Advances in Research of Extraction Processes and Anti-aging Effect of Flavonoids in Traditional Chinese Medicines

Yining MA, Liwei BI

1. Department of Pharmacy, Xiamen Medical College, Xiamen 3611023, China; 2. Xiamen Key Laboratory of Traditional Chinese Medicine Bioengineering, Xiamen 361023, China

Abstract Flavonoids are secondary metabolites in plants and possess extensive pharmacological activities. Flavonoids have remarkable effect in prevent anti-tumor and cancer, but flavonoids can’t be synthesized directly in the body. According to the different properties and structures of flavonoids in Traditional Chinese medicines, different extraction methods should be selected. Besides, flavonoids have good anti-aging effects. Based on three theories of modern aging, the anti-aging effect of flavonoids was discussed. It is expected to provide a beneficial reference for further development and utilization of natural flavonoid resources.

Key words Flavonoids, Extraction process, Anti-aging effect, Advances in research

1 Introduction

Flavonoids are secondary metabolites of plants and widely distributed in nature. In plants, most of them are combined with sugars as glycosides or carbosaccharides, and a few exist in free form. Flavonoids have anti-aging effects, and their active ingredients include polysaccharides, saponins, flavonoids and alkaloids,etc.[1]. They have a wide range of pharmacological activities, such as antioxidant, anti-aging, anti-tumor, antibacterial, anti-inflammatory, anti-cancer and regulation of vascular penetration[2]. Natural flavonoids contain many different substituent groups, their physical and chemical properties also have significant differences, so different extraction processes will affect the extraction efficiency of traditional Chinese medicine flavonoids, and accordingly affect the clinical efficacy. Anti-aging has become a research hot-spot issues in the fields of medicine, health and health products in recent years. Active components of flavonoids contained in Traditional Chinese medicine can delay the aging of nervous system, immune organs, reproductive system, liver and skin[3]. In this study, we compared and analyzed the advantages and disadvantages of different extraction methods of flavonoids in Traditional Chinese medicines, and reviewed the research on the anti-aging effect of flavonoids in Traditional Chinese medicines through three mechanisms.

2 Study on extraction processes of flavonoids from Traditional Chinese medicines

Flavonoids have high application value and have significant effects in anti-cancer and anti-tumor,etc.However, flavonoids cannot be synthesized directly in the human body and can only be obtained from food intake, so it is particularly important to study their extraction, separation and purification methods. According to the different properties and structures of flavonoids in Traditional Chinese medicines, extraction methods mainly include hot water extraction, solvent extraction, and ultrasonic extraction.

2.1 Hot water extractionFlavonoid glycosides have high solubility in hot water solution, and flavonoids are extracted according to their properties. The method is simple, the solvent is not toxic to human body, but the extraction time and subsequent purification process is long, and the extraction rate is not high.

2.2 Solvent extractionIn recent years, most flavonoids have been extracted with organic solvents. The reason is that flavonoids are easily soluble in organic solvents. By using its characteristics, flavonoids can be dissolved from plant tissues, and the higher the concentration of organic solvents, the better the extraction effect. However, there are a lot of alcohol-soluble impurities in plants, and too high concentration will easily increase the solubility of impurities, so the organic solvent should not be too high to avoid interference[4].

2.3 Ultrasonic-assisted extractionThis method mainly uses the strong vibration, high-speed cavitation effect and agitation generated by ultrasound to destroy plant tissue cells, so that the extraction solvent can quickly penetrate into the cells and accelerate the leaching of flavonoids. Ultrasonic-assisted extraction (UAE) has the advantages of high extraction rate, fast extraction speed and small loss of active components[5]. Using the ultrasonic-assisted extraction of flavonoids in jujube, Liu Xiuminetal.[6]constructed a DES system with a molar ratio of choline chloride and urea of 2∶1, adding 45% water, and a solid-liquid ratio of jujube and DES of 1∶40 (g/mL) at 70 ℃ for 44 min. The effect was the best, and the flavonoid yield reached 5.79 mg/g under these conditions.

2.4 Microwave-assisted extractionMicrowave-assisted extraction (MAE) is a new technique for extracting the active components of natural products in recent years. In this method, the plant cells expand rapidly through local heating, which causes the cell wall to burst and promotes the active components in the cells to penetrates into the solvent from the broken cells. The technology has fast, "green and clean" performance[7-8].

Zhang Lujieetal.[9]obtained the optimal extraction conditions by microwave-assisted extraction of total flavonoids fromChrysanthemumcoronariumleaf: microwave temperature at 73 ℃, microwave power at 400 W for 8 min, solid-liquid ratio at 30∶1 and volume fraction of 68% ethanol. Under these conditions, the extraction rate of total flavonoids was 0.55%.

2.5 Other extraction methodsMeng Qinghuanetal.[10]extracted total flavonoids from peony seed coat by enzyme-assisted ethanol, and found that the extraction rate of flavonoids by enzyme-assisted ethanol was higher than that by pure ethanol solvent extraction. Lu Xiaojianetal.[11]used supercritical carbon dioxide to extract dried tangerine peel’s polymethoxylated flavones. The optimum extraction conditions were obtained as follows: extraction pressure was 28 MPa, extraction temperature was 55 ℃, and extraction time was 67 min. Under these conditions, the total yield of polymethoxy flavonoids was 1.89 mg/g. Wang Jianhong[12]used internal boiling method to extract flavonoids from blood orange peel. The optimal extraction conditions were as follows: ethanol concentration was 60%, solid-liquid ratio was 1∶40 g/mL, extraction temperature was 70 ℃, extraction time was 30 min. Under these conditions, the yield of flavonoids was 6.234 mg/g.

3 Study on the anti-aging effect of flavonoids in Traditional Chinese medicines

Aging is a natural phenomenon in which the body’s function declines with age. Under the influence of heredity and environment, stress and homeostasis gradually decline, and the body’s structure and function appear irreversible decline[13-14]. Flavonoids in Traditional Chinese medicines have a good anti-aging effect. Researches on the anti-aging effect of flavonoids in Traditional Chinese medicines based on modern aging theory mainly include free radical theory, immune theory and inflammatory theory[15].

3.1 Delaying aging through antioxidantHypothesis, also called the oxidative damage of aging free radical theory, was put forward for the first time in 1956[16]. Free radicals induced damage accumulation according to the hypothesis leads to aging, follow-up, and oxidative stress hypothesis merger[17]. It is concluded that the body due to oxidation and antioxidant imbalance, cause the metabolism of reactive oxygen species (ROS) cannot eliminate and cause oxidative damage accumulation, Is one of the causes of aging and aging-related diseases.

Shi Haoetal.[18], using flavonoids from 0.3 mg /mLActinidiaargutato treat human immortalized epidermal cells with hydrogen peroxide damaged, and found that SOD activity in cells increased by about one time compared with model group, and there was a significant difference. Compared with model group, ROS level was significantly decreased by about 13%, indicating that flavonoids inActinidiaargutahad a certain pre-protective effect on oxidative damage caused by hydrogen peroxide. The results showed that flavonoids in plants could decrease the content of 8-OHDG in serum, increase the activities of SOD, GSH-Px, CAT, T-AOC and mitochondrial ATPASE in serum and organs, and significantly decrease the content of MDA and the activity of MAO. The expression levels of Nrf2 and ho-1 proteins in liver were improved to alleviate liver lesions in d-galactose aging model mice[19-20]. Liu Sitongetal.[21], by studying the effect of Genistein (Gen) on antioxidant effect of aging H9c2 cells induced by tert-butyl hydroperoxide (t-BHP) peroxide and its mechanism, found that Gen inhibited the senescence of H9c2 cells by increasing the antioxidant capacity of H9c2 cells, the mechanism of which may be related to the up-regulation of P53, P21 and Keap1 proteins and Nrf2 and HO-1 proteins. Transforming growth factor-β (TGF-β) can not only maintain the normal structure of vascular wall, but also have anti-inflammatory, anticancer and arterial protection effects. In TGF-β/Smad3 signaling pathway, Smad3 is a downstream TGF-β transduction factor, and its expression level increased beyond a certain value will lead to vascular smooth muscle cells, endometrial hyperplasia and other diseases[23-24]. Gai Liyueetal.[22], studied the effects of medium, high and low dose groups of total flavones of epimedium (TFE) on renal fibrosis in naturally aging rats and the possible mechanism of action respectively. The results showed that TFE could improve renal tissue morphology and reduce collagen fiber in aging SD rats. Meanwhile, the mRNA and protein expressions of TGF-β1 and Smad3 were significantly down-regulated to improve renal fibrosis in naturally aging rats.

3.2 Delaying aging through anti-inflammatoryInflamm-aging refers to the chronic progressive increase of pro-inflammatory response in the body during the natural aging process, and the cytokine theory holds that pro-inflammatory cytokines play a core role in the occurrence and development of inflammatory aging[25]. Inflammatory cytokines are soluble proteins or glycoproteins with high biological activity secreted by nerve, immune, endocrine and tissue cells, including IL, IFN, TNF, colony stimulating factor and transforming growth factor (TGF),etc.[26]. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases, which are important signal transduction enzymes involved in regulating cell apoptosis, cell proliferation, differentiation and aging[27]. Silencing information regulator 2 related enzyme 1 (SIRT1) is an NAD+ dependent deacetylase involved in physiological activities such as cell proliferation, metabolism and apoptosis. Studies[28]have shown that activation of SIRT1 signaling pathway can prolong biological life span, directly regulate NF-κB signaling pathway to participate in the regulation of aging process, and also delay aging by regulating mitochondrial function and reducing oxidative stress damage, which has a certain connection with the free radical theory and inflammatory theory involved in modern aging theory. Han Guifangetal.[29]found that total flavonoids of Herba medium can promote the expression of P-AMPK and SIRT1 proteins in the testicular tissue of naturally aging rats, significantly reduce the expression levels of acetyl-NFκBp65 and its downstream inflammatory factors IL-1β and TNFα, and reduce the inflammatory response in the testicular tissue of naturally aging rats. Fu Yiyietal.[30]found that flavonoids from sea buckthorn significantly increased phagocytosis and inhibited the production of NO, IL-6, TNF-α and COX-2 in Raw264.7 cells in a dose-dependent manner at 50-250 μg/mL.

3.3 Delaying aging by regulating immunityThe immune senescence hypothesis was first proposed by Walfordetal.[31], who believed that many diseases in the normal aging process of human and all animals are related to the disorder of the immune system with the increase of age, and the functions of the main immune cells in the immune system, such as T lymphocytes, B lymphocytes and macrophages, will be aging.

Xia Shijinetal.[32], found that NF-κB is involved in the regulation of immune aging in elderly rats by inhibiting apoptosis of lymphocytes. Total flavonoids of Epimedium may inhibit apoptosis of lymphocytes by increasing the activity of NF-κB and inducing the high expression of P65. Zeng Manhongetal.[33]found that rhodiola flavone can significantly increase the contents of SOD, GSH-Px and IL-2 in serum of aging rats, reduce the contents of MDA and IL-6, and significantly improve the indexes of thymus and spleen and proliferation of T and B lymphocytes to enhance antioxidant and immune functions and delay aging in aging rats. Yan Jianhuaetal.[35], through administering dendrobium officinale total flavonoids to d-galactose induced aging mice, found that the contents of HYP, typeⅠ collagen and type III collagen in skin tissues were higher than those in the model group, as well as the contents of thymus, spleen and kidney index, thus playing a certain protective role in skin aging of mice. Wang Mengetal.[34]found that TOTAL flavonoids ofAstragalusmembranaceuscould moderately activate NF-κB signaling pathway and inhibit overactivation of NF-κB signaling pathway by regulating the expression of key proteins in normal and LPS-stimulated RAW264.7 cells, respectively, and increase the content of cytokines and mRNA transcription levels in normal cultured RAW264.7 cells, and inhibit LPS-stimulated RAW264 cells.7 Excessive transcription of proinflammatory factor content and mRNA level in cells, thus exerting its bidirectional regulation of anti-inflammatory immunity.

4 Conclusions and prospects

There are many kinds of flavonoids and their physical and chemical properties vary greatly. Therefore, appropriate extraction methods are of great importance to ensure the effective composition of high content. At present, the extraction methods of flavonoids in Traditional Chinese medicine include thermal reflux extraction, ultrasonic extraction, supercritical extraction, microwave extraction,etc.These methods can improve the extraction rate of flavonoids in traditional Chinese medicine from different aspects. These methods lay a good foundation for further development of abundant flavonoid resources in China and promote full and reasonable utilization of natural flavonoid resources. Researchers at home and abroad have done a lot of work on the anti-aging effect of flavonoids in Chinese medicine, which will contribute to the research and development of health food, cosmetics and anti-aging drugs containing flavonoids in Chinese medicine, and even assist clinicians to treat some diseases related to aging, so as to slow down the aging process.