Antioxidant Effects of Apocynum venetum Tea Extracts on d-Galactose-Induced Aging Model in Mice

As a traditional Chinese medicinal drink, Apocynum venetum, a local tea from Xinjiang, China, is favored for its rich flavor and biological functionality. This study looked at aging mice induced by d-galactose to determine the in vivo anti-aging effect of Apocynum venetum tea extracts (AVTEs) and its bioactive components. We evaluated the weight of major organs (via organ index) and pathological changes in the liver. We also detailed the effects of AVTE (250 mg/kg in the low dose group, 500 mg/kg in the high dose group) on biochemical parameters (malondialdehyde, superoxide dismutase, glutathione, glutathione peroxidase, catalase, total antioxidant capacity, and nitric oxide) and cytokines (IL-6, IL-12, TNF-α and IL-1β) in the serum of aging mice. We investigated the anti-aging effects of AVTE in d-galactose-induced aging mice via quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) assay. In addition, we analyzed the biological components of AVTEs by high performance liquid chromatography (HPLC). The results were remarkable, suggesting that AVTE significantly improved d-galactose-induced aging mice, with the high dose group showing the best results among other groups. ATVE can effectively alleviate hepatocyte edema, as well as inflammatory cell infiltration and injury in mice, induce a protective effect via up-regulation of glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) antioxidant related factors, and play an important role in the up-regulation of anti-inflammatory factors (IL-10) and the down-regulation of pro-inflammatory factors (IL-6, TNF-α and IL-1β). At the same time, HPLC analysis showed that AVTEs contain neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, isoquercitrin, isochlorogenic acid B, isochlorogenic acid A, astragalin, isochlorogenic acid C, rosmarinic acid, and trans-cinnamic acid. Thus, AVTE appears to be an effectively functional drink due to its rich functional components and anti-aging activities.


Introduction
Senescence mainly refers to the degradation due to age of the cell, tissue, and organ structure and function, a process gradually increasing the body's vulnerability to death, and can be caused by many factors, including genetics, environment, and diet [1,2]. At present, the mechanism of aging is not clear. Table 1. The sequences of reverse transcription-polymerase chain reaction primers.

HPLC Analysis
The AVTE was dissolved in dimethyl sulfoxide (DMSO, for HPLC, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to obtain a solution with a concentration of 10 mg/mL and diluted with 50% methanol to produce a final concentration of 2.5 mg/mL. The sample was passed through a 0.22-µm organic filter before testing. About 5 µL of the diluted AVTE sample solution was analyzed using an UltiMate3000 HPLC System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and separated using an Accucore C18 column (2.6 µm, 4.6 × 150 mm; Thermo Fisher Scientific, Inc., USA). Mobile phase A was water containing 0.5% acetic acid, and mobile phase B was acetonitrile. The flow rate was 0.5 mL/min with a column temperature of 30 • C, and the detection wavelength was 328 nm. The gradient elution conditions were as follows: Equilibrium stage was set for 10 min with 12% B (isocratic) then, 0-30 min with 12%-45% B (linear gradient), 30-35 min with 45%-100% B (linear gradient), and 35-40 min with 100% B (isocratic).

Statistical Analysis
The SPSS version 20.0 statistical software (SPSS Inc., Chicago, Illinois, USA) was used to analyze the experimental data. The results were analyzed using one-way analysis of variance (ANOVA) with Duncan's multiple range tests, and p < 0.05 was considered statistically significant. All experiments were repeated 3 times, and the data were presented as the mean ± standard deviation (SD).

Organ Coefficient
The organ coefficient can directly reflect the structural changes of the organ and indirectly reflect changes in organ function. Therefore, observing the changes of the organ coefficient in the mice had important reference value for judging mouse aging [20]. Compared with the normal group (Table 2), the coefficients of cardiac, liver, splenic, and kidney tissue in the aging model group decreased to a certain extent (p < 0.05). After treatment with different doses of AVTE, the coefficient of cardiac, liver, splenic, and kidney tissue were reduced to a certain extent, the degeneration of the organs in the mice was delayed, and the atrophy of the organs was antagonized to a certain extent [21].

Histological Analyses
The liver plays an important role in the detoxification of potentially harmful chemicals and the regulation of a stable internal environment. It was found that the morphology of the liver tissue also changed during aging in the mice [22]. As shown in Figure 1, the liver cells of the normal group were regular in morphology and uniform in size and staining, while the hepatocyte cords were arranged in an orderly manner with clear boundaries and distributed radially around the central vein. In the aging group, the liver cells were not arranged regularly, the staining was uneven, the central venous traits were irregular, the hepatic cell lines were disordered, and the boundaries were unclear. Additionally, the cells were swollen, the cytoplasm was loose, and the cells showed some balloon-like changes, inflammatory cell infiltration, and some cell necrosis. After treatment with AVTE (250 and 500 mg/kg), the liver cells of the mice were again arranged in an orderly manner, with slight changes in morphology, while the hepatocyte edema, necrosis, and inflammation were all reduced [16]. central vein. In the aging group, the liver cells were not arranged regularly, the staining was uneven, the central venous traits were irregular, the hepatic cell lines were disordered, and the boundaries were unclear. Additionally, the cells were swollen, the cytoplasm was loose, and the cells showed some balloon-like changes, inflammatory cell infiltration, and some cell necrosis. After treatment with AVTE (250 and 500 mg/kg), the liver cells of the mice were again arranged in an orderly manner, with slight changes in morphology, while the hepatocyte edema, necrosis, and inflammation were all reduced [16].

MDA, SOD, GSH, GSH-Px, CAT, T-AOC, and NO Concentrations in Serum
The activity of the antioxidant enzymes in the body decreases as the body ages while the accumulation of oxygen increases from base levels, eventually leading to abnormal cell structure and functional degeneration, resulting in a series of diseases [23]. As shown in Figure 2, compared with the normal group, the MDA and NO content in the serum of the model group mice were significantly increased with a difference that was statistically significant (p < 0.05). Compared with the model group, the MDA and NO content in the serum of the mice treated with the AVTE intervention groups (250 and 500 mg/kg) showed a statistically significant (p < 0.05) decrease. Antioxidant enzymes (such as SOD, CAT, GSH-Px) can convert peroxides into low toxic substances or non-toxic water through redox action [8]. As shown in Figure 3, compared with the normal group, the SOD, GSH, GSH-Px, CAT, and T-AOC in the serum of the model group mice were significantly decreased (p < 0.05). Compared with the model group, after treatment with

MDA, SOD, GSH, GSH-Px, CAT, T-AOC, and NO Concentrations in Serum
The activity of the antioxidant enzymes in the body decreases as the body ages while the accumulation of oxygen increases from base levels, eventually leading to abnormal cell structure and functional degeneration, resulting in a series of diseases [23]. As shown in Figure 2, compared with the normal group, the MDA and NO content in the serum of the model group mice were significantly increased with a difference that was statistically significant (p < 0.05). Compared with the model group, the MDA and NO content in the serum of the mice treated with the AVTE intervention groups (250 and 500 mg/kg) showed a statistically significant (p < 0.05) decrease.
Antioxidants 2019, 8 6 of 16 central vein. In the aging group, the liver cells were not arranged regularly, the staining was uneven, the central venous traits were irregular, the hepatic cell lines were disordered, and the boundaries were unclear. Additionally, the cells were swollen, the cytoplasm was loose, and the cells showed some balloon-like changes, inflammatory cell infiltration, and some cell necrosis. After treatment with AVTE (250 and 500 mg/kg), the liver cells of the mice were again arranged in an orderly manner, with slight changes in morphology, while the hepatocyte edema, necrosis, and inflammation were all reduced [16].

MDA, SOD, GSH, GSH-Px, CAT, T-AOC, and NO Concentrations in Serum
The activity of the antioxidant enzymes in the body decreases as the body ages while the accumulation of oxygen increases from base levels, eventually leading to abnormal cell structure and functional degeneration, resulting in a series of diseases [23]. As shown in Figure 2, compared with the normal group, the MDA and NO content in the serum of the model group mice were significantly increased with a difference that was statistically significant (p < 0.05). Compared with the model group, the MDA and NO content in the serum of the mice treated with the AVTE intervention groups (250 and 500 mg/kg) showed a statistically significant (p < 0.05) decrease. Antioxidant enzymes (such as SOD, CAT, GSH-Px) can convert peroxides into low toxic substances or non-toxic water through redox action [8]. As shown in Figure 3, compared with the normal group, the SOD, GSH, GSH-Px, CAT, and T-AOC in the serum of the model group mice were significantly decreased (p < 0.05). Compared with the model group, after treatment with Antioxidant enzymes (such as SOD, CAT, GSH-Px) can convert peroxides into low toxic substances or non-toxic water through redox action [8]. As shown in Figure 3, compared with the normal group, the SOD, GSH, GSH-Px, CAT, and T-AOC in the serum of the model group mice were significantly decreased (p < 0.05). Compared with the model group, after treatment with AVTE (250 and 500 mg/kg), the levels of antioxidants in the serum of the mice were significantly improved, and SOD GSH, GSH-Px, CAT, and T-AOC were significantly (p < 0.05) increased.
Antioxidants 2019, 8 7 of 16 AVTE (250 and 500 mg/kg), the levels of antioxidants in the serum of the mice were significantly improved, and SOD GSH, GSH-Px, CAT, and T-AOC were significantly (p < 0.05) increased.

IL-6, IL-10, TNF-α and IL-1β Concentrations in Serum
Reactive oxygen species in the mice were activated in large amounts, which induced the expression of various cytokines such as tumor necrosis factor (TNF-α) and interleukins (IL-6, IL-10, and IL-1β) [24]. As shown in Figure 4, compared with the normal group, the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of model group (D-galactose induced) increased (p < 0.05), especially the decrease of TNF-α, it can promote the production of various inflammatory factors by T cells, which is the key link of various signaling pathways [12]. In contrast, the expression of anti-inflammatory factor IL-10 decreased (p < 0.05). Compared with model group, low dose AVTE group (250 mg/kg) and high dose AVTE group (500 mg/kg) all had down-regulation effect on TNF-α, IL-6, and IL-1β, but up-regulation effect on IL-10, and the effect of the high dose AVTE group was more significant (p < 0.05), indicating that AVTE could reduce the expression of inflammation in the mice.

IL-6, IL-10, TNF-α and IL-1β Concentrations in Serum
Reactive oxygen species in the mice were activated in large amounts, which induced the expression of various cytokines such as tumor necrosis factor (TNF-α) and interleukins (IL-6, IL-10, and IL-1β) [24]. As shown in Figure 4, compared with the normal group, the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of model group (d-galactose induced) increased (p < 0.05), especially the decrease of TNF-α, it can promote the production of various inflammatory factors by T cells, which is the key link of various signaling pathways [12]. In contrast, the expression of anti-inflammatory factor IL-10 decreased (p < 0.05). Compared with model group, low dose AVTE group (250 mg/kg) and high dose AVTE group (500 mg/kg) all had down-regulation effect on TNF-α, IL-6, and IL-1β, but up-regulation effect on IL-10, and the effect of the high dose AVTE group was more significant (p < 0.05), indicating that AVTE could reduce the expression of inflammation in the mice. Antioxidants 2019, 8 8 of 16

Effects of AVTE on the Gene Expression of SOD1, SOD2, GSH-Px, and CAT in the Liver of Aging Mice (RT-qPCR Assay)
As shown in Figure 5, the expression levels of SOD1, SOD2, GSH-Px, and CAT in the D-galactose-induced aging model group were significantly lower than those in the normal group. After treatment with different concentrations (250, 500 mg/kg) of AVTE, the expression of SOD1, SOD2, GSH-Px, and CAT in liver tissue was significantly improved, and the high dose of AVTE had the best effect (p < 0.05).

Analysis of the Chemical Composition of AVTE
HPLC analysis of the AVTE is shown in Figure 6A. Compared with the retention time of the chemical standards ( Figure 6B), 11 peaks were identified from the AVTE, including neochlorogenic acid (peak 1, 3.743 min), chlorogenic acid (peak 2, 4.947 min), cryptochlorogenic acid, (peak 3, 5.270 min), rutin (peak 4, 10.273 min), isoquercitrin (peak 5, 11.213 min), isochlorogenic acid B (peak 6, As shown in Figure 5, the expression levels of SOD1, SOD2, GSH-Px, and CAT in the d-galactose-induced aging model group were significantly lower than those in the normal group. After treatment with different concentrations (250, 500 mg/kg) of AVTE, the expression of SOD1, SOD2, GSH-Px, and CAT in liver tissue was significantly improved, and the high dose of AVTE had the best effect (p < 0.05).

Effects of AVTE on the Gene Expression of SOD1, SOD2, GSH-Px, and CAT in the Liver of Aging Mice (RT-qPCR Assay)
As shown in Figure 5, the expression levels of SOD1, SOD2, GSH-Px, and CAT in the D-galactose-induced aging model group were significantly lower than those in the normal group. After treatment with different concentrations (250, 500 mg/kg) of AVTE, the expression of SOD1, SOD2, GSH-Px, and CAT in liver tissue was significantly improved, and the high dose of AVTE had the best effect (p < 0.05).

Discussion
Many kinds of plants have been used as tea drinks in China since ancient times. Traditionally, "tea" refers to the buds, tender leaves, or leaves of the plants from the Camellia genus of the Theaceae family, while non-Camellia tea consists of different parts of the plants from different families and genera [25]. According to the degree of fermentation, the biologically active ingredients of tea vary greatly, but the main active ingredient in tea is polyphenol, a chemical known to act as a preventive agent against malignant tumors and have strong free radical scavenging and reducing activity. Non-fermented tea (e.g., green tea) has a higher polyphenol content, causing astringency and irritation to the stomach. Total fermented tea (e.g., black tea) has a greater loss of polyphenols, but its components are more complex (e.g., aromatic components), causing a better effect on stomach maintenance [26]. Studies have shown that Apocynum venetum, a non-Camellia tea, contains phenolic acids, flavonoids, amino acids, and other chemicals that have been shown to lower blood pressure, blood lipids, and blood sugar levels [15], have antioxidant and anti-inflammatory properties [16], and anti-cancer properties [17].
In this study, eleven bioactive components including chlorogenic acid and its five isomers were detected in AVTE by HPLC. Chlorogenic acids (CGAs) are phenolic acids with vicinal hydroxyl groups on aromatic residues, derived from the esterification of trans-cinnamic acids (including caffeic, ferulic and p-coumaric acids) with quinic acid. There are several CGA subgroups, including neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. These compounds have many pharmacological actions, such as scavenging free radicals, lowering blood pressure and blood lipids, and protecting the liver and gallbladder. CGAs are abundant in the human diet, and epidemiological studies have shown that consumption of tea, coffee, wine, different herbal preparations, and some fruits (such as apples, pears, and certain berries) can reduce the risk of various chronic diseases [27,28].

Discussion
Many kinds of plants have been used as tea drinks in China since ancient times. Traditionally, "tea" refers to the buds, tender leaves, or leaves of the plants from the Camellia genus of the Theaceae family, while non-Camellia tea consists of different parts of the plants from different families and genera [25]. According to the degree of fermentation, the biologically active ingredients of tea vary greatly, but the main active ingredient in tea is polyphenol, a chemical known to act as a preventive agent against malignant tumors and have strong free radical scavenging and reducing activity. Non-fermented tea (e.g., green tea) has a higher polyphenol content, causing astringency and irritation to the stomach. Total fermented tea (e.g., black tea) has a greater loss of polyphenols, but its components are more complex (e.g., aromatic components), causing a better effect on stomach maintenance [26]. Studies have shown that Apocynum venetum, a non-Camellia tea, contains phenolic acids, flavonoids, amino acids, and other chemicals that have been shown to lower blood pressure, blood lipids, and blood sugar levels [15], have antioxidant and anti-inflammatory properties [16], and anti-cancer properties [17].
In this study, eleven bioactive components including chlorogenic acid and its five isomers were detected in AVTE by HPLC. Chlorogenic acids (CGAs) are phenolic acids with vicinal hydroxyl groups on aromatic residues, derived from the esterification of trans-cinnamic acids (including caffeic, ferulic and p-coumaric acids) with quinic acid. There are several CGA subgroups, including neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. These compounds have many pharmacological actions, such as scavenging free radicals, lowering blood pressure and blood lipids, and protecting the liver and gallbladder. CGAs are abundant in the human diet, and epidemiological studies have shown that consumption of tea, coffee, wine, different herbal preparations, and some fruits (such as apples, pears, and certain berries) can reduce the risk of various chronic diseases [27,28].
Rosemarinic acid is a polyphenolic hydroxyl compound formed by the condensation of caffeic acid and 3,4-dihydroxyphenyl lactic acid. The biosynthesis pathway includes two parallel branching pathways: Phenylalanine and tyrosine. Its antioxidant activity is stronger than caffeic acid and chlorogenic acid. It helps to prevent cell damage caused by free radicals, reducing the risk of cancer and atherosclerosis. It also demonstrates anti-melanin production and anti-inflammatory, anti-mutagenic, and anti-peeling activity [29]. Trans-cinnamic acid is mainly used to treat coronary atherosclerosis and other diseases. It can inhibit the formation of black tyrosinase and significantly inhibit the proliferation of lung adenocarcinoma cells [30]. Rutin is a hydrogen transporter, which may participate in the role of oxidoreductase in vivo, affect thyroid activity, protect adrenaline from oxidation, enhance and promote vitamin C accumulation, maintain vascular elasticity, reduce vascular permeability and fragility, promote cell proliferation, and prevent blood cell agglutination. It has also been shown to have hypolipidemic and anti-inflammatory effects [31].
Isoquercitrin, a flavonol compound obtained by hydrolyzing rutin, widely exists in mulberry leaves, Apocynum venetum, Cyclocarya paliurus, Thalictrum angustifolia, and other medicinal plants. As an important active ingredient of Apocynum venetum, it has been shown to be anti-inflammatory [16] in addition to possessing anti-cancer [17] and anti-oxidation effects [32]. Astragalin is also a natural flavonoid widely existing in medicinal plants. It has anti-inflammatory and anti-hepatotoxic effects [33].
In conclusion, the presence of these bioactive chemicals in AVTE may be the main reason for their various pharmacological effects.
The long-term injection of d-galactose prevents the mice from completely metabolizing. When the excess d-galactose accumulates in the body, it can be reduced to galactitol, which has toxic effects on the body. The galactitol can then be further oxidized to galactose aldehyde and hydrogen peroxide by the action of its enzyme. Hydrogen peroxide, as a reactive oxygen species (ROS), can produce hydroxyl radicals after reaction, resulting in the decrease of antioxidant activity in vivo. The accumulation of free radicals in vivo is further aggravated by reactive oxygen species. d-galactose can also induce up-regulation of the intracellular Ca 2+ /Mg 2+ ratio, mitochondrial dysfunction, and phospholipase A2 activation. In conclusion, d-galactose induces oxidative stress, induces the expression of many inflammatory cytokines and irreversible cell apoptosis, leading to aging and functional deterioration [34]. In most publications, the mice or rat aging model was established by intraperitoneal injection of 50-500 mg/(kg·d) d-galactose daily for 6-8 weeks [35]. We established an aging model of mice through intraperitoneal injection of 120 mg/(kg·d) d-galactose for six weeks in this research, and the effect was significant (sore whiskers). Therefore, we recommend low-dose, long-term modeling, which is more similar to natural aging. As the most vigorous organ of the human body, the liver is also the most important detoxification organ and is very sensitive to drug metabolism. Studies have shown that aging cells cause aging and age-related diseases by producing a low-grade inflammatory state [36][37][38]. d-galactose-induced aging in mice can cause hepatic cell swelling, necrosis, inflammatory cell infiltration, and other pathological changes, leading to liver damage, mainly oxidative stress damage and inflammatory response [39].
Under normal circumstances, the body's oxidation and anti-oxidation systems are in dynamic equilibrium, and the body's own antioxidant enzymes such as SOD, CAT, and GSH-Px play an important role in scavenging free radicals. When external stimuli cause oxidative stress damage, the body's metabolism is disrupted, and the initial equilibrium state cannot be maintained; lipid peroxidation causes the body to produce a large amount of peroxidation products [5]. We found that the activities of antioxidant enzymes SOD, GSH-Px, and CAT in aged liver tissue were significantly lower than normal. On the contrary, the content of MDA and NO in peroxidation products was significantly higher than those in the normal group and the antioxidant system in the mice was unbalanced. MDA is the final metabolite of membrane lipid peroxidation in vivo, which can better reflect the degree of tissue peroxidation. The MDA released on the cell membrane can react with proteins and nucleic acids to cause cross-linking polymerization and also inhibit the synthesis of proteins, mainly by damaging the membrane structure and function and changing its permeability, thereby affecting the biochemical reaction of normal organisms [40]. NO is a highly reactive free radical in the body that relaxes vascular smooth muscle, inhibits platelet aggregation, and mediates cytotoxic effects and immune regulation. Its abnormality is closely related to the development of certain diseases, and its concentration increases with the age of the organism [41].
SOD is an important enzyme in the antioxidant system of the body. According to the combination of different metal ions, SOD is divided into Mn-SOD, Fe-SOD, and Cu/Zn-SOD. It can convert the excess superoxide anion radical of the body into hydrogen peroxide, which is converted into H 2 O by CAT and GSH-Px to protect it from damage. With the aging of the body, the activity of SOD is continuously decreasing [10]. Compared with the aging group, the levels of SOD in the AVTE group (250 mg/kg and 500 mg/kg) increased, and the high dose group were significantly better, indicating that AVTE can alleviate lipid peroxidation in mice and alleviate the degree of aging damage in mice. CAT can regulate superoxide anion radicals in the body and has a high affinity for hydrogen peroxide. It can reduce toxic hydrogen peroxide to H 2 O, and coordinate with SOD to delay the aging of the body [42]. Under the action of GSH-Px, GSH can reduce intracellular hydrogen peroxide to form H 2 O, and GSH is oxidized to GSSG, which generates GSH under the catalysis of glutathione reductase. GSH-Px works together with GSH to protect the body from ROS damage and maintain the normal function of the body [43]. T-AOC can scavenge reactive oxygen species to put the body in a redox state, making it a good indicator of the comprehensively reflecting enzymes and non-enzymatic antioxidants [44]. The interaction of multiple compounds in AVTE may be the main reason for its ability to significantly regulate changes in these indicators in aged mice.
d-galactose-induced mouse aging not only causes oxidative stress damage in the body, resulting in low immune function, but also secretes inflammatory cytokines such as TNF-α and IL, causing apoptosis and necrosis of hepatocytes [45]. The immune system is associated with Th1 or Th2 cells and contains both anti-inflammatory and pro-inflammatory cytokines, which play an important role in the inflammatory response. Orchestrating cell-mediated immunity is a vital function of Th1 cells, which secrete INF-γ, IL-2, and IL-12. In contrast, regulation of humoral responses is a key capability of Th2 cells, which secrete IL-4, IL-6, and IL-10. These subpopulations are regulated by important cytokines [46]. INF-γ can suppress the development of Th2, while IL-4 and IL-10 cells inhibit the Th1 response; their mutual regulation creates a normal state. This homeostasis is disrupted during inflammation. Therefore, we screened pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, which cause immune disorders and amplify inflammation, as well as anti-inflammatory cytokine IL-10. IL-1β is an important pro-inflammatory factor, which can induce multiple signaling pathways in cells and promote the production of cytokines. It is considered to be one of the most potent inflammatory factors [47]. IL-6 can promote the accumulation of acute proteins and T cells in the inflammation site, and TNF-α is the earliest and most important mediator in the inflammatory process-they both play an important role in the pathological process of liver injury. They can act on hepatocyte surface related receptors, have obvious toxicity to the liver, and cause massive necrosis of liver cells; inhibition of TNF-α and IL-6 expression can attenuate liver aging damage caused by d-galactose [48][49][50]. In this study, AVTE can attenuate the expression of inflammatory factors in aging mice by down-regulating TNF-α, IL-6 and IL-1β, and up-regulating IL-10.
Cardio-vascular functions changes are also major symptoms of aging. The most significant effect was manifested in left ventricular hypertrophy and left atrial dilatation. Fibrosis occurred with long-term myocardial insufficiency, arrhythmia, and myocardial degenerative lesions occurred, and the final outcome was heart failure [51]. It can also cause damage to the intima of the aortic valve, causing blood eddy, which in turn leads to endocarditis. This leads to mitral annular calcification (MAC), which is closely related to conduction system disease, atherosclerosis, and adverse cardiovascular disease [52]. The roots of cannabis Apocynum cannabium and Apocynum androsaemifolium in the first half of the 20th century were used to treat heart disease in Europe [53]. In recent years, an in vitro study showed that Apocynum venetum extract increased the contractile force and pulses of isolated guinea pig atria [54]. Although the cardiovascular function in this study has not been directly tested and proved, the changes of MDA, SOD, GSH, GSH-Px, CAT, T-AOC, and NO in the serum of the mice were analyzed, which are related to cardiovascular function. Further, those compounds identified by HPLC from AVTE have been shown to improve cardiovascular function.
In addition, we clarify the limitations and precautions of this research, and give the direction for future research. The AVTE treatment group was designed into the mice grouping scheme and could serve the entire experiment better. Although the solubility of DMSO to AVTE is stronger than water, ethanol, etc., some insoluble compounds with DMSO are still neglected when detected by HPLC. Mass spectrometry analysis is more convincing than single HPLC analysis for the identification of compounds. The biological activity and functional expression of AVTE at the protein level are still unclear. However, the research on Apocynum venetum tea (non-Camellia tea) is relatively rare compared with the typical tea, especially as the functional evaluation in vivo of AVTE is less. We evaluated the effect of AVTE on d-galactose-induced aging mice, and the improvement was significant. The compounds were analyzed, and some meaningful compounds were found, which were not found in other studies. We will further improve them in future experiments, and we plan to study the effects and specific mechanisms of AVTE on cardiovascular function in aging mice, and to evaluate the pharmacodynamics of multiple compounds combination in AVTE.

Conclusions
In summary, anti-aging effects of AVTE (250, 500 mg/kg) include the regulation of the weight of the major organs; improvement of hepatocyte morphology, edema, and inflammation; up-regulation of SOD, CAT, GSH, GSH-Px, T-AOC; down-regulation of NO and MDA levels; reductions in the concentration of pro-inflammatory factors (IL-6, TNF-α and IL-1β), and increases in the concentration of anti-inflammatory factor IL-10. In addition, the 11 compounds tested by HPLC were previously shown to have many biological activities including anti-oxidant, anti-inflammatory, and anti-cancer, etc.