

hypobranchial gland of P. pansa. Like in marine algae the peroxide-induced

bromination reactions may be to scavenge excess hydrogen peroxide during oxidative

stress (Gribble 1999). We could determine by IR spectroscopy the chromogen IV,

described by Fouquet (1970) as 6-bromo-2 methylsulfonylindoxylsulfate, as a



substance responsible the for the free radical scavenging activity. Using new analytical

techniques to determine the structure of chromogen IV, the structure as described by

Fouquet (1970) should be treated with caution. It is much more likely to be a 2-

methylthio compound with a trace of the 2-methyldithio compound (Cooksey, pers.

comm.).

A reinvestigation of the biologic functions of the hypobranchial gland, and the

chemical properties of its secretions, especially of the purple precursors would seem

to be overdue.

ACKNOWLEDGMENTS

The authors thank Irma Castanon-Estrada and Anabel Rosales-Maldonado (U. A.

Nayarit) for their help to determine the sex of the snails, and Carlos Augusto Aguilar

and Jorge Lopez Rocha (CICIMAR) for their assistance during the fieldwork. Special

thanks to Jorge Lopez-Rocha and Oscar Armendariz-Ruiz for improving the

drawings. This work was supported by grants from CGPI, COFAA and EDI (Instituto

Politecnico Nacional, Mexico). The advice from Chris Cooksey (London) during the

process of the research helped us significantly. Mabelle Delgado (Tecnologico de

Culiacan) collaborated with the preparation of organic extracts and the toxicity tests

and Lorena Leon Deniz (CINVESTAV/IPN) with the bacteriological assays. The

comments of the reviewers helped to improve this work.

LITERATURE CITED

Andrews, E. B., M. R. Elphick & M. C. Thorndyke. 1991. Pharmacologically active

constituents of the accessory salivary and hypobranchial glands of Nucella lapillus, J.

Molluscan Studies 57(1): 136-138.

Anonymous. 1968. Documenta Geigy. Wissenschaftliche Tabellen. 7th. ed. Basle,

Switzerland: J. R. Geigy. 798 pp.

Baker, J. T. & C. C. Duke. 1973. Chemistry of the indoleninones. II. Isolation from the

hypobranchial glands of marine mollusks of 6-bromo-2,2-dimethylthioindolin-3-one

and 6-hromo-2-methylthioindoleninone as alternative precursors to Tyrian purple.

Aust. J. Chem 26:2153-2157.

Benkendorff, K., J. B. Bremner & A. R. Davis. 2000. Tyrian purple in precursors on

the egg masses of the Australian muricid, Dicathais orbita: A possible defensive role.

J. Chemical Ecology 26(4):1037-1050.

Benkendorff, K., J. B. Bremner & A. R. Davis. 2001. Indole derivates from the egg

masses of muricid mollusks. Molecules 6:70-78.

Clench, W. J. 1947. The genera Purpura and Thais in the Western Atlantic.



Johnsonia 2(23):61-91.

Cole, W. 1685. A letter from Mr. William Cole of Bristol to the Phil. Society of Oxford,

containing his observations on the purple fish. Philos. Trans. Royal Soc. London

15(178):1278-1286.

Cooksey, C. 2001. Tyrian Purple: 6,6'-dibromoindigo and related compounds,

Molecules 6:736-769.

Cooksey, C. & R. Withnall. 2001. Chemical studies on Nucella lapillus. Dyes in History

and Archaeology 16/17:91-96.

Cuendet, M., K. Hostettmann & O. Potterat. 1997. Iridoid glucosides with free radical

scavenging properties from Fagraea blumei. Helvetica Chimica Acta. 80:1144-1152.

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers & F. Smith. 1956. Colorimetric

method for determination of sugars and related substances. Analytical Chemistry

28:350-356.

Dubois, R. 1909. Recherches sur la pourpre et sur quelques autres pigments animaux.

Arch. Zool. Exptl. Gen. 5(2):471-590.

Erspamer, V. 1946. Ricerche chimiche e pharmacologiche sugli estratti de ghiandola

ipobranchiale di Murex (Truncularia) trunculus (L.), Murex (Bolinus) brandaris (L.)

e Tritonalia erinacea (L.). Pubbl. Staz. ZooL Napoli 20:91-101.

Erspamer, V. 1952. Wirksame Stoffe der hinteren Speicheldrtise der Oktopoden und

der Hypobranchialdruse der Purpurschnecken. Arzneimittel Forschung 2(6):253-

258.

Erspamer, V. & O, Benati. 1953. Isolierung des Murexins aus

Hypobranchialdrusenextrakten von Murex trunculus und seine Identifizierung als

[beta]-[Imidazolyl-4(5)]-acryl-cholin. Biochemische Zeitschrift 324:66-73.

Fouquet, H. 1970. Bau und Reaktionen naturlicher Chromogene indigoider

Farbstoffe bei Purpurschnecken. PhD. Thesis. University of Saarbrucken, Germany.

106 pp.

Fretter, V. & A. Graham. 1994. British prosobranch molluscs. Their functional

anatomy and ecology. Revised and updated ed. London: The Ray Society. 820 pp.

Friedlander, P. 1909. Uber den Farbstoff des antiken Purpurs aus Murex brandaris.

Ber. dtsch. Chem. Ges. 42:765-770.

Ghiretti, F. 1994. Bartolomeo Bizio and the rediscovery of Tyrian purple. Experientia



50:802-807.

Gribble, G. W. 1998. Naturally occurring organohalogen compounds. Accounts of

Chemical Research 31 (3): 141-152.

Gribble, G. W. 1999. The diversity pf naturally occurring organobromine compounds.

Chem. Soc. Rev 28:335-346.

Hoessel, R., S. Leclerc, J. A. Endicott, M. E. M. Nobel, A. Lawrie, P. Tunnah, M.

Leost, E. Damiens, D. Marie, D. Marko, E. Niederberger, W. Tang, G. Eisenbrand &

L. Meijer. 1999. Indirubin, the active constituent of a Chinese antileukaemia

medicine, inhibits cyclin-dependent kinases. Nat. Cell Biol. 1:60-67. Website

cellbio.nature.com Supplementary Information.

Huang, C. L. & G. N. Mir. 1971. Pharmacological properties of hypobranchial gland

of Thais haemastoma (Clench). J. Pharmacological Sciences 60(12):1842-1846.

Jannun, R. & E. L. Coe. 1987. Bromoperoxidase from the marine snail Murex

trunculus. Comparative Biochemistry and Physiology B--Comparative. Biochemistry

88:917-922.

Keen, A. M. 1971. Seashells of tropical West America: Marine molluscs from Baja

California to Peru. Stanford University, Stanford, CA. 1064 pp.

Koren, Z. C. 1994. HPLC analysis of the natural scale insect, madder, and indigoid

dyes. J. Society of Dyers and Colourists 110:273-277.

Malaszkiewicz, J. 1967. Chromogene und Farbstoff-Komponenten der

Purpurschnecke Murex trunculus L." PhD. Thesis, University of Saarbrucken,

Germany. 75 pp.

Meyer, B. N., N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen, D. E. Nichols & J. L.

McLaughlin. 1982. Brine shrimp: a convenient general bioassay for active plant

constituents. J. Medical Plant Research Planta Medica 45:31-34.

Michel, R. H., J. Lazar & P. E. McGovern. 1992. The chemical composition of the

indigoid dyes derived from the hypobranchial glandular secretions of Murex

mollusks. J. Society of Dyers and Colourists 108: 145-150.

Paredes, C., P. Huaman, F. Cordoso & V. Vera. 1999. Estado actual del conocimiento

de los moluscos acuaticos en el Peru. Revista Peruana Biologia 6(1):5-47.

Pena, G. G. M. 1970. Zonas de distribucion de los gasteropodos marinos del Peru.

Anales Cientificos de la Universidad Nacional Agraria 8: 153-170.



Rahalison, L., M. Hamburger & K. Hostettmann. 1991. A bioautographic agar overlay

method for the detection of antifungal compounds from higher plants.

Phytochemical Analysis 2:199-203.

Roller, R. A., J. D. Rickett & W. B. Stickle. 1995. The hypobranchial gland of the

estuarine snail Stramonita haemastoma canaliculata (Gray)

(Prosobranchia:Muricidae): a light and electron microscopical study. American

Malacological Bulletin 11(2):177-190.

Roseghini, M., C. Severini, G. Falconieri-Erspamer & V. Erspamer. 1996. Choline

esters and biogenic amines in the hypobranchial gland of 55 species of the

neogastropod muricoidea superfamily. Toxicon 34(1): 33-55.

Schunck, E. 1880. LII. Notes on the purple of the ancients (continuation). 3. Purple

dyeing in modern times. J. Chemical Society (London) 37: 613-617.

Shiomi, K., K. Sasaki, H. Yamanaka & T. Kikuchi. 1983. Volatile sulfur compounds

responsible for a fetid odor of the hypobranchial gland of muricid gastropods:

Reishia Thais clavigera and R. T. bronni. Bulletin of the Japanese Society of Scientific

Fisheries 48(9): 1353-1356.

Shiomi, K., M. Ishii, K. Shimakura, Y. Nagashima & M. Chino. 1998. Tigloycholine: a

new choline ester from the hypobranchial gland of two species of muricid gastropods

(Thais clavigera and Thais bronni). Toxicon 36(5):795-798.

Sleet, R. B. & K. Brendel. 1983a. A flow-through hatching and cold storage system for

continuous collection of freshly hatched Artemia nauplii. J. Aquaricul. Aqua. Sci.

3(4):76-83.

Sleet, R. B. & K. Brendel. 1983h. Improved methods for harvesting and counting

synchronous populations of Artemia nauplii for use in developmental toxicology.

Ecotoxicol. Environm. Safety 7:435-446.

Solis, P. N., C. W. Wright, M. M. Anderson, M. P. Gupta & J. D. Phillipson. 1993. A

microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Med 59:250-

252.

Storey, K. B. 1996. Oxidative stress: animal adaptations in nature, Brazilian J. of

Medical and Biological Research 29:1715-1733.

Verhecken, A, 1989. The indole pigments of mollusca. Annls. Soc. R. Zool. Belg.

119(2):181-197.

Whittaker, V. P. 1960. Pharmacologically active choline esters in marine gastropods,

Ann. N.Y. Acad. Sci 90:695-705.



Withnall, R., D. Patel, C. Cooksey & L. Naegel. 2003. Chemical studies of the purple

dye from Purpura pansa. Dyes in History and Archaeology 19:107-115.

Wouters, J. 1992. A new method for the analysis of blue and purple dyes in textiles.

Dyes in History and Archaeology 10:17-21.

LUDWIG C. A. NAEGEL * AND JESUS I. MURILLO ALVAREZ

Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional

(CICIMAR-IPN) Apdo. Postal 592 La Paz, B.C.S. 23000 Mexico

* Corresponding author. E-mail: [email protected]

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