Is Sodium Copper Chlorophyllin Safe?

Chlorophyll is a natural pigment that is safe and has certain physiological functions. Modern research has found that chlorophyll can not only be used as a natural coloring agent in food or cosmetics, but also has important physiological activities, such as anti-mutagenic, cholesterol-lowering and constipation-relieving effects [1]. In addition, because the molecular structure of chlorophyll is very similar to that of human hemoglobin, with the only difference being the central ions, chlorophyll is also used to treat anemia [2].

In addition, chlorophyll can also promote the growth of normal red blood cells, increase the body's oxygen content, and promote cell division, thereby helping the body's metabolism [3]. However, chlorophyll is not soluble in water and is easily decomposed under light and certain temperature conditions, which limits its application. Studies have shown that the product formed by replacing the magnesium ion in the center of chlorophyll with metal ions such as copper, iron, and zinc is more stable and water-soluble than chlorophyll, and still has a similar color and physiological function as chlorophyll. Therefore, there has been a lot of research on chlorophyll derivatives, and the method of replacing the magnesium ion with a copper ion to form a copper sodium chlorophyll salt is the most widely used method [2].

This paper provides an overview of the structure and properties of sodium copper chlorophyllin, focusing on the preparation process. It also summarizes the problems and possible improvements, providing a theoretical basis for further research and the development of related products.

1 Structure and properties of sodium copper chlorophyllin

1.1 Structure of sodium copper chlorophyllin

The molecular formulas of chlorophyllin copper sodium salt are C34 H31 O6 N4 CuNa3 and C34 H30 O5 N4 CuNa2, and the relative molecular masses are about 724.17 and 684.16. Chlorophyllin copper sodium salt is a product prepared from chlorophyll through a series of reactions. Chlorophyll contains four pyrrole rings linked to each other by methylene groups (= C –), forming a stable conjugated system. A magnesium ion is bound to the centre of the conjugated system, and two esterified carboxyl groups are attached to the side chains of the conjugated system, which are esterified with methanol and phytol [4]. Sodium copper chlorophyllin is a product of saponification of chlorophyllin to remove phytol and methanol, and the replacement of Mg2+ by Cu2+ under acidic conditions. The molecular structures of chlorophyllin and sodium copper chlorophyllin are shown in Figure 1.

1.2 Properties of sodium copper chlorophyllin

Sodium copper chlorophyllin is easily soluble in water, slightly soluble in alcohols, and insoluble in oils, fats and petroleum ether. It is dark green in color and in powder form. Its aqueous solution is transparent blue-green. If Ca2+ is present, it will precipitate. Scanning with UV-Vis spectroscopy shows that there are maximum absorption peaks in the wavelength ranges 405 nm ± 3 nm and 630 nm ± 3 nm.

2 Functional applications of sodium copper chlorophyllin

2.1 Food applications

Sodium copper chlorophyllin is approved for use in products such as jelly, canned vegetables, candies, beverages, fruit and vegetable juice drinks, baked goods, prepared wines, etc. [5].

2.2 Medical applications

Studies have found that sodium copper chlorophyllin has the effect of protecting and promoting the liver, and can also treat jaundice and other diseases [6]; moreover, sodium copper chlorophyllin can enhance hematopoietic function, promote the production of hemoglobin, and treat symptoms such as anemia [7]; sodium copper chlorophyllin has the effect of regulating oral microorganisms, preventing tooth decay and periodontitis, and eliminating bad breath in the mouth and respiratory tract [8]. In addition, sodium copper chlorophyllin is also used to treat eczema, frostbite, acute pancreatitis and other diseases [9].

2.3 Dyeing applications

Sodium copper chlorophyllin can be used for dyeing and is an environmentally friendly and energy-saving dye. Using it for dyeing not only makes full use of biomass resources, but also conforms to the current concept of pursuing ecological and environmental protection. Wang Na, Yang Ruiling et al. [10-11] found through research that sodium copper chlorophyllin dye is suitable for dyeing wool, silk and nylon under acidic conditions, and the color fastness of these materials after dyeing with sodium copper chlorophyllin can also reach level 3 or above.

2.4 Other applications

Ruan [12] found that all-solid-state supercapacitors with chlorophyllin copper sodium salt electrodes have good bending ability and flexibility. This also shows the potential application of chlorophyllin copper sodium salt in all-solid-state supercapacitors.

3 Research on the preparation process of sodium copper chlorophyllin

The preparation of sodium copper chlorophyllin includes the extraction of chlorophyll and the use of chlorophyll to prepare sodium copper chlorophyllin.

3.1 Extraction of chlorophyll

Studies have shown that the methods for extracting chlorophyll mainly include solvent extraction, ultrasonic-assisted extraction, supercritical fluid extraction, and other methods [13]. The most commonly used method is solvent extraction. This method is based on the principle of like dissolves like. The more similar the chemical properties of the extraction solvent and the extracted substance, the greater the solubility of the extract in the solvent and the easier it is to extract. Chlorophyll contains a hydrophilic porphyrin group and a lipophilic chlorophyllol structure [13].

The lipophilic chlorophyllin has 20 carbon atoms, and the longer carbon chain determines its low polarity, strong lipophilicity and weak hydrophilicity. On the contrary, the polar porphyrin structure enhances its polarity. Therefore, the best solvent for extracting chlorophyllin is a moderately polar organic solvent, such as acetone, ethanol, ether, etc. The polarities of common solvents are shown in Table 1 below.

Yang Jun [14] experimentally compared the effect of more than ten solvents such as 100% anhydrous ethanol and 100% acetone on the extraction rate of chlorophyll. The result was that the best extraction solvent was a mixture of acetone and anhydrous ethanol (1:2, v/v) with a mass fraction of 85%. The mixed solvent has a better extraction effect than a single solvent, which can be considered as a synergistic extraction effect. It can also be considered that the properties of the mixed solvent are more similar to those of the extract, resulting in a higher extraction rate. Although the mixed solution of acetone and other solvents has a high chlorophyll extraction rate, acetone has a low flash point, is explosive and highly volatile, and is therefore dangerous to use on a large scale in industrial applications. Therefore, it is necessary to replace acetone with a reagent that is highly safe and low in toxicity as the solvent for extracting chlorophyll. Ethanol is low in volatility, low in toxicity and highly safe, and it has a high extraction rate for chlorophyll, making it the best reagent for industrial chlorophyll extraction.

Chlorophyll is located between the protein and lipid bilayer of the chloroplast. The hydrophilic porphyrin group is linked to the protein, while the hydrophobic chlorophyllol is linked to the lipid layer. When extracting chlorophyll, the addition of a small amount of water is beneficial for separating the hydrophilic group of chlorophyll from the protein, thereby facilitating the extraction of chlorophyll. Fang Jiayang [15] found that the maximum extraction rate of chlorophyll was 12.8 g/kg when the ethanol-to-water concentration ratio was 4:1. The extraction rate decreased when 100% ethanol was used.

Supercritical fluid extraction technology is a new separation technology that has low operating temperatures, high separation efficiency and high solvent recovery rates. It has been used in recent years to extract the active ingredients of plants and Chinese herbal medicines. Lefebvre [16] found that chlorophyll can be obtained by adding 30% polar modifier to carbon dioxide by supercritical fluid extraction.

Ultrasonic-assisted extraction technology is also often used in separation and extraction. The cavitation effect of ultrasonic vibrations can promote cell lysis, thereby facilitating solvent extraction. Choi [17] showed that the extraction rate of chlorophyll using ultrasonic-assisted extraction was higher than that using organic solvents.

3.2 Preparation of sodium copper chlorophyllin

The preparation of sodium copper chlorophyllin involves four reaction steps: saponification, acidification, copper substitution and salt formation. In addition, because the chlorophyll content in the raw material is extremely low, it will contain many impurities after extraction. Therefore, in addition to these necessary reaction steps, a purification and impurity removal step is also added. In fact, the existing process has certain disadvantages, such as incomplete saponification, “green loss” during copper substitution, poor purification results, and low product quality. Therefore, further improvements are needed.

3.2.1 Principle of sodium copper chlorophyllin preparation (1) Saponification

The two ester groups on the chlorophyllin molecule react with sodium hydroxide to form a saponification reaction, which removes phytol and methanol to form a water-soluble sodium chlorophyllin salt (using chlorophyllin a as an example, see Figure 2).

(2) Acidification

In an acidic environment, the hydrogen ions replace the magnesium and sodium ions in the sodium chlorophyllin salt to form chlorophyllic acid and magnesium and sodium sulfates (see Figure 3).

(3) Copper generation

In an acidic medium, a certain amount of CuSO4 solution is added, and the hydrogen ions in the chlorophyll molecule are replaced by copper ions to form the dark green copper chlorophyllin (see Figure 4).

(4) Salt formation

Dissolve chlorophyllin copper acid and react with sodium hydroxide solution to obtain a water-soluble sodium copper chlorophyllin salt (see Figure 5).

3.2.2 Problems and improvements in the preparation process

The degree of saponification of chlorophyllin not only affects the progress of the copper substitution reaction, but also affects the yield, color and texture of the sodium copper chlorophyllin salt. Some studies have explored the effect of pH on the saponification reaction and concluded that the optimal saponification conditions are pH = 11 or 12 [3, 18-21]. However, most pH meters and pH test strips on the market are only suitable for use in aqueous solutions, while the extraction solvent for chlorophyll is a high concentration of organic reagents such as ethanol and acetone. Under these conditions, the measured pH value is subject to certain deviations and is not stable. Therefore, the saponification reaction should be explored based on the actual amount of NaOH added, not just the pH value.

Chlorophyll is a fat-soluble substance that can be dissolved in petroleum ether before saponification. After saponification, the water-soluble sodium chlorophyllate is formed and is insoluble in petroleum ether. Therefore, after the saponification reaction, petroleum ether is added for extraction, and the completeness of the saponification reaction can be predicted by the layering and state of the petroleum ether layer. The reaction is complete when the two phases separate clearly and the ether layer is yellow [21].

When acidifying sodium copper chlorophyllin, many studies add a certain concentration of sulfuric acid to the sodium chlorophyllin solution to adjust the pH to about 2.5, and then add copper sulfate after reacting for a certain period of time [22-24]. In fact, directly adjusting the pH to 2.5 may destroy the porphyrin structure of the sodium salt of chlorophyll, which causes the resulting copper chlorophyllate to lose its green color and further affects the quality of the sodium copper chlorophyllate. The purpose of acidification is to make the replacement of copper easier and more convenient. Acidification also avoids the reaction of copper sulfate with sodium hydroxide to form other substances such as copper hydroxide. Therefore, when acidifying the copper substitute, the pH is first adjusted to neutral, an appropriate amount of copper sulfate is added to react, and then the solution is adjusted to 2.5. This can prevent the destruction of the porphyrin structure in the sodium salt of chlorophyll that may be caused by a too acidic environment.

Since chlorophyll is present in very small amounts in the raw materials, there are relatively many impurities after extraction, so a purification step is necessary. In the preparation of sodium copper chlorophyllin, a saponification reaction is carried out to form sodium chlorophyllin, which is then added to petroleum ether for solvent extraction. The purpose is to remove fat-soluble substances such as fat, carotene, lutein and phytol in order to obtain a higher product quality [25]. During solvent extraction, the greater the difference in the partition coefficients of the components in the two-phase solvent, the better the separation effect and the higher the impurity removal rate.

The traditional process is to carry out a saponification reaction in ethanol to form a sodium chlorophyllin salt, and then use petroleum ether to extract and remove impurities. In fact, the effect of solvent extraction using an ethanol-petroleum ether two-phase system is poor, because some lipophilic impurities also have a high solubility in ethanol, which makes the impurity removal effect unsatisfactory. If the ethanol is recovered, the sodium salt of chlorophyll is only soluble in water, and the polarity difference between water and petroleum ether is large, so a better decontamination effect can be obtained. In addition, multiple extractions with a single solvent can only remove a small amount of impurities that are highly soluble in the solvent. However, multiple extractions with reagents of different polarities can separate and extract a variety of impurities, thereby enhancing the decontamination effect. Therefore, 3 to 4 solvents of different polarities, such as ethyl acetate, butanol, chloroform, and petroleum ether, are used for solvent extraction. The aqueous sodium chlorophyll solution is extracted stepwise from low polarity to high polarity to remove impurities with different polarities.

In addition, after the copper-substitution reaction to form copper chlorophyllate, impurities are removed by washing with water, low-concentration alcohol, petroleum ether, etc. Washing with water can remove excess water-soluble impurities such as sodium ions and copper ions. Washing with low-concentration alcohol can remove unsaponified polar substances, and washing with petroleum ether can remove fat-soluble impurities. Finally, the crude copper chlorophyllate is washed to form a dark green, loose, granular, high-quality product with a metallic luster. These steps are also used to purify and remove impurities to obtain a high-quality product.

4 Summary and outlook

Currently, due to increased awareness of food safety, many synthetic pigments have been banned, and safe, natural products are more popular, thus providing good opportunities for the development of the natural pigment market. Sodium copper chlorophyllin, as a safe natural pigment, can not only be added to food as a coloring agent, but also has good effects and applications in medicine. However, due to the low chlorophyll content in the raw material, there are many impurities after extraction, and there are also certain defects in the existing preparation process, which results in the low quality of most of the sodium copper chlorophyllin products sold on the market. Therefore, there is an urgent need to improve the preparation process and purification method of sodium copper chlorophyllin from different perspectives.

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