Sie sind auf Seite 1von 4

Research Articles

Capsaicin

Research Articles Determination of Optimal Water Solubility of Capsaicin for its Usage as a Non-toxic Antifoulant
Cafer Turgutl, Bi-min Newby2, and Teresa J. Cutright3*
1

Adnan Menderes University, 09100 Aydin, Turkey

23Department of Chemical Engineering2 and Department of Civil Engineering\ The University of Akron, Akron OH 44325-3905, USA

Corresponding author (tcutright@uakron.edu)

DOl: http://dx.doi.org/1 0.1 065/espr2003.12.180


Abstract

Background. Capsaicin, a non-toxic natural product antifoulant,


is an attractive alternative to the currently used toxic metal based antifoulants for protecting the submerged surfaces. The problem associated with conducting capsaicin experiments is its limited aqueous solubility. Methods. A procedure was developed to obtain the maximum capsaicin concentration in water. Capsaicin was first dissolved in one of the three organic solvents, methanol, ethanol, and ac erone. The organic solvent was replaced by gradually adding distilled water and evaporating the organic solvent. Results. The best solvent for obtaining highest concentration of capsaicin in water was ethanol. A maximum capsaicin in water 6400 ppm was achieved by using 16 mg capsaicin dissolved in 0.8 ml ethanol followed with the addition of 2.5 m! distilled water in 0.2 ml increments and then slow evaporation of the solvent.

have exhibited a toxic response to TBT concentrations be low 1 ng/1 [4]. There have also been reports of oyster shell deformities, mutagenic effects in gastropod mollusks, and accumulation in salmon tissue [5]. As such, many govern ments have banned the use of TBT and other metal based antifoulant coatings [2,4,5]. International bans on the use of metal-based antifoulants have increased interest in the development of alternative biocides. Most of the current interest centers on natural compounds that act as nontoxic repellent such as capsaicin. The active ingredient in capsaicin is obtained by grinding dried ripe fruits of Capsicum frutescens L. (Chili peppers) into a fine powder. Once obtained it can be processed via normal manufactured processes into repellents or coatings. In 1995, the first capsai cin antifouling coating composition was patented [6). Recent research has documented that capsaicin's effective ness is dependent on the concentration used, application method (attached, in solution, etc.), and the species present. For instance, when 25 pg capsaicin was coated on a 7.5 em x 2.5 em glass slide, it inhibited the microbial growth of P. solanaius [7]. In another study using similar concentra tions, capsaicin was toxic to Desulvibrious vulgaris and Geobacter metalreducens [8]. However, capsaicin was not effective for gram positive bacteria even at aqueous con centrations greater than 300 pg/ml [7). Thus even natural antifoulants can be toxic depending on the concentration used and species present. The primary obstacle for using capsaicin as an antifoulant at the aforementioned concentrations is its limited water solubility. Even under saturated conditions, capsaicin has only slight water solubility ( - 60 mg/1}. In patent applica tion and research studies mentioned above, capsaicin had to be predissolved in an organic solvent in order to be easily processed [9]. For instance, previous studies first dissolved capsaicin in benzene, which can present its own set of envi ronmental concerns [10]. The amount of benzene used was just as toxic to sensitive microorganisms as TBT based com pounds. In addition, benzene could interfere with assays used to evaluate capsaicin as a natural antifoulant. Therefore the aim of this study was to determine how to obtain elevated concentrations of capsaicin into an aqueous environment without the use of toxic solvent.

Conclusion. The concentration obtained using our approach is


well above the threshold concentration of capsaicin in deterring the marine organism attachments.
Keywords: Antifoulants; biocides; capsaicin; non-toxic anti

foulant; water solubility

Introduction

Biofouling on ship-hulls, oil platforms, pipelines, and har bor construction equipment has always posed a major prob lem for maritime industries. The attached organisms not only increase the weight and frictional drag of ships, leading to reduced speeds and increased fuel consumption, but also result in corrosion related repairs [1,2]. The annual estimated cost for removing organisms from ship-hulls, platforms and pipelines is in billions of dollars. Applying an antifouling coating to the submerged surface can reduce the biofouling problem. Over the past several decades, many chemicals, including organa-mercury compounds, lead, arsenic and DDT were used as antifoulants [3]. Unfortunately, the most effective metal-based compounds such as tributyl tin (TBT) are not selective in the organisms that they kill to reduce the number of attached fouled or ganisms. Sensitive marine species such as Nucella lapillus,

ESPR - Environ Sci & Pollut Res 1 1 ( 1) 7 - 10 (2004) ecomed publishers. D-86899 Landsberg, Germany and Ft. Worth(fX

Tokyo

Mumbai

Seoul

Melbourne

Paris

Capsaicin

Research Articles

2 2.1

Materials and Methods Reagent sources

Capsaicin was purchased from the Aldrich Chemical Co. The specific formulation contained 65% capsaicin and 35% dihydrocapsaicin as active ingredients. Ethanol, methanol, and acetone were purchased from Fisher Scientific in technical grade.
2.2
Saturated solubility study

studies [13,14] have reported that these two peaks corre spond, respectively, to capsaicin and dihydrocapsaicin, with relative peak areas of 66.28 % and 29.13%. Based on the peaks observed in the HPLC chromatograph for the satu rated capsaicin solution, the concentration of capsaicin was determined to be 60 mg/1.
3.2
Optimization of aqueous capsaicin solubility

The solubility of capsaicin in water was determined by a saturation study. The saturated solution of capsaicin was prepared by adding 200 mg of capsaicin and 10 ml distilled water into a glass vial. The mixture was then subjected to constant stirring for 1 week under ambient conditions. Af ter one week, the stirring was stopped and the vial sealed. The sealed vial was then stored undistributed for two days at room temperature to allow the un-dissolved capsaicin to settle to the bottom. After settling, 200 J.ll of the superna tant was extracted and mixed with an equal volume of pure acetonitrile. The new solution was then analyzed via High Performance Liquid Chromatograph (HPLC).
2.3
HPLC method

The HPLC (Shimadzu LC-10AT), equipped with a C-18L column (Waters Scientific), was operated isocratically with a 50:50 acetonitrile:water mobile phase. The flowrate and sample volume was 1 mVmin and 10 J.Ii, respectively. Calibration curves were used to convert chromatogram ar eas to mg/1 of capsaicin. The curves were generated using capsaicin standards (88000 mg/1). Each standard was pre pared by dissolving the required amount of capsaicin in acetontrile, followed by the addition of an equal volume of HPLC grade water.
2.4
Experimental approach to optimize aqueous solubility

As indicated by the saturation study, capsaicin has limited wa ter solubility. In order to increase its aqueous concentration, capsaicin is normally pre-dissolved in an organic solvent that is miscible with water, and then transferred from the organic so lution to the aqueous solution. In this study, three organic sol vents, methanol, ethanol, and acetone were used. The solubil ity parameters for methanol, ethanol, and acetone were found to be 29.7 (M]fm3) 112, 26.2 (MJfm 3) 112, and 19.7 (M]!m3) 112, respectively, whereas water had a solubility parameter of 48 (MJ!m3) t/2. Capsaicin, a stable alkaloid, is an organic compound. The solubility parameter of capsaicin, estimated using the group contribution method based on its formula (see Fig. 1), its den sity (1.15 g!cm 3), its molecular weight (M 305.4 g!mol) , and the group molar attraction constants [15], is 23.7 (MJ/m3) 112. By comparing this value to the solubility parameters of metha nol, ethanol, acetone, and water, ethanol (o 2.5 (M]fm3) 111) appears to be the best solvent for capsaicin, followed with ac etone, then methanol. Water apparently is an extremely poor solvent for capsaicin with a o value of 24.5 (M]fm3) 112.
= =

Fig. 1: Chemical structure of capsaicin

Capsaicin was added to different volumes of organic sol vent to achieve final concentrations ranging from 6.5 J.Ig/ml to 100 mg!ml. The solvents investigated included methanol, ethanol and acetone. These solvents were selected since they pose minimum toxicity to microorganisms at low concen trations [11,12]. In some instances, methanol and ethanol can be used as an auxiliary carbon source for microbial growth. However, too much of either chemical can alter the microbial community's respiration mode and/or dynamics. The experimental approach was first conducted with direct additions of capsaicin to each solvent. This was followed by small increments of distilled water with mixing and gentle heating. The evaporation of solvent before and after all water additions was evaluated, and the amount of total solution was measured to estimate the quantity of solvent evaporated. The optimal rate of water additions was also as.::ertained. Details and observations regarding the solution clarity, color, precipitate formation for each combination were recorded.
3 3.1
Results and Discussion Saturated water solubility

The first set of experiments investigated the dissolved cap saicin concentrations used by previous researchers in each of the three solvents [16]. When capsaicin was dissolved in pure methanol, ethanol or acetone, neither color changes nor precipitate formation was present, as summarized in the first three rows of Table 1. After adding 1 ml of distilled water, capsaicin still remained in solution. However after the solvent was evaporated, the solution color turned to white, indicating a slight phase change. When more distilled water was added, capsaicin started to precipitate out of so lution as evidenced by the formation of insoluble particles and the experiment was terminated. However it is impor tant to note that both acetone and methanol systems had capsaicin particles after only 1 ml of distilled water was added. The ethanol system did not form precipitates until after 3 ml of water had been added, confirming the predic tion, based on solubility parameter, that ethanol is the best of the three solvents to be used for dissolving capsaicin. The next experimental set investigated the effects of addition of water on the behavior of capsaicin prior to evaporating the solvent. The exact procedure starting with an initial 10 mg/ml capsaicin stock solution is outlined in Table 2. The capsaicin concentration was diluted via increments of distilled water. Initially, 0.2 ml increment was used until the desired concen-

HPLC analysis of the capsaicin standards exhibited two main peaks at retention times of 12.6 and 18.0 minutes. Other

ESPR - Environ Sci & Pollut Res 1 1

( 1) 2004

Research Articles

Capsaicin

Table 1: Solubility of capsaicin in different solvents Color EtOH


6 mg Capsaicin + 1 ml Ethanol .5 6 mg Capsaicin + 1 ml Acetone .3 4 mg Capsaicin .5
+1

Particles MeOH
No No No No

Acet
No No No No

EtCH
No No No No No No No Yes

Acet
No No No No No Yes Yes Yes

MeOH
No No No No No Yes Yes Yes

No No No No

ml Methanol

1 ml distilled water added


Heated and solvent evaporated

w w w w

w w w w

w w w w

1 ml distilled water added


2 ml distilled water added 3 ml distilled water added EtOH: Ethanol, MeOH: Methanol, Acet: Acetone, W: Wh1te

Table 2: Solubility of capsaicin in different solvents with water addition prior to solvent evaporation Color EtOH
2 mg Capsaicin + 0.2 ml Ethanol 2 mg Capsaicin + 0.2 ml Acetone 2 mg Capsaicin + 0.2 ml Methanol 0.2 ml distilled water added 0.4 ml distilled water added 0.6 ml distilled water added 1.6 ml water-0.5 ml increments 2.6 ml distilled water added 10.6 ml water in2 ml increments 15.6 ml distilled water added 25.6 ml distilled water added 4 5.6 ml distilled water added 6 5.6 ml distilled water added Heating and evaporation of solvents W: White, T: Turb1d1ty, EtOH: Ethanol, MeOH: Methanol, Acet: Acetone No No No No No

Particles MeOH
No No No No

Acet
No No No No

EtCH
No No No No No No No No No No No No No No

Acet
No No No No No No No No No No No No No No

MeOH
No No No No No No No No No No No No No No

w w w w w w w
T No No

w w w w w w w
No No No

w w w w w w
T No No

..

trarion, the maximum value in Table 1, was obtained. Then larger increments of distilled water were added. This was fol lowed by solvent evaporation at 40C with gentle mixing. Using this approach, the final concentration for ethanol and methanol was 32 11g/ml (11-lg/ml H=1 ppm) in distilled water, which was too low to be utilized for the subsequent antifoulant assays to determine the effectiveness of capsaicin on antifoul ing behaviors or its effects on microbial activities. As men tioned earlier, studies where capsaicin was effective used aque ous concentrations ranging from 100 to 1000 ppm. Therefore, a higher concentration stock solution may be needed to ob tain a higher capsaicin aqueous concentration. In the efforts to achieve higher aqueous capsaicin concentra tions, we first attempted to generate a concentrated capsaicin methanol stock solution. When 100 mg capsaicin was dis solved in 1 ml methanol and followed by 1 ml distilled water, the addition of water precipitated the capsaicin (shown in Table 3 ). The same procedure was repeated with lower amounts of capsaicin until the addition of 1 ml distilled water did not facilitate observable precipitants. The highest capsaicin concentration that did not form precipitates after adding water was 80 mg/ml. Although this approach did achieve the desired capsaicin concentration, approximately 12% of the methanol remained. For small-scale experiments, the impact of the re sidual methanol could be easily and cost effectively handled. However, for large scale applications, this methanol concentra-

Table 3: Solubility of capsaicin in methanol Color 100 mg Capsaicin + 1 ml Methanol 1 ml distilled water added
No No

Particles
No Yes

tion would preferentially increase the amount of methanotrophs, substantially altering the microbial ecosystem [12]. The viability of ethanol was further evaluated since it would not increase the amount of methanotrophs. The maximum aqueous capsaicin concentration that could be obtained from the most concentrated capsaicin-ethanol stock solution (16 mg of capsaicin in 0.2 ml) was 40 mg/ml (Table 4a). Afterwards, more dilute stock solutions, 16 mg capsaicin in 0.4 ml etha nol (Table 4b) and 16 mg capsaicin in 0.8 rnl ethanol (Table 4c) were used. For the case of using 0.4 ml ethanol, a total of 0.6 ml distilled water was added in 0.2 ml increments , fol lowed by two 0.5 ml additions. As shown in Table 4b, the first 0.2 ml additions resulted in change in solution clarity (clear to opaque), but insoluble particles formed only after a 0.5 ml distilled water addition was used. Thus, when water is added in greater than 0.2 ml increments, it may cause capsai cin to precipitate out of solution. In order to verify this specula tion, a slow increment (0.2 ml) of water addition to the 16 mg capsaicin in 0.8 ml was conducted, and a total of 3 ml distilled water was added (Table 4c). Although there was a color 9

ESPR - Environ Sci & Poilu! Res

11 ( 1) 2004

Capsaicin

Research Articles

Table 4: Evaluation of capsaicin solubility in ethanol


Color
(a) 16 mg Capsaicin+ 0.2 ml Ethanol 1 ml distilled water added (b) 1 mg Capsaicin+0.4 ml Ethanol 6 0.2 ml distilled water 0.2 ml additional distilled water 0.2 ml additional distilled water 0.5 ml additional distilled water 0.5 ml additional distilled water (c) 16 mg Capsaicin + 0.8 ml Ethanol Total3 ml distilled water in 0.2 ml increments Evaporation of solvent W: Wh1te, EtOH: Ethanol, MeOH: Methanol, Acet: Acetone No

Particles
No Yes No No No No Yes/No Yes No No No

w
No

w w w w w
No

w w

change in the solution upon addition of water, no capsaicin was precipitated out from the solution as water was being added. These results indicate that a slow addition of water to a more dilute capsaicin-ethanol solution, under continu ous stirring, would be most favorable for generating more concentrated capsaicin in water. Ethanol was evaporated via gentle heating (50C) as the fi nal step of the approach to generate a high concentration capsaicin-aqueous solution. As ethanol was evaporated, the solution went from clear to slightly opaque. However, cap saicin did not precipitate out of solution until the concen tration of ethanol was less than 2 wt. %. At 2 wt% ethanol would not be toxic to most aquatic organisms. Maintaining the ethanol concentration at 2 wt% would prevent the for mation of precipitates while minimizing solvent toxicity.
4
Conclusion

References

[1) [2)

Clare A (1995): Natural ways to banish barnacles. New Scien tist, 28-41 Ponasik J, Conova S, Kinghorn D, Kinney WA, Rittschof D, Ganem B (1998): Pseudoceratidine, a marine natural product with antifouling activity: Synthetic and biological sruciies. Tet rahedron 54 (25) 6977-6986 Lewis JA (1998): Marine biofouling and its prevention on un derwater surfaces. Materials F orum 22, 41-61 Callow ME ( 1999): The status and future of biocides in marine biofouling prevention. In: (Thompson MF, Nagarhushanam R, eds), Recent advances in marine biotechnology 109-126, Sci ence Publisher Perez M, Carcia M, Vetere V, Deya M, del Amo B, Stupak M ment Resin Techno! 30 ( 1) 34-38 Watts JL (1994): Anti-fouling coating composition using cap saicin. US Patent 5,397,385 Tsuchiya H (2001 ): Biophasic membrane effects of capsaicin, as an active component in Capsicum species. J Ethnopharmacology 75,295-299 Paterek JR, Cepuritis A, Srivastava VJ (1998): Prevention and control of microbiologically influenced corrosion using environ (2001): Benzoates a new approach to non-toxic biofouling. Pig

[3) [4]

[5]

[6] [7)

Numerous studies have documented the environmental con cerns associated with metal-based antifoulants. The attack on non-target organisms by TBT-based antifoulant paints have led to complete international ban on their use by 2008. There fore environmentally friendly, natural compounds, such as capsaicin, have become the preferred alternatives. In this study, we reported a procedure use to increase capsaicin's water solu bility by first dissolving capsaicin in organic solvents, such as methanol, ethanol, and acetone, then gradually replacing the organic solvent by adding known amount water and evapo rating the organic solvent. Using this approach, the exact amount of capsaicin in water can be determined. Amount the three solvents used, acetone was found to re-precipitate cap saicin easier than the other two. Methanol was not selected as the final solvent since it has a greater tendency to change the microbial population. Overall, the best results were obtained when ethanol was used as the initial solvent. The optimum cap saicin concentration in water was found to be 6.4 mglml or 6400 ppm. This concentration was obtained by using a 16 mg of capsaicin dissolved in 0.8 ml ethanol followed with a gradual addition of distilled water up to 2.5 ml without precipitating capsaicin. Then, the ethanol was allowed to evaporate slowly immediately prior to any precipitation occurs in the solution. This procedure enabled over 98% of ethanol to be evaporated to avoid the presence of too much ethanol, which could facili tate a change in the population dynamics, thus preventing any negative impacts of ethanol on the aquatic ecosystem.

[8)

mentally benign natural products. Proceedings International Gas Research Conference 2, 681-691 (9) US Environmental Protection Agency (1999): Report on Cap saicin Registration Eligibility. Washington DC [10] Zhang W.,Bouwer EJ (1997): Biodegradation of benzene, tolu ene, and naphthalene in soil-water slurry microcosms. Biodeg radation 8, 167-175 [11) Cookson JT,Jr (1995): Bioremediation Engineering: Design and Application. McGraw-Hill, New York [12) Errett DJ, Chin Y P, Xu Y, Yan Y (1996): The sorption and des orption kinetics of PAHs in methanol!water mixtures. Haz Waste Haz Mad 13 (2) 177-19 [13] Kurian AL, Starks AN (2002): HPLC analysis of capsaicinoids extracted from whole orange habanero chilli peppers. J Food Sci 67,957-962 [14) Weaver KM, Awde DB (1986): Rapid high performance liquid chromatographic method for the determination of very low cap saicin levels. J Chromatogr 367, 438-442 [15) Rheineck AE, Lin KF (1968): Solubility parameter calculations based on group contributions.] Paint Technol 40 (527) 611-618 [16) Jacobson AH, Willingham GL (2000): Sea-nine antifoulant: An environmentally acceptable alternative to organotin antifoulants. Sci Total Environ 258, 103-110
Received: April 28th. 2003 Accepted: December 11th, 2003 OnlineFirst: December 15th, 2003

10

ESPR - Environ Sci & Pollut Res 11

( 1) 2004