What Is Sodium Copper Chlorophyllin Powder?

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 different central ions, chlorophyll is also used to treat anemia [2]; in addition, chlorophyll can also increase 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 chlorophyll center 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 have been a large number of studies on chlorophyll derivatives, among which the use of copper ions to replace magnesium ions to form copper chlorophyll sodium 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 formula of copper sodium chlorophyll is C₃4H₃₁O₆N₄CuNa₃ and C34H₃₀O₅N₄CuNa₂, with a relative molecular mass of approximately 724.17 and 684.16. Copper sodium chlorophyll is a product prepared from chlorophyll using 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 center 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, which involves the removal of 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 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 jelly, canned vegetables, confectionery, beverages, fruit and vegetable juice drinks, baked goods, prepared wines, and other products [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 anemia and other symptoms [7]; sodium copper chlorophyllin has the effect of regulating oral microorganisms, preventing and treating 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 conditions [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 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 a full solid-state supercapacitor with chlorophyllin copper sodium salt electrodes has good bending ability and flexibility. This also shows the potential application of chlorophyllin copper sodium salt in full solid-state supercapacitors.

3 Research on the preparation process of chlorophyllin copper sodium salt

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

3.1 Chlorophyll extraction

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 substance to be extracted, 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 long 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 acetone-anhydrous ethanol (1:2, v/v) mixture with a mass fraction of 85% was the best extraction solvent. 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 higher 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 industry. Therefore, it is necessary to replace acetone with a reagent that is highly safe and low in toxicity as the chlorophyll extraction solvent. 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 lipophilic phycobilin is linked to the lipid bilayer. When extracting chlorophyll, adding a small amount of water is beneficial for separating the hydrophilic groups of chlorophyll from the protein, thereby facilitating the extraction of chlorophyll. Fang Jiayang [15] found that the chlorophyll extraction rate is greatest when the ethanol to water concentration ratio is 4:1 – 12.8 g/kg. When 100% ethanol is used to extract chlorophyll, the extraction rate decreases.

Supercritical fluid extraction technology is a new type of separation technology with low operating temperatures, high separation efficiency and high solvent recovery rates. In recent years, it has been applied to the extraction of active ingredients from plants and Chinese herbal medicines. Lefebvre [161] 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 chlorophyll includes 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 Preparation principle of sodium copper chlorophyllin

(1) Saponification

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

(2) Acidification

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

(3) Copper substitution

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 chlorophyll acid (see Figure 4).

(4) Salt formation

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

3.2.2 Problems and improvements in the preparation process

The degree of saponification of chlorophyll not only affects the progress of the copper substitution reaction, but also the yield, color and texture of the sodium copper chlorophyllinate. 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 currently 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. Therefore, the saponification reaction should be explored based on the actual amount of NaOH added, and not just the pH value. Chlorophyll is fat-soluble and can be dissolved in petroleum ether before saponification. After saponification, the water-soluble sodium salt of chlorophyll 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. A clear separation of the two phases and a yellowish appearance of the petroleum ether layer indicate a complete reaction [21].

When acidifying sodium copper chlorophyllin, many studies add a certain concentration of sulfuric acid to the sodium chlorophyllin aqueous solution to adjust the pH to about 2.5, and then add copper sulfate solution 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 sodium chlorophyllin, causing the resulting copper chlorophyllin to lose its green color and affecting the quality of the sodium copper chlorophyllin. 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 replacement, 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 copper sodium chlorophyllin, the 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]. In 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 involves saponification in ethanol to produce sodium chlorophyllin, and then extraction with petroleum ether to remove impurities.

In fact, the use of ethanol-petroleum ether two-phase solvent extraction is not very effective, 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 that solvent. However, multiple extractions with various reagents of different polarities can separate multiple impurities and thus enhance the decontamination effect. Therefore, solvent extraction with 3 to 4 solvents of different polarities, such as ethyl acetate, butanol, chloroform, and petroleum ether, is used. The sodium salt of chlorophyll aqueous 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 colors 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.

References

[1] Wang Min, Liu Linwei. Research progress of chlorophyll and derivatives and analysis of green protection technology [J]. Journal of Zhengzhou Institute of Light Industry, 2001(1): 63-67.

[2] Liu Taoli. Stepwise preparation of pectin, zinc sodium chlorophyllin and leaf protein from silkworm excrement [D]. Nanning: Guangxi University, 2014.

[3] Liu Dan. Preparation of sodium copper chlorophyllin from microalgae and extraction and separation of other bioactive substances [D]. Nanchang: Nanchang University, 2014.

[4]Rodriguez-Amaya,Delia B,Natural  food pigments and colorants [J].Current Opinion in Food Science,2016, 7:20-26.

[5] GB 26406-2011, Food safety national standard – Food additive – Sodium copper chlorophyllin [S].

[6]Mahmoud Y   I,Shehata   AMM,Fares N   H,et   al.Spir- ulina  inhibits  hepatocellular  carcinoma  through  activating p53  and  apoptosis  and  suppressing  oxidative  stress and angiogenesis [J].Life Sci,2021,265:118827.

[7] Wang Shoujun, Wei Kemin. Effect of sodium copper chlorophyllin combined with traditional Chinese medicine on T lymphocyte subsets in immune-mediated aplastic anemia mice [J]. Chinese Journal of Traditional Chinese Medicine Science and Technology, 2013(20):618-619.

[8] Luo Huaiyu. Research on Sodium Copper Chlorophyllin Fresh Breath Toothpaste [J]. Oral Care Products Industry, 2014(24):18-19.

[9]Kunihara Mineo,Kanbayashi Miyuki,Ohshima Takao. Opposite effects of morphine on feeding and drinking in rats  relative  to  administration  time   [J].Japanese  Journal of  Pharmacology,1983,33:829-835.

[10] Wang Na, Du Yuanyuan, Huang Haidong, et al. Dyeing of wool, silk and nylon knitted fabrics with sodium copper chlorophyllin [J]. Knitting Industry, 2020(10):37-40.

[11] Yang Ruiling. Research on the Mechanism and Properties of Dyeing Silk Fabrics with Sodium Copper Chlorophyllin [D]. Wuxi: Jiangnan University, 2012.

[12]Ruan  Kaibin,Hu Qichang,Wang Yuzhu;et al.Allsolid-state flexible supercapacitors based  on sodium copper chlorophyllin with ultrahigh  rate  capability [J].Materials  Letters,2019,236:383-386.

[13] Li N. Study on the extraction of chlorophyll from filter mud in sugar cane sugar factory and the preparation of zinc sodium chlorophyllin [D]. Dalian: Dalian Polytechnic University, 2014.

[14] Yang J, Cao JX, Yang WH, et al. Study on the extraction of chlorophyll from spirulina by cell disruption [J]. Science, Technology and Innovation, 2019(17):7-9.

[15] Fang Jiaxiang, Li Yuebin, Qiu Qinglian, et al. Study on the process of ultrasonic-assisted extraction of chlorophyll a from spirulina powder [J]. Journal of Food Safety and Quality Testing, 2016(7):4198-4202.

[16]Lefebvre T,Destandau E,Lesellier E.Sequential extraction of carnosic  acid,rosmarinic  acid  and  pigments (carotenoids and  chlorophylls)from Rosemary  by  online supercritical fluid extraction-supercritical fluid chromatography[J].J Chromatogr A,2021,1639:461709.

[17]Choi Woon,Lee Hyeon.Enhancement of Chlorophyll a Production  from  Marine  Spirulina  maxima  by   an  Optimized  Ultrasonic Extraction Process  [J].Applied    Sci- ences,2017,8:26-36.

[18] Ding Huanxing. Research on the chemical composition of lotus seed sugar and the preparation process of copper sodium chlorophyll [D]. Lanzhou: Lanzhou University, 2012.

[19] Han Yaoling. Comprehensive utilization of sisal [D]. Nanning: Guangxi University, 2004.

[20] Yang Guizhi. Study on the process and stability of extracting and preparing sodium copper chlorophyllin from seaweed [D]. Tianjin: Tianjin University of Science and Technology, 2005.

[21] Luan Qianqian. Study on the separation and purification of effective components in fresh tobacco leaves [D]. Dalian: Dalian University of Technology, 2018.

[22] ZHONG Yali. Study on the preparation of sodium ferric chlorophyllin [D]. Xi'an: Shaanxi University of Science and Technology, 2014.

[23] WEN Xing. Study on the color protection process of endive and the preparation of sodium ferric chlorophyllin [D]. Xi'an: Shaanxi University of Science and Technology, 2013.

[24] Liu Ling. Preparation of sodium copper chlorophyllin from silkworm excrement and extraction of its active ingredients [D]. Nanning: Guangxi University, 2007.

[25] Yin Teng. Study on the extraction technology of natural pigments from cyanobacteria in Taihu Lake [D]. Wuxi: Jiangnan University, 2010.