Effective Antioxidants for Stabilization of Chlorophyll Adsorbed on Silica Surface

Abstract

This study investigates effective antioxidants to stabilize chlorophyll, a valuable and most abundant but unstable natural green pigment, adsorbed on a silica surface. Although fixing chlorophyll on silica offers some protection, significant photo-induced oxidative degradation still occurs. To enhance photostability, the prepared chlorophyll–silica composites were combined with various well-known antioxidants. The stability of these samples was evaluated by the deterioration ratio of the chlorophyll under visible light irradiation. The results showed that gallic acid provided the most significant stabilization effect. This was attributed to its moderate hydrophilicity, allowing it to be positioned near the chromophore part of the chlorophyll molecule adsorbed on the silica surface. Further tests with the derivatives of gallic acid revealed that smaller molecular size and less steric hindrance were also crucial for effectiveness as an antioxidative stabilizer. Pyrogallol and gallic acid, being the smallest molecules, performed best. It was concluded that the ability of an antioxidant to approach a chlorophyll molecule is essential for stabilization. This requires an appropriate balance of hydrophilicity and a small molecular size. Considering the nontoxicity together, gallic acid is recommended as a superior stabilizer for chlorophyll on silica surfaces.

1. Introduction

Chlorophyll is a green pigment found in plants, and it is the most widely distributed natural pigment on Earth. Chlorophyll is found in all photosynthetic organs, from plants to bacteria [1]. Including the degradation of chlorophyll in nature, the amount of chlorophyll products on Earth is estimated to be about 1000 million tons per year [2].

Chlorophyll is classified as chlorophyll a, chlorophyll b, etc. The concentration of chlorophyll a present in plant leaves is considered two to three times that of chlorophyll b [3]. The structure of chlorophyll a, which is more abundant in nature, is shown in Scheme 1. Chlorophyll has a chlorin ring where four pyrroles are linked by methine bridges. In addition, the Mg²⁺ cation is coordinated to the center of the tetrapyrrole chlorin ring [4]. The chlorin ring is a chromophore originating from the green color of chlorophyll and is regarded as relatively hydrophilic [5]. On the other hand, chlorophyll has a long-chain alkyl group called a phytyl group, resulting in a hydrophobic nature. Therefore, chlorophyll can be seen as an amphipathic substance that has both a hydrophilic chromophore and a hydrophobic alkyl chain [6].

Chlorophyll is used as a natural green colorant because of its vivid green color [7]. In general, natural pigments obtained from plants and animals in nature have low toxicity and are highly safe for the human body for consumers, although there are many exceptions. Chlorophyll is no exception; it can be extracted from plant leaves as a highly safe pigment for the human body. In particular, chlorophyll has excellent properties for health, such as antioxidant, antibacterial, anti-inflammatory, and deodorizing effects, in addition to its vivid green color and low toxicity [8].

However, despite these various advantages, chlorophyll is a very unstable compound, especially once extracted from the plant body. Chlorophyll is quite rapidly decomposed and loses its color under oxygen, acid, and light. Under visible irradiation in the air, chlorophyll absorbs light energy and reacts with oxygen to generate unstable peroxy radicals. As the generated radicals are highly reactive, they attack other chlorophyll to cause a chain reaction of chlorophyll oxidation, resulting in severe deterioration and fading [3]. This inferiority mainly limits the application of chlorophyll on a commercial scale as a natural colorant.

Various studies have been conducted to prevent chlorophyll fading [9,10,11,12,13]. The most common and traditional method to maintain the green color of chlorophyll is the replacement of the metal ion coordinated at the center of the chlorin ring [4]. The central metal ions coordinated to the chlorin ring of chlorophyll are rather easily removed. When the central metal ion is removed, the green chlorophyll is converted to yellowish-to-olive brown pheophytin, followed by quick fading [14]. Replacement of the central metal ion can contribute to the preservation of the green color of chlorophyll. One typical example is zinc chlorophyll, where the original Mg²⁺ coordinated to the chlorophyll is replaced by Zn²⁺ [15]. Ngo et al. reported that when heat-treated green pears were immersed in a Zn²⁺ solution of 1300 ppm to 5200 ppm for 18 min, the Mg²⁺ coordinated to the chlorin ring of chlorophyll was substituted by Zn²⁺, and the green pears maintained their green color for 19 weeks at 38 °C in the dark and for 35 weeks at 10 °C [16]. However, the U.S. Food and Drug Administration (FDA) has set the upper limit for the amount of zinc added to food at 75 ppm [17]. This means there are some possible concerns about the safety of foods containing high concentrations of zinc for the human body.

On the other hand, encapsulation is another stabilization method of chlorophyll that does not involve the replacement of the central metal ion. Encapsulation is the preparation method of so-called core–shell materials; generally, an unstable substance such as chlorophyll is used as a core material, and a more stable shell material surrounds the core to serve as physical protection [18]. Kang et al. reported that when chlorophyll was encapsulated with maltodextrin, the green color of the chlorophyll could be maintained for 10 days at 4 °C [19]. Typical encapsulation steps are (1) dissolving chlorophyll in oil as the core compound, (2) dissolving maltodextrin in water as the shell compound, and (3) drying by spraying the emulsion of (1) and (2) under a hot air stream (spray drying). Protection by encapsulation does not change the molecular structure of chlorophyll, so chlorophyll can be stabilized while maintaining a high level of safety [20]. However, the spray drying used for encapsulation requires heating at high temperatures, which causes concern in terms of the degradation of the chlorophyll. In addition, encapsulation requires a lot of complicated processes [21].

Fixation and incorporation of chlorophyll molecules on inorganic host materials are yet another method for the stabilization of chlorophyll [11,22]. In particular, silica-based materials have been employed for the adsorption of chlorophyll [23,24,25]. Stability enhancement has been reported for chlorophyll molecules tightly fixed through covalent bonding with amino groups planted by chemical modification of a silica surface [26,27]. Itoh et al. reported that the photostability of chlorophyll was enhanced by the incorporation into the mesopore of FSM-type ordered mesoporous silica [28,29]. Adsorption of chlorophyll on a silica surface can be conducted by simply mixing the chlorophyll solution with silica powder, possibly at room temperature. Unlike encapsulation, this method does not require any complicated steps or heat treatment at elevated temperatures. Because the chlorophyll molecules are not subjected to any chemical conversion as the metal ion substitution during the adsorption process, the safety of chlorophyll will not be compromised. Since the silica is also nontoxic, especially without any chemical modification, the composite of chlorophyll and silica will maintain chlorophyll’s characteristics as a safe material. However, in general, due to the attack of atmospheric oxygen, the photo-induced oxidative degradation of chlorophyll under visible irradiation is still significant and not negligible, particularly without tight fixation on silica by chemical bonding.

A common method to prevent the oxidative deterioration of naturally occurring dyes is the addition of antioxidative agents. Visible irradiation of natural dye generates radical species from dye molecules due to the singlet oxygen. The generated radical species attack other dye molecules, causing deterioration by the radical chain reaction. The antioxidative agent plays the role of a radical scavenger to seize the chain reaction. Wu et al. reported the stability enhancement of lutein, a yellow natural dye, by the addition of some phenolic compounds showing antioxidative properties. Phenolic antioxidants are generally added as a stabilizer for oils, petrochemicals, or synthetic rubbers. Organic sulfur compounds also present as a radical scavenger and are especially known to work in vivo to prevent radical chain reaction, causing the degradation of unsaturated fatty acids [30,31], as well as to decompose the hydroperoxide species generated during the oxidation of the fatty acids. As mentioned above, the degradation and discoloration of chlorophyll also proceed through radical chain reaction, causing destructive oxidation, just as with oils and fatty acids. Therefore, we can also expect the effect of phenolic and sulfur-containing antioxidants on the stabilization of chlorophyll.

In this study, we tried to search for effective antioxidative agents for chlorophyll fixed on the surface of silica, targeting the more expanded use of chlorophyll as a colorant. Chemical modification of the silica surface was prevented, because the chemical process would hurt the nontoxicity of the silica as the host material of a natural dye-based colorant. For antioxidants, we first investigated the effect of the addition of vitamins (vitamin C and vitamin E), α-lipoic acid, and gallic acid on the stabilization of the chlorophyll adsorbed on silica. Vitamins are widely used as antioxidants in foods. The α-lipoic acid was adopted as a sulfur-containing antioxidant. Gallic acid is one of the phenolic compounds known to have a strong antioxidative effect. Next, we examined the influence of hydrophilicity and steric hindrance of the antioxidants on the degree of stabilization. By these investigations, we attempted to find the conditions for an effective antioxidant for the chlorophyll–silica composite material.

2. Materials and Methods

2.1. Materials

Chlorophyll purchased from Tokyo Chemical Ind. Co. (Tokyo, Japan) was used in this study. Since this reagent contains hydrophilic impurities like arabic gum and lactose to increase the affinity to water, purification was carried out before use as follows: the purchased chlorophyll powder (1 g) was dispersed in 200 cm³ of water by sonication, followed by extraction with 200 cm³ of ethyl acetate. The deep green ethyl acetate layer was collected and stored at 277 K in the dark as a mother liquor of the purified chlorophyll. For each experiment using the chlorophyll, a portion of the mother liquor was dried up to obtain powdery chlorophyll for the intended usage. Hereafter, the purified chlorophyll was denoted as Chl. Based on the comparison of the absorbance of the purified and unpurified chlorophyll solution, the purity of the chlorophyll increased by at least 4 times through this purification process.

For vitamins as the antioxidative agents, ascorbic acid (vitamin C, denoted as VC) and α-tocopherol (vitamin E, VE) were employed. Those were purchased from Tokyo Chemical Ind. and Fujifilm Wako Co. (Osaka, Japan), respectively. As a sulfur-containing antioxidant, α-lipoic acid (denoted as LA) from Tokyo Chemical Ind. was used. For the phenolic antioxidants, gallic acid (Fujifilm Wako, denoted as GA), ethyl gallate (Fujifilm Wako, denoted as C2), dodecyl gallate (Tokyo Chemical Ind., C12), and pyrogallol (Tokyo Chemical Ind., PG) were used. All antioxidative agents were used without further purification. The molecular structures of the antioxidants were illustrated in Scheme 2.

2.2. Sample Preparation

As an inorganic host material of chlorophyll, silica (CARiACT Q-10, with a mean pore diameter of 10 nm) was kindly supplied by Fuji Silicia Chem. Co. (Kasugai, Japan). The specific surface area and pore volume of the silica were 300 m²/g and 1.1 mL/g, respectively. Before use, the silica was ground in a mortar to pass a 100-mesh sieve and calcined at 773 K to clean up its surface. Thus, the obtained powder was denoted as S hereafter.

The purified Chl (1.35 mg) was dissolved in 0.50 cm³ of ethanol. The Chl solution was dropped down onto 0.10 g of S in a mortar and mixed well. After drying under reduced pressure at 313 K, the composite sample of the chlorophyll adsorbed on silica was obtained. The composite sample was denoted as Chl/S. To prepare the Chl/S samples with an antioxidative agent, 1.2 mmol of each antioxidant was mixed with the ethanolic solution of Chl prior to the addition to S in a mortar. In case that LA was used as an antioxidant, aqueous dispersion of the Chl and LA was used instead of an ethanolic solution, because LA was hardly dissolved in ethanol.

2.3. Measurements

The UV-vis spectrum of the Chl ethanolic solution was collected with a JASCO V-730 spectrophotometer. The diffuse-reflectance UV-vis spectra of the composite samples were collected using a JASCO V-750 spectrophotometer equipped with an ISV-922 integrating sphere. The powdery composite samples were packed in a home-made polystyrene cell and covered with a thin quartz glass.

The light fastness of each composite sample was evaluated as follows: the samples were packed in the cell for the diffuse-reflectance UV-vis measurement. The packed samples were irradiated with visible light from a 100 W halogen lamp (Schott, Megalight 100) for 180 min. During irradiation, diffuse-reflectance spectra of the samples were measured every 5 min until 30 min irradiation, every 10 min until 60 min, and every 30 min after that. The light fastness was evaluated from the retention of the absorption at around 665 nm (Q band of the chlorophyll). A burst decrease in the absorption of the chlorophyll was observed in every sample at the very initial stage of the photoirradiation. Therefore, to evaluate the photostability of each sample, the retention ratio was calculated by the absorption value at each given time divided by that at 5 min irradiation instead of 0 min irradiation.

3. Results and Discussion

3.1. The Impact of the Addition of Antioxidants on the Color of Chl

Figure 1 represents the UV-Vis spectrum of the Chl ethanolic solution (6 mg/10 cm³) together with the diffuse-reflectance UV-Vis spectrum of the Chl/S composite sample. The Chl solution showed absorption peaks at 412 nm and 664 nm, assigned to the Soret band and the Q band of chlorophyll, respectively [32]. The spectrum of Chl/S quite resembled that of the Chl solution, as seen in Figure 1. The peak positions of the two bands were 416 nm and 664 nm, respectively. The similar spectral shape and the near-same wavelengths of the two bands of Chl/S and the Chl solution suggested that Chl was adsorbed on the silica surface without any change in its molecular structure. This result supports the possible utilization of the Chl composites as a colorant.

To investigate the influence of the addition of antioxidative reagents on the color of Chl/S, the diffuse-reflectance spectra of the composite samples before and after the addition of antioxidants were compared. The thick curves in the left and right panels of Figure 2 illustrate the spectra of Chl/S without and with the addition of VC, respectively (before irradiation). The spectral shapes of Chl/S and VC/Chl/S resembled each other. In addition, the two absorption peaks of VC/Chl/S were observed at 414 nm and 662 nm, indicating no significant wavelength shift by the addition of VC. It was confirmed that the addition of all other antioxidative agents (VE, LA, GA, and their derivatives) to Chl/S did not cause noticeable changes in the spectra. This means that the presence of those antioxidants caused no interference with the electronic transition of Chl responsible for the signals observed in the UV-Vis region. From these results, it was concluded that the addition of those antioxidants did not obstruct the utilization of Chl/S as a colorant.

3.2. Stabilization of the Adsorbed Chl by Various Antioxidative Agents

Figure 2 also illustrates the spectral changes in Chl/S and VC/Chl/S during visible light irradiation for 180 min. In both samples, the light absorption of Chl decreased along with the irradiation time. Although the difference was very small, the ratio of the degradation of Chl was found to be suppressed in VC/Chl/S to a small degree. It showed the effect of the addition of VC on Chl/S as an antioxidant.

To make clear the difference in the degradation of Chl during irradiation of Chl/S with various antioxidants, the retention ratio of the absorption value at the Q band (ca. 665 nm) is represented in Figure 3. The absorption values (A) were normalized to the initial value (A₀); the larger value of A/A₀ means a more stabilizing effect of Chl by the antioxidant.

From Figure 3, we can see that the stabilization effect of VC and LA was only slight, and that of VE was almost none, compared with the A/A₀ value of Chl/S without an antioxidant. The hydrophobicity can explain the reason why VE did not show any stabilization effect. As can be recognized from the molecular structure, VE is quite a hydrophobic compound. Although Chl also shows a hydrophobic nature mainly due to the phytyl groups in its structure, the hydrophobicity of VE results in hindrance from the chlorin ring part of Chl responsible for its green color. Since VE molecules cannot approach the chromophore part of the Chl molecules when adsorbed on the silica surface, VE cannot work effectively as an antioxidative reagent to protect the color of Chl. Similarly, even though the hydrophobicity may be lower than VE, LA is also a hydrophobic compound, so the stabilization effect was small on Chl/S.

On the contrary, GA exhibited a significantly higher stabilization effect than others. GA is rather hydrophilic, which urges the approach of GA near the chromophore part of the Chl molecules during the adsorption process on the silica surface. Therefore, the antioxidative effect was sufficiently seen on GA/Chl/S. However, VC did not show such a significant stabilization effect, even though VC is a hydrophilic compound. This result can be explained by the degree of hydrophilicity. The VC molecules are too hydrophilic, so the compatibility with rather hydrophobic Chl molecules is not so good, resulting in the adsorption at a separate position from the Chl on the silica surface. The hydrophilicity of LA, GA, and VC can be quantitatively compared by the partition coefficient between water and octanol (P_ow). The log(P_ow) value of LA, GA, and VC are reported as 2.1, −0.53, and −2.0, respectively [33,34,35]. Therefore, the largest stabilization effect was found to be obtained by the addition of the antioxidative agent with moderate hydrophilicity, which would match the hydrophilic property of the chromophore part of the Chl molecule.

Taking these into consideration, we concluded that GA was the superior antioxidant for the Chl adsorbed on the silica surface, because its hydrophilicity was appropriate to take a position near the chromophore part of the Chl molecule.

3.3. Effectiveness of Various Gallic Acid Derivatives as an Antioxidant

From the viewpoint of hydrophilicity, GA was found to be the most effective antioxidant for the Chl adsorbed on the silica surface. Because hydrophilicity was expected to be an important parameter for the effectiveness of the antioxidant, various gallic acid derivatives with different lengths of alkyl chains as a substituent were employed as the stabilizer for the adsorbed Chl. Figure 4 represents the stabilization effect of those GA derivatives on the adsorbed Chl under visible irradiation. All the GA derivatives exhibited a stabilization effect, since the retention ratio of the light absorption by Chl was superior with the addition of those GA derivatives. However, the effectiveness of the GA derivatives was quite different from each other. The most effective one was PG with no substituent other than three hydroxyl groups on the phenyl ring. The stability of GA/Chl/S was only slightly inferior to that of PG/Chl/S. Indeed, Chl is generally reported to be unstable under acidic conditions, but the existence of the carboxyl group on GA did not apparently influence the stability of Chl. The slight difference between PG and GA was possibly due to the difference in molecular size; as PG is smaller than GA due to the lack of a carboxyl group, PG can more easily access near the Chl molecule on the silica surface without strong steric hindrance. On the contrary, C2 and C12 showed a smaller stabilization effect on Chl. The large alkyl group of C2 and C12, in addition to the carboxyl group of GA, probably causes strong steric hindrance to the Chl molecule, resulting in a smaller stabilization effect on Chl. Since C2 and C12 have both hydrophilic and hydrophobic parts in their molecular structure, the affinity to the amphipathic Chl molecule would be fundamentally larger. However, the disadvantage deriving from the large steric hindrance by the alkyl chain canceled out the affinity, so that the C2 and C12 molecules could not be in touch with the Chl molecule adsorbed on the silica surface.

4. Conclusions

Taking the above results into consideration, we concluded that the degree of the approach of the antioxidative agent molecule to the chlorophyll molecule adsorbed on the silica surface should be a very important factor to exhibit enough of a stabilization effect. To access the adjacent position of the adsorbed chlorophyll molecule on the silica surface, an appropriate hydrophilicity of the antioxidant similar to the chlorophyll molecule would be advantageous. In addition, the molecular size of the antioxidant is also important. Large molecular sizes would interrupt the approach of the antioxidant to the chlorophyll due to steric hindrance, causing insufficient effectiveness as a stabilizer. Since they have a moderately hydrophilic nature together with a small molecular size, we propose gallic acid and pyrogallol as the best antioxidative stabilizers for the chlorophyll fixed on the silica surface. Between the two, gallic acid would be more recommendable because gallic acid is more nontoxic than pyrogallol. The information obtained through this study will contribute not only to the wide use of naturally occurring chlorophyll as a safe and stable green colorant but also to expanding the usage of the chlorophyll composite as various photo-functional materials.