Antioxidant, Anti-Aging And Organ Protective Effects Of Total Saponins From Cistanche Ⅱ

Results 

Analysis of Cistanche Composition 

As shown in Figure 1, HPLC analysis revealed that Cistanche contained six types of saponins, including tara saponin IV, arabinoside C, stipuleanoside R2, pseudo ginsenoside RT1, arabinoside A, and chipsets saponin Iva. Table 1 shows the content of six saponins in Cistanche.

Figure 1 Saponins constituents of Cistanche. (A) Standard chromatograms; (B) Saponins of Cistanche chromatograms. 1: Tarasaponin IV; 2: Araloside C; 3: Stipuleanoside R2; 4:Pseudoginsenoside RT1; 5: Araloside A; 6: Chikusetsu saponin IVa.

Antioxidant Capacities in vitro 

As shown in Figure 2A, Cistanche provided single electrons for pairing with DPPH radicals, and as the concentration of Cistanche increased (0.01–1 mg/mL), the color of the reaction system gradually became lighter, obeying a significant dose-dependent relationship. The reaction system was almost colorless when Cistanche concentrations reached 1 mg/mL, reaching a scavenging rate of 90.5 ± 1.4%, close to that of VC. In addition, the IC50 values of DPPH radical scavenging by Cistanche and VC were 0.27 mg/ mL and 5.19 μg/mL, respectively. The results indicate that Cistanche has the ability to scavenge DPPH free radicals.

The scavenging effect of Cistanche on ABTS free radicals is shown in Figure 2B. When the concentration increased from 0.01 to 1 mg/mL, the scavenging rate increased from 1.56 ± 3.46% to 56.01 ± 3.40%. It was found that the scavenging effect of Cistanche on ABTS free radicals was dose-dependent. The IC50 value was 0.78 mg/mL. This shows that increasing the concentration of Cistanche can significantly improve the scavenging ability of ABTS free radicals. Under the same conditions, the IC50 value of VC scavenging ABTS free radicals was 15.48 μg/mL.

The tyrosinase inhibitory activity of Cistanche was evaluated with L-DOPA as a substrate in vitro. As shown in Figure 2C, the inhibition rate of tyrosinase activity by Cistanche obeyed a dose dependence at concentrations of 0.01–1 mg/mL. The inhibition rate of tyrosinase activity by Cistanche was 48.55 ± 2.62%, and the IC50 value was 1.18 mg/mL at 1 mg/mL. Cistanche was proven to be a potential tyrosinase activity inhibitor in the present study. In this study, it was proven to be a potential tyrosinase activity inhibitor for the first time; thus, it may have a potential whitening effect.

Figure 2D shows that the clearance of HO• by Cistanche was only 3.04 ± 1.93% at a concentration of 0.01 mg/mL and reached 56.86 ± 1.31% at 1 mg/mL with increasing Cistanche concentration. The IC50 value was 0.528 mg/mL. In conclusion, Cistanche has a good scavenging capacity for HO•. It is speculated that Cistanche may act as a metal ion chelating agent to block the formation of HO•. In this study, the scavenging rate of •O2- by Cistanche was 39.94 ± 0.49% at a concentration of 1 mg/mL, with an IC50 value of 2.43 mg/mL. In addition, Figure 2E shows that the scavenging rate in •O2- quenching correlated with the concentration of Cistanche.

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Anti-Aging Activities Assaying

Daily Behaviour Observations and Organ Indices During the eight weeks of our experiment, the daily appearance and behavioral activities of the rats were observed and recorded weekly. The rats in the normal group were not only in good mental condition but were also lively, active and agile and had a healthy diet. As shown in Figure 3A, after the 5th week of D-gal injection, the rats in the model group showed a decline in body weight accompanied by obvious signs of senescence, including slow motion, gradually reduced food intake, depression and dry fur, indicating that the senescence model triggered by intraperitoneal injection of D-gal had been successfully established. The above senescence features were regressed to different degrees in both the VC group and the treated rats compared with the model group. In particular, in the high-dose Cistanche-treated group, the abovementioned aging characteristics of the rats were significantly reduced compared with those of the model group. Aging causes degenerative atrophy of organs, leading to a decrease in organ indices.27,28 In this study, thymus, liver, spleen, kidney and heart organ indices were highest in normal control rats and lowest in the model group (p<0.05) (Figure 3), indicating that D-gal caused organ atrophy in rats. Notably, the Cistanche-induced increase in organ indices may reflect the prevention of atrophy of body tissues.

Morris Water Maze (MWM) Test 

We used the MWM test to assess the effects of Cistanche on spatial learning and memory abilities in a D-gal-induced mouse model. As shown in Figures 4A and B, rats in the D-gal group suffered a significant impairment in spatial learning ability during the 5-day location navigation training, as evidenced by a longer escape latency compared to control rats, whereas rats treated with Cistanche had a significantly shorter escape latency, which was closer to the normal level. To assess spatial memory more directly, rats were subjected to another test in which the target platform was removed the day after navigation training. As shown in Figure 4C, the rats given D-gal crossed the location less often than the control group. However, compared to those in the model group, the rats in the treatment group had less time to escape the latency and crossed the platform more often, with the higher dose group being more significant (p<0.05). These results suggest that the aging model impaired spatial learning and memory, while oral administration of Cistanche restored age-related cognitive deficits induced by D-gal exposure.

Oxidation-Related Index Assays 

SOD levels in serum, brain, heart, lung, spleen and kidney were significantly lower in the untreated model group than in the normal group (p<0.01) (Figure 5), indicating that the organism was damaged by oxidative stress to different degrees. However, the SOD levels in the aging rats administered different doses of Cistanche were significantly higher than those in the model group (p<0.05). The results indicated that Cistanche could reverse the decrease in SOD content in aging tissues and serum, with the best effect in the high-dose group.

Compared with the MDA value of normal rats, the MDA levels in the serum, brain, heart, lung, spleen, and kidney of the untreated model group were significantly increased (p<0.01) (Figure 6), indicating that organs suffer from an oxidative stress injury. In contrast, Cistanche treatment downregulated MDA accumulation (p<0.05). The results indicate that the potential anti-aging properties of Cistanche may be related to its reduction in MDA content.

The levels of CAT were significantly lower (p<0.01) in the serum, brain, heart, lung, spleen and kidney of rats in the untreated model group than in the normal group (Figure 7), indicating that the accumulation of H2O2 in the organism led to structural cell damage. However, CAT levels improved after treatment in both the VC and Cistanche administration groups (p<0.05), especially in the serum and organ CAT levels of rats in the high-dose Cistanche group, which were significantly higher than those in the model group. 

GSH-Px was significantly lower (p<0.01) in the serum, brain, heart, lung, spleen and kidney of untreated aging rats (Figure 8) than in the normal group of rats. In contrast, GSH-Px activity was significantly increased after Cistanche administration, with a significant difference compared to that in the model group (p<0.05), possibly acting by blocking free radical damage to cell membrane lipids.

As shown in Figure 9, serum, brain, heart, lung, spleen and kidney T-AOC levels were significantly lower in the untreated aging group than in the normal group (p<0.01). However, Cistanche treatment significantly increased T-AOC levels (p<0.05). This may be due to the long-term consumption of antioxidant enzymes caused by high levels of oxidative stress, which was reversed by oral Cistanche. This result strongly indicates that Cistanche has antioxidant potential and can increase antioxidant capacity, thus reducing the damage caused by aging.

ALT and AST are effective markers for evaluating liver function injury.29 As shown in Figure 10, compared with those in the normal group of rats, the levels of ALT and AST in the untreated model group were significantly increased (p<0.01), indicating that the liver was damaged by D-gal stimulation. In contrast, after administration of Cistanche, the levels of ALT and AST in the livers of aging rats were significantly reduced (p<0.05). The results show that TAST has a protective effect on the liver.

Histopathological Observations 

In this study, histopathological observations were performed on the brain, heart, lung, kidney, liver and spleen to confirm the evidence from the biochemical analysis. Figure 11 shows the effect of Cistanche on the histopathology of D-gal-induced aging rats. It is obvious that organ injury is mainly manifested by the degradation of cells and nuclei. In addition, senescent rats exhibited other pathological patterns in these organs, such as partial edema, fragmented arrangement of nerve cells and atrophy of nuclei in brain tissue (Figure 11A), broken, loosely arranged and increased intercellular fibers in the myocardium (Figure 11B), alveolar hemorrhage, thickened alveolar walls and inflammatory cell infiltration in the alveolar space and around blood vessels (Figure 11C), glomerular atrophy and possible proximal tubules of the kidney balloon dilatation lumen (Figure 11D), binucleation of hepatocytes, hepatic cord disorder and swelling of hepatocytes (Figure 11E), and old hematopoiesis in the spleen with increased fibrous septa and fused lymphoid follicles (Figure 11F). Interestingly, Cistanche treatment significantly attenuated the adverse organ lesions caused by D-gal, with the best results seen in the high-dose group. Moreover, the vitamin C-treated control rats had a similar recovery effect. The results suggest that Cistanche has poten

tial protective effects on the brain, heart, lung, kidney, liver and spleen against acute D-gal-induced injury 

Figure 6 Effect of Cistanche on MDA levels in (A) Serum, (B) Brain, (C) Heart, (D) Lung, (E) Spleen and (F) Kidney. ***p<0.0001 vs normal group; #p<0.05, ##p<0.01 and ###p<0.0001 vs model group. All the data are expressed as the means ± SD of 6–8 rats in each group

Discussion

In recent years, Cistanche has been shown to have high anti-oxidant activity and contribute to the prevention of ROS-induced oxidative damage in the body. Based on the free radical theory of aging, long-term injection of D-gal causes oxidative stress by stimulating excessive production of ROS, leading to metabolic disorders and inducing oxidative damage to organs in vivo associated with aging.30,31 Therefore, D-gal-induced aging models have been widely used in anti-aging and organ injury studies, for which antioxidant supplementation may be a potential therapeutic strategy for D-gal-induced oxidative and aging damage. In this study, the antioxidant activity of Cistanche was consolidated by scavenging DPPH, ABTS, hydroxyl radicals and superoxide anion radicals and inhibiting tyrosinase activity in vitro. Furthermore, behavioral evaluation, organ indices, biological indicators of oxidative stress and analysis of pathological changes revealed that D-gal-induced aging rats had reduced major organ indices and reduced learning memory capacity with varying degrees of oxidative damage, but this effect was inhibited by Cistanche intervention.

HPLC was used to analyze Cistanche in this study, and the results showed that Cistanche contained six triterpene saponins, including araloside C, Chikusetsu saponin IVa, araloside A, pseudoinsenoside RT1, tarrasaponin IV and stipuleanoside R2. Araloside C and Chikusetsu saponin IVa have been shown to have antioxidant properties, reduce the accumulation of ROS in cells, inhibit apoptosis and reduce Ca2+ overload.32,33 It has been reported that mitochondrial dysfunction generates excess reactive oxygen species and contributes to aging-related diseases.34 Araloside C reduces mitochondrial ROS levels by increasing cell viability and improving disruption of mitochondrial membrane potential.35 However, whether other saponins have similar effects needs to be further investigated. These results suggest that the anti-aging effect of Cistanche is related to the scavenging effect on ROS associated with its active saponins.

Increasing evidence has shown that ROS such as superoxide anion, hydrogen peroxide and hydroxyl free radicals can attack life macromolecules and cell walls, thus inducing oxidative stress, causing various oxidative damage and pathological changes, and accelerating aging.10,36,37 The present work evaluates the antioxidant activity of Cistanche by measuring the scavenging of DPPH radicals, ABTS radicals, tyrosinase, hydroxyl radicals and superoxide anions in vitro. Among them, DPPH is one of the stable and mature organic nitrogen free radicals and has the maximum absorption at 517 nm. After adding antioxidants, electrons or hydrogen atoms are transferred to DPPH to form stable DPPH-H compounds, which reduces its absorption at 517 nm.38 In ABTS analysis, ABTS was converted to ABTS+ by adding K2S2O8. In the presence of antioxidants, active ABTS+ was converted into a colorless natural form. Tyrosinase plays a key role in melanin formation and browning. Under ultraviolet radiation stimulation, the excessive production and accumulation of melanin in the skin may lead to pigmentation disorders and skin aging, and the down-regulation of tyrosinase is considered to be the reason for the decrease in melanin activity. Tyrosinase has also been reported to be associated with neurodegenerative diseases. When produced in excess, tyrosinase is able to increase intracellular dopamine production, followed by the induction of melanin formation, causing cell death.39,40 Interestingly, some studies have proved that saponins could be potential natural tyrosinase inhibitors. For example, saponins from Xanthoceras sorbifolia nutshell were reported to inhibit 52.0% of tyrosinase activity at a concentration of 0.96 mg/ mL.41 Furthermore, some transition metals including Fe2+,   Cu2+ and Co2+ can trigger the free radical reactions in magnifying the cellular damage. Among these transition metals, Fe2+ is known as the most powerful prooxidant on stimulating lipid peroxidation by generating hydroxyl radicals through the Fenton reaction.42 •O2- is a product of biological and photochemical stimuli. It is highly toxic when combined with HO•, causing lipid peroxidation and damage to the normal structure of cells.43 Hence, the antioxidant and anti-aging properties of Cistanche may be due to its free radical scavenging, tyrosinase inhibition and metal ion chelating. Additionally, there has report that the scavenging properties of Cistanche were mainly associated with structural differences such as the category and the sequence of the oligosaccharide chain at C-3 position in such saponins.20

Figure 10 Effect of Cistanche on (A) ALT and (B) AST levels in liver. ***p<0.0001 vs normal group; #p<0.05, ##p<0.01 and ###p<0.0001 vs model group. All the data are expressed as the means ± SD of 6–8 rats in each group

Learning and memory deficits are considered to be clinical manifestations of neurodegenerative diseases associated with aging.26 Increasing evidence suggests that oxidative stress in the brain may play a key role in the pathological process of cognitive decline.44 This was confirmed in the MWM test study, where our results showed that escape latencies were longer in a D-gal-induced aging rat model, suggesting that D-gal impaired spatial learning in rats. Furthermore, the results across plateau counts also demonstrate that Cistanche treatment improves spatial memory impairment in the D-gal-induced model. Moreover, cellular damage to hippocampal neurons has been shown to contribute to cognitive decline in AD, and we used H&E to evaluate the effects of Cistanche on hippocampal neurons.45 Our results suggest that oral administration of Cistanche has a neuroprotective effect, improving learning memory capacity and attenuating neuronal damage. This is consistent with a previous result that Aralia taibaiensis total saponin attenuates I/R-induced mitochondrial dysfunction and oxidative stress, thereby protecting brain cells from damage.46

Organ indices are indicators to assess the health status of animals.47 With aging, the body’s immune system undergoes degenerative changes that not only reduce the sensitivity of non-specific immunity but also present a general imbalance in immune function, which ultimately leads to the development of diseases. Therefore, body immunity is one of the main tools in anti-aging research. The thymus and spleen are two important immune organs in the body, and their organ indices can initially reflect the strength of the nonspecific immune system.48,49 Compared with the normal group, the organ indices of the thymus and spleen in the model group of rats were reduced. However, there was an overall increase in organ indices in the drug group, but the variability was not significant, which may be related to the individual variability of the rats. The liver is also considered to be the immune organ of the animal, and alterations in the liver index can affect the body’s immune function.50 The results showed that Cistanche significantly increased the D-gal-induced decrease in the organ index, which is consistent with its traditional hepatoprotective effect. These results suggest that D-gal successfully induces oxidative aging in rats, leading to a reduction in the organ index and that Cistanche produces anti-aging effects by regulating immune organs.

It is well established that antioxidant enzyme activity decreases during aging. In the present study, SOD activity was significantly reduced during D-gal-induced aging. As the most important enzyme in living organisms, SOD protects the structural integrity of cells by blocking damage to cells by •O2-. In a state of oxidative stress, the inward flow of intra-cellular Ca2+ leads to the irreversible conversion of xanthine dehydrogenase to xanthine oxidase, which in turn catalyzes the oxidation of hypoxanthine, generating large amounts of •O2- that damage cellular structure and accelerate aging.51 Free radical overload leads to an overconsumption of SOD and reduced activity. CAT is another important antioxidant enzyme that maintains H2O2 homeostasis in the body, rapidly breaking down H2O2 into H2O and O2 and preventing the abnormal accumulation of H2O2 in cells. The main role of GSH-Px is to interrupt the chain reaction of lipid peroxidation and catalyze the conversion of lipid peroxides into nontoxic hydroxyl compounds.28 In this study, we found that CAT and GSH-Px levels were significantly lower in the serum and organs of aged rats than in those of normal rats. This has been reported that this may be related to increased utilization of CAT and GSH-Px by the body’s antioxidant system.10 MDA is a product of free radical chain reaction-induced lipid peroxidation and has been used as a marker to assess oxidative damage. In the current study, free radical action on cell membranes leading to membrane lipid damage may be responsible for the significant increase in MDA levels in the serum and organs of aging rats.10,51 The results of this study are consistent with earlier reports in the literature. We found that when Cistanche was administered to aging rats, the levels of SOD, CAT and GSH-Px were significantly increased, and the levels of MDA were significantly decreased. This may be due to the active components of Cistanche after metabolism in vivo, which restores the activity of these enzymes by providing single electrons to pair with free radicals, thereby scavenging them. This restoration contributes to cellular integrity, reduces oxidative stress and mitigates lipid peroxidation. These results strongly confirm the ability of Cistanche to delay aging by scavenging free radicals, which was consistent with the results of in vitro experiments. Compared to single anti-oxidants, T-AOC can reflect the collective effect of enzymatic or nonenzymatic systems involved in antioxidant defense in biological fluids and is a comprehensive measure to evaluate the total antioxidant capacity of the body.52 Thus, higher levels of T-AOC may indicate an increased protective response of individuals and decreased susceptibility to oxidative damage.53 In this study, the level of T-AOC decreased in rats exposed to D-gal for a long time, which may be due to the long-term consumption of antioxidant enzymes caused by high levels of oxidative stress, which was reversed by oral Cistanche. The liver is the most vigorous metabolic organ in the body and an important detoxification organ. It plays an important role in regulating the health of the body.54 ALT is mainly found in the hepatocyte cytoplasm, while AST is mainly found in the mitochondria of hepatocytes. When liver cells are damaged, serum ALT levels increase, and when liver cells die, serum AST levels increase significantly.55 Therefore, serum ALT and AST levels can reflect the extent of liver injury. The results showed that ALT and AST levels were elevated in liver tissue after D-gal injury, but Cistanche intervention inhibited the elevation of these transaminases. These results strongly suggest that Cistanche attenuates oxidative damage to organs during aging to some extent by inhibiting oxidative stress. These can be demonstrated in histological observations.

Conclusion

In this study, we consolidated the antioxidant, anti-aging and organ protective effects of Cistanche in vitro and in vivo.Cistanche is a class of natural saponins with good scavenging effects on DPPH, ABTS, hydroxyl radicals and superoxide anion radicals and inhibits tyrosinase activity. In addition, Cistanche exerted anti-aging effects in D-gal-induced aging rats by improving learning and memory capacity, preventing immune organ atrophy, increasing the activity of key antioxidant enzymes in the serum, brain, heart,lung, spleen and kidney, attenuating lipid peroxidation and inhibiting transaminase levels in liver tissues, thereby reducing oxidative and aging damage and exerting antiaging effects. However, although our study is important in consolidating the antioxidant, anti-aging and organ protective effects of Cistanche, its molecular regulation on key antioxidant enzymes needs to be further investigated. Furthermore, due to the use of total saponins in this study, it should be further explored which specific components of Cistanche contribute to these effects. Further investigation of these effects will provide supportive preclinical evidence for the potential clinical application of Cistanche as a product for the treatment of age-related diseases.

University of Traditional Chinese Medicine (2019-YL11).

Disclosure

The authors report no conflicts of interest in this work.

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