Abstract
To study the extraction technology of polysaccharides (AAP) from Chinese herbal medicine formula and its mechanism of delaying aging. First, L9(3)4 orthogonal test was used to optimize the optimal enzyme-assisted extraction parameters of polysaccharides. And the anti-aging effects was evaluated by detecting mitochondrial function, protein, DNA, adhesion molecules and cell cycle in aging rats. The optimal extraction process parameters were the cellulase concentration of 1.5%, the pH at 5, the enzyme temperature at 50°C and the extraction time of 180 min. The anti-aging results showed that AAP can effectively increase the activities of malate dehydrogenase, succinate dehydrogenase and superoxide dismutase. It also can decrease the activity of monoamine oxidase and methane dicarboxylic aldehyde levels in the brain tissue. Meanwhile, the polysaccharides enhanced telomerase activity while reduced p16 protein expression of the brain mitochondria. In addition, the polysaccharides continued to improve heart damage and significantly lessen mitochondrial DNA concentrations. For a certain period of time, it also enhanced the activity of superoxide dismutase, reduced glutathione, glutathione peroxidase and decreased protein carbonyl and methane dicarboxylic aldehyde content of kidney in D-galactose-induced aging rats. Furthermore, the polysaccharides restored the number of cells in the peripheral blood lines and BMNC through inhibiting the drop of the number of red blood cells, white blood cells, platelets in the peripheral blood and bone marrow mononuclear cell of the aging rats. At the same time, AAP accelerated G1 phase cell to enter S phase in cell cycle in aging rats. Our research suggests that the polysaccharides may be a potential anti-aging agent and can be further developed as a functional food or new drug to delay aging or treat aging-related diseases.
Keywords: polysaccharide, optimization, orthogonal design, anti-aging, D-galactose, aging rats, mechanism
Introduction
Senescence, or, aging, is an age-dependent process characterized by gradual damage and physiopathological changes in different organs [1, 2]. Many widespread theories as to why aging takes place abound, including telomere shortening, DNA damage, mitochondria dysfunction, reactive oxygen species (ROS) and immunological functions and so on [3]. Among these factors, mitochondria have been demonstrated to be a key in driving numerous of the pro-aging process [4] and mitochondrial dysfunction has been regarded as one of the hallmarks of aging [5]. It is well established that mitochondrial function deteriorates with age in different tissues, such as heart [6], muscle [7] and liver [8].
Cardiac and kidney aging are often accompanied by a general decline in mitochondrial function, clonal expansion of dysfunctional mitochondria, increased production of ROS, suppressed mitophagy [9] and dysregulation of mitochondrial quality control processes [10, 11]. Accordingly, development of novel therapeutic approaches for the attenuation of mitochondrial damages and rejuvenation of mitochondrial holds promise for postponing aging. In recent times, several compounds that target mitochondria have been discovered for delaying aging [12] and the treatment of metabolic disorders diseases [13].
Plant polysaccharides, especially from medicinal herbs, have attracted considerable attention for their pharmacological effects, which not only possess antioxidant, anti-hyperlipidemia [13], anticancer and immune activities [14, 15] but also show low cytotoxicity and side effects [15, 16].
Angelica sinensis and astragalus membranaceus are herbs widely distributed in the northwest of China. They exhibit pharmacological effects like hematopoietic effect, antitumor, immunomodulatory and antioxidant activities [17]. The polysaccharides from angelica and astragalus (AAP) were prepared according to Danggui Buxue Decoction, a well-established Chinese medicine formula, has been used to invigorate the circulation of blood and enhance immunity for centuries. There are many researches on the extraction and isolation of single plant polysaccharides and their biological activities. But the preparation and structural characterization of composite plant polysaccharides (extracted from more than two plant tissues) are rarely reported. Nevertheless, the structural characterization [18] and the pharmacological functions of AAP have been investigated in our laboratory [19, 20], and the results have displayed that AAP possessed obvious antioxidant and anti-aging effects both in vitro [21, 22] and in vivo [19]. But the related molecular mechanisms underlying the postponing aging of AAP were not thoroughly researched at home and aboard.
Hence, in this paper, the extraction conditions of AAP were optimized by employing an L9(3)4 orthogonal array in order to obtain a higher yield and concentration of polysaccharides. Then, we further studied the anti-aging effect of AAP and its related mechanism in rats induced by D-galactose.
Materials and Methods
Plants and reagents
Angelica and astragalus were purchased from Minxian (North latitude N34°26′21.62″，East longitude E104°02′45.90″) Shunfa Medicinal Material Company (Gansu Minxian City, China), and were identified as angelica sinensis (voucher number LutZ 20140410) and astragalus membranaceus (voucher number LutZ 20140411) by Associate Professor L.Yang, College of Life Science and Engineering, Lanzhou University of Technology. Cellulase (15 000 U/g) (Enzyme) was obtained from Nanning Pangbo Biotechnology Co. (Guangxi, China). Ethanol, chloroform and n-butanol were obtained from Tianjin Reagent Co. (Tianjin, China). All other reagents were of analytical grade.
Extraction of polysaccharides
The roots of angelica and astragalus (360 g, 1:5, w/w) were ground in a blender to obtain a fine powder. The powder was extracted twice with 80% ethanol under heat reflux (2 h each) to remove the impurities and lipophilic molecules [23, 24]. The degreased powder was extracted in a constant temperature water bath at 95°C for 2 h at a solid–liquid ratio of 1:10. In this extraction process, the enzyme-assisted method (cellulase) was applied; a given concentration of cellulase enzyme was added to the extraction solvent and maintained at the designed temperature, time and pH.
The aqueous extract was centrifuged at 3000 × g for 10 min to remove the pellets, then concentrated and precipitated via addition of a 4-fold volume of anhydrous ethanol, and incubated at 4°C for 24 h. After centrifugation (3000 × g, 10 min), the precipitate was sequentially washed with anhydrous ethanol and acetone to yield the crude angelica and astragalus polysaccharides (CAAP). CAAP were re-dissolved in distilled water, extracted using the enzyme-assisted method to remove the dissociated protein, and precipitated via addition of ethanol. The light yellow dry precipitate is named angelica and astragalus polysaccharides (CAAP). The polysaccharide extraction yield was calculated according to the following equation:
 |
Orthogonal test design for optimization of polysaccharide extraction
Four factors at three levels of orthogonal experimental design was used to optimize the parameters of the enzyme-assisted extraction of polysaccharides. The orthogonal factors and their levels were chosen based on the results of the single-factor experiments (Table 1). The total polysaccharide content was determined according to the phenol-sulfuric acid method, using glucose as the standard reference [25].
Table 1.
Factors and levels of orthogonal experimental design
| Levels | (A) Concentration of cellulase/% | (B) T/°C | (C) Time/minutes | (D) PH |
|---|---|---|---|---|
| 1 | 1 | 40 | 60 | 4.0 |
| 2 | 1.5 | 50 | 120 | 5.0 |
| 3 | 2.0 | 60 | 180 | 6.0 |
Purification of CAAP
The crude polysaccharides from angelica and astragalus (CAAP) was re-dissolved in distilled water and extracted by the sevag method to remove the dissociative protein and precipitated by addition of ethanol [26]. Then precipitation collected was applied to a DEAE cellulose-52 column and size-exclusion chromatography on a Sephadex G-100 column to further be purified in turn. This polysaccharide was concentrated, dialyzed and lyophilized to obtain a white power (it was called AAP) [27].
Characterization of AAP
The structural characterization of AAP was carried out systematically. Firstly, the total polysaccharide content of AAP was determined by the phenol sulfuric acid method [28]. The protein was quantified according to the Bradford method [29]. Uric acid content was determined using m-hydroxybiphenyl colorimetry. An elemental analyzer was used to analyze carbon (C), hydrogen (H), nitrogen (N) and sulfur (S) in AAP. Meanwhile, infrared analysis also was performed by Fourier transform infrared (FTIR) spectrometer [30]. The homogeneous were examined by high-performance gel permeation chromatography (HPGPC). And HPGPC coupled with multi-angle laser light scattering was applied to test the molecular weight (MW) of AAP. Gas chromatography was used to assay monosaccharide composition. Methylation analysis was performed according to the previous method [31]. NMR was used to record 1H and 13C NMR spectra to assay the structure of AAP. Last, morphological characteristics was observed by scanning electron microscope.
Experimental design and D-gal-induced aging rats
In total, 50 young specific-pathogen-free Wistar rats (3 months old, body weight (BW) = 180–200 g, 25 males and 25 females) were obtained from the Experimental Animal Center of Medical College at Gansu University of Traditional Chinese Medicine (certificate number: SYXK (Gan) 2017–0004). The rats were kept in open-top cages under the conditions of 14 h light/10 h dark cycles, temperature (22 ± 2°C) and humidity (50% ± 10%). They had free access to water and food. All procedures involving animals were approved by the Institutional Animal Ethical committee. After acclimatization for 5 days, the rats were weighed and randomly divided into five groups (n = 10 for each group) as follows: Group 1 = normal control group; Group 2 = D-gal model group; Group 3 = positive control group; Group 4 = low-dose AAP groups; and Group 5 = high-dose AAP group. D-gal was subcutaneously injected at a dose of 150 mg/kg BW daily [32], except for the normal control group. While modeling, vitamin (Vit) E (100 mg/kg BW daily) was intragastrically administrated to each rat in the Group 3. The rats of Groups 4 and 5 were treated via oral gavage with AAP at doses of 0.5 and 2 g/kg BW, respectively, once a day for 6 weeks [32]. Rats of Group 1 were administrated an equal volume of 0.9% normal saline on the same days. During the experiment, all rats had free access to water and food, and the BW was measured once a week. After 6 weeks, all rats were weighed and then euthanized on the 42th day according to the international act of animal welfare and other relevant guidelines. Serum samples were collected and heart, brain and kidneys were excised, cleaned and dried with a filter paper. Then they were weighed and stored at −20°C until further assays. The part of heart tissue was fixed with 10% neutral-buffered formalin for histopathological examination.
Determination of telomerase activity and p16 protein expression of the brain tissue
Experiments were conducted to investigate effects of AAP on telomerase activity and p16 protein expression of the brain, which were analyzed using double-antibody sandwich ELISA kits (Shaihai Meilian Enzyme-linked Biotechnology, Co., Ltd. Shaihai, China). According to the manufacturer’s instructions, the absorbance was measured at a wavelength of 450 nm. Then, the concentration was calculated. Telomerase activity was expressed in U/L while p16 protein expression was expressed in pg/mL.
Assay of oxidative damage of the brain mitochondria
To study the influence of AAP on the oxidative damage of mitochondria in the brain of D-gal-induced aging rats, experiments were carried out to investigate whether AAP could relieve the oxidative damage and improve the energy metabolism.
First, the brain mitochondria were extracted using sucrose density gradient centrifugation method. Briefly, brain homogenates (10%, w/v) were prepared in Tris-HCl buffer (0.1 M, pH 7.4) containing 0.25 M sucrose and centrifuged at 3000 × g for 10 min. Then, the supernatants (1 ml) were used in the ELISA assays. The rest of the supernatant was subjected to centrifugation at 11 000 × g for 20 min at 4°C in a cooling centrifuge. The mitochondrial pellets were washed twice with Tris-HCl buffer (0.1 M, pH 7.4) containing 0.34 M sucrose. The mitochondrial fractions were broken down by a tissue grinder, which was used for indexes of inspection.
The activities of monoamine oxidase (MAO), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD), as well as the levels of methane dicarboxylic aldehyde (MDA) were determined in the mitochondrial fractions by chemical colorimetry using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The activities of MAO, SDH, MDH and SOD were expressed as U/mg protein while MDA levels were expressed as nmol/mg protein.
Evaluation of the heart mtDNA
To evaluate the effects of AAP on the heart of D-gal-induced aging rats, the heart mtDNA (n = 10 for each group) were extracted, and its concentration were determined. In addition, a histopathological examination of the heart was carried out. Briefly, the heart mitochondria were extracted using the same method as described above. Then, the mtDNA was extracted according to the standard sodium dodecyl sulfate (SDS)-potassium acetate (KAc) method with modifications [33]. mtDNA concentrations were calculated as follows:
 |
where A260 is the absorbance of the nucleic acid at 260 nm. Moreover, the other portion of the heart specimen was fixed in 10% neutral-buffered formalin, embedded in paraffin, sliced (4 μm thickness) and stained with hematoxylin and eosin. The histopathological changes were observed, and the sections were photographed using an optical microscope (BXP-102, Tu ming Optical Instrument Co., Shanghai, China).
Analysis of GSH, SOD and GSH-PX activities as well as MDA and PC levels in kidneys
To investigate the effect mechanisms of AAP on kidney, the activities of glutathione (GSH), SOD and glutathione peroxidase (GSH-PX) as well as MDA and protein carbonyl (PC) levels in the kidneys were analyzed using biochemical analytical methods. In brief, kidney homogenates were prepared in ice-cold 0.9% isotonic normal saline (0.1 g tissue/mL solution). The samples were centrifuged at 1000 × g at 4°C for 10 min. The activities of GSH, GSH-PX and SOD, as well as MDA and PC levels were determined in the supernatants of the kidney homogenates using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). GSH-PX and SOD activities were expressed as U/mg protein. GSH activity was expressed as mg/g protein, whereas MDA and PC levels were expressed as nmol/mg protein. The protein content was measured according to the Bradford method [26].
Influence of AAP on peripheral blood and bone marrow hematopoietic function in aging rats
Automatic hematology analyzer was used to study the effect of AAP on peripheral blood WBC, RBC and PLT of aging rats. The number of bone marrow mononuclear cells (BMNCs) was calculated by automatic cell counter.
Effect of AAP on BMNC cell cycle
After trypsinization, about 1 × 106 BMNC cells were collected, washed with pre-chilled PBS and centrifuged again to discard the supernatant. The centrifuged cells were resuspended in 1 ml of pre-chilled 75% ethanol and fixed at 4°C overnight. After the fixation, the supernatant was discarded by centrifugation, and 1 ml of staining solution (containing 50 mg/L pyridine iodide and 50 mg/L ribonuclease) was slowly resuspended to the pellet and incubated for 30 min in the dark. Data were collected by flow cytometry for cell cycle analysis, and the cell proliferation index (PI) was calculated. The calculation formula for PI is:
 |
Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS v19.0 (International Business Machines Corporation). Differences were considered statistically significant if P < 0.05.
Results
Optimization of crude polysaccharides extraction
The cellulase concentration, ideal temperatures for enzymatic action, extraction time and pH value were important factors affecting the extraction efficiency and the CAAP yield, as shown in Fig. 1. The extraction yield of CAAP was enhanced with the increase of the enzyme concentration, and it reached the peak when the cellulase concentration was approximately 1.5%. The maximum yield of CAAP was obtained after 180 min at a temperature of 50°C (ideal temperature for enzymatic action) and pH of 4.0–5.0. Thus, the optimal extraction conditions were as follows: cellulase concentration of 1.5%, extraction pH at 5.0, temperature for enzymatic action at 50°C and extraction time of 180 min.
Figure 1.
Optimization of crude polysaccharides extraction.
Orthogonal experimental design for polysaccharide extraction
The orthogonal experimental results showed that the orthogonal factors and their degrees affected the extraction yield in a decreasing order: A > B > D > C, where A, B, C and D are the orthogonal factors representing the concentration of cellulase, optimal temperature for enzymatic action, extraction time and liquid pH, respectively. Factors A, B, C and D at the levels of 2, 2, 3 and 2, respectively, gave the maximum polysaccharide extraction yield of 13.87% (Table 2). Therefore, the optimum conditions for crude polysaccharide extraction from the defatted powder (mixture of Angelica and Astragalus at a ratio of 1:5) using the enzyme-assisted extraction method were as follows: Factor A at level 2 (cellulase concentration of 1.5%), Factor B at level 2 (an optimal temperature for enzymatic action of 50°C), Factor C at level 3 (an extraction time of 180 min) and Factor D at level 2 (a pH value of 5). The polysaccharide content of CAAP was 82.82% when the optimum extraction conditions were employed.
Table 2.
The analysis of range of orthogonal test through enzyme-assisted extraction method
| Number | A (%) | B (°C) | C (min) | D (pH) | Extraction yield (%) |
|---|---|---|---|---|---|
| 1 | 1 | 1 | 1 | 1 | 7.44 |
| 2 | 1 | 2 | 2 | 2 | 11.90 |
| 3 | 1 | 3 | 3 | 3 | 8.20 |
| 4 | 2 | 1 | 2 | 3 | 11.20 |
| 5 | 2 | 2 | 3 | 2 | 13.87 |
| 6 | 2 | 3 | 1 | 1 | 12.13 |
| 7 | 3 | 1 | 3 | 2 | 11.40 |
| 8 | 3 | 2 | 1 | 3 | 10.17 |
| 9 | 3 | 3 | 2 | 1 | 8.15 |
| k1 | 9.180 | 10.013 | 9.913 | 9.820 | |
| k2 | 11.400 | 11.980 | 10.417 | 11.810 | |
| k3 | 9.907 | 9.493 | 11.157 | 9.857 | |
| R | 3.220 | 2.487 | 1.244 | 1.990 |
Purification of polysaccharides
Three fractions were, respectively, obtained by chromatography on DEAE-52 column. The main fraction was further purified on Sephadex G-100 column to obtain one fraction named AAP-2A and AAP-2B [27].
Structural Characterization of AAP
The results showed that AAP-2A contained no protein and was a neutral polysaccharide. The mass fractions of C, H, O, N and S were 38.79, 6.95, 54.26, 0.00 and 0.00%, respectively. Moreover, AAP-2A was not absorbed at 260 and 280 nm, indicating that AAP-2A did not contain nucleic acid and protein. The IR spectrum showed that AAP-2A was a typical polysaccharide spectrum. And it was a homogeneous polysaccharide, whose MW, root mean square radius (RMS) and polydispersity index (MW/Mn) were 2.252, 2.252 × 103 kda, 28.4 nm and 1.038, respectively. AAP-2A was composed of rhamnose (Rha), galactose (Gal), arabinose (Ara) and glucose (Glc) with a molar ratio of 1:2.13:3.22:6.18. They were linked by eight types of linkages 1,3-linked Rhap, 1,3-linkedGalp, 1,3-linked Araf, 1,5-linked Araf, 1,3,5-linked Araf, terminal Glcp, 1,4-linked Glcp and 1,4,6-linked Glcp at a molar ratio of 1.01:2.06:1.07:1.08:1.06:3.04:1.02:1.98 (nearly 1:2:1:1:1:3:1:2). The surface morphology of AAP-2A was curly, loose and flaky, which was an amorphous solid under the scanning electron microscopy [27].
Effects of AAP on telomerase activity and p16 protein expression in the brain
We examined the effects of AAP on the activity of telomerase and expression of p16 protein in the brain of D-gal-induced aging rats. The results showed that telomerase activity in the brain tissue of aging rats (4.75 ± 0.03 U/L) was significantly lower (P < 0.01) than that of the normal rats (6.94 ± 0.02 U/L). However, p16 protein level showed an opposite tendency. After administration of AAP and Vit E, the expression of p16 protein were significantly decreased, whereas the activity of telomerase was noticeably increased than that of the D-gal model group (P < 0.05 or P < 0.01) (Fig. 2). These results indicated that AAP could enhance the activity of telomerase and reduce the expression of p16 protein in the brain tissues of D-gal-induced aging rats.
Figure 2.
Effects of AAP on telomerase activity and p16 protein expression in the brain.
Influence of AAP on oxidative damage and energy metabolism in the brain mitochondria
The activities of SOD, MAO, SDH and MDH, as well as the levels of MDA were measured in the brain mitochondria to investigate the protective mechanism of AAP on brain tissue of D-gal-induced rats (Table 3). Compared with the normal control group (11.94 ± 3.71 U/mg protein), SOD activity was significantly decreased in the D-gal model group (8.77 ± 1.44 U/mg protein, P < 0.05). However, the level of MDA was dramatically increased in the D-gal model group (11.63 ± 1.11 nmol/mg protein) compared with the normal control group (8.90 ± 1.02 nmol/mg protein, P < 0.01). In addition, the activity of SOD was enhanced to 12.84 ± 0.98 U/mg proteins after treatment with the higher-dose AAP (P < 0.01). Meanwhile, the level of MDA significantly decreased to 9.74 ± 1.64 nmol/mg proteins (P < 0.05) in the same group.
Table 3.
Effects of polysaccharides on SOD, MAO, SDH, MDH activities and MDA level in brain mitochondria of aged rats
| Groups | SOD (U/mgprot) | MDA (nmol/mgprot) | MAO (U/mgprot) | SDH (U/mgprot) | MDH (U/mgprot) |
|---|---|---|---|---|---|
| Normal | 11.94 ± 3.71 | 8.90 ± 1.02 | 146.09 ± 5.84 | 23.39 ± 4.10 | 61.0 ± 13.53 |
| D-gal model | 8.77 ± 1.44 | 11.63 ± 1.11 | 162.48 ± 1.79 | 15.39 ± 2.61 | 52.51 ± 1.59 |
| Vit E | 10.20 ± 2.24 | 10.09 ± 1.76 | 111.45 ± 17.00** | 22.33 ± 5.50** | 59.53 ± 9.96* |
| Lower-dose AAP | 10.09 ± 1.76 | 9.89 ± 0.93* | 111.07 ± 18.11** | 23.83 ± 6.11** | 56.24 ± 5.12 |
| Higher-dose AAP | 12.84 ± 0.98** | 9.74 ± 1.64* | 109.58 ± 13.22** | 25.50 ± 4.66** | 61.63 ± 9.42** |
All values represent the mean ± SD (n = 10).
* P < 0.05 and **P < 0.01 compared with the D-gal model group.
The activities of MAO, SDH and MDH of the brain mitochondria in the higher-dose, lower-dose and positive control groups were significantly altered as compared with those of the D-gal model group (P < 0.01 or P < 0.05). The activities of SDH and MDH in the higher-dose group (1 g/kg BW) were 25.5 ± 4.66 and 61.63 ± 9.42 U/mg protein, respectively, which were significantly higher than those of the D-gal model group (15.39 ± 2.61 U/mg protein, P < 0.01; 52.51 ± 1.59 U/mg protein, P < 0.05, respectively). In addition, MAO activity of the rats in the two AAP groups was significantly decreased as compared with that of the D-gal model group (P < 0.01). However, there were no significant differences between the two treatment groups (P > 0.01).
Protective effects of AAP on heart mtDNA of aging rats induced D-galactose
The concentration of the heart mitochondrial DNA (mtDNA) was measured to investigate the protective effects of AAP against heart toxicity of D-gal-induced rats. As shown in Fig. 3, mtDNA concentration was significantly higher in the D-gal model group than that of the normal control group (P < 0.01). However, the administration of Vit E and AAP lessened mtDNA concentration of the heart samples. The heart mtDNA concentrations of the lower-dose and higher-dose AAP groups were 359.40 ± 35.22 μg/mL and 271.8 ± 33.86 μg/mL, respectively, which were significantly lower than those of the D-gal model group (P < 0.01). And there was a significant difference between mtDNA concentration of the AAP groups and that of the Vit E group (P < 0.01).
Figure 3.
Protective effects of AAP on heart mtDNA of aging rats induced D-galactose.
AAP dramatically ameliorates D-gal-induced cardiac injury in rats
Histopathological examination of the heart tissue of the normal control rats showed that the cardiac structures were normal (Fig. 4A). However, the heart tissues of the D-gal-treated rats showed more extensive injuries, the cell nuclei were dim and the chromatin was unevenly dispersed throughout the nucleus. Moreover, the cardiac structures were not clear (Fig. 4B). It is noteworthy that these cardiac lesions were significantly ameliorated by treatments of Vit E (Fig. 4C) and AAP (Fig. 4E). These findings were consistent with the results of the heart mtDNA concentrations, demonstrating that AAP dramatically ameliorated D-gal-induced heart injury. And this effect of the higher-dose AAP was better than that of Vit E.
Figure 4.
AAP dramatically ameliorates D-gal-induced cardiac injury in rats.
Protective effects of AAP on the kidney tissue of D-gal-induced aging rats
The ROS-scavenging effects of AAP were investigated on the kidney tissue of each rat. MDA and PC contents were significantly increased (P < 0.05) in the kidney tissue of D-gal model rats, whereas an obvious decrease in SOD, GSH-PX and GSH activities was observed relative to those of the normal control group (P < 0.01 or P < 0.05). The administration of AAP resulted in a sharp decrease of MDA and PC contents and an increase of the activities of SOD, GSH-PX and GSH (P < 0.05 or P < 0.01). In addition, there were no significant differences between the two AAP groups (P > 0.05) for SOD, GSH-PX and GSH. And the two AAP groups and Vit E group did not show significant differences in MDA and PC (P > 0.05) (Table 4). Therefore, we concluded that AAP and Vit E exhibited similar effects on reducing the ROS damage of the kidneys in D-gal-induced aging rats.
Table 4.
Effects of polysaccharides on GSH, SOD and GSH-PX activities and PC, MDA levels in kidney of aging rats
| Groups | SOD (U/mgprot) | GSH-PX (U/mgprot) | MDA (nmol/mgprot) | GSH (mg/gprot) | PC (nmol/mgprot) |
|---|---|---|---|---|---|
| Normal | 4.26 ± 0.63 | 9.94 ± 1.04 | 2.61 ± 0.72 | 1.41 ± 0.33* | 2.60 ± 0.81 |
| D-gal model | 2.72 ± 0.36 | 7.70 ± 2.04 | 3.06 ± 0.19 | 0.91 ± 0.21 | 3.86 ± 0.77 |
| Vit E | 3.88 ± 0.72** | 8.21 ± 1.17 | 2.10 ± 0.23** | 1.22 ± 0.18* | 2.57 ± 0.88* |
| Lower-dose AAP | 4.03 ± 0.59** | 9.51 ± 0.83* | 2.60 ± 0.26* | 1.21 ± 0.13* | 2.86 ± 0.45 |
| Higher-dose AAP | 4.19 ± 0.30** | 9.14 ± 1.26* | 2.19 ± 0.30** | 1.29 ± 0.14** | 2.83 ± 0.56* |
All values represent the mean ± SD (n = 10).
* P < 0.05 and **P < 0.01 compared with the D-gal model group.
Effect of AAP on peripheral blood and bone marrow hematopoietic function in aging rats
The results showed that the number of WBC, RBC, PLT and BMNC in the peripheral blood of the aging group rats decreased sharply; however, AAP restored the number of cells in the peripheral blood and BMNC by inhibiting the decline of the cells of each line. (Table 5).
Table 5.
Effect of AAP on blood routine and BMNC in aging rats (n = 10, 
±s)
| Groups | White blood cell(109/L) | Red blood cell (1012/L) | Platelets (109/L) | BMNC(106/Root femur) |
|---|---|---|---|---|
| Normal | 5.54 ± 0.70** | 3.01 ± 0.77* | 453.4 ± 43.92** | 7.94 ± 0.46** |
| D-gal model | 3.98 ± 0.42 | 2.10 ± 0.43 | 381.00 ± 49.62 | 2.31 ± 1.66 |
| Vit E | 7.01 ± 0.31** | 4.20 ± 0.20** | 412.25 ± 17.39** | 4.73 ± 0.69** |
| Higher-dose AAP | 8.00 ± 0.35**# | 5.56 ± 0.19**## | 492.67 ± 21.22**## | 6.75 ± 0.37**# |
| Lower-dose AAP | 7.07 ± 0.42** | 4.65 ± 0.54** | 465.00 ± 51.32**** | 7.11 ± 0.88**** |
Notes: *P < 0.05 compared with aging model group; **P < 0.01 compared with aging model group; #P < 0.05 compared with positive drug group, ##P < 0.01 compared with positive drug group.
Influence of AAP on BMNC cell cycle
Flow cytometry analysis of BMNC cell cycle was studied, we found that BMNC occurs G0/G1 block in the bone marrow of aging rats, AAP accelerated G1 phase cell to enter S phase in cell cycle in aging rats (Table 6). It is verified from the organism level that AAP delay the aging mechanism by accelerating cell division and proliferation, which also indirectly reflected the effective hematopoietic function of AAP.
Table 6.
Effect of AAP on BMNC cycle in bone marrow of aging rats
| Groups | G0/G1(%) | G2/M(%) | S(%) |
|---|---|---|---|
| Normal | 61.47 ± 2.51** | 7.90 ± 0.33** | 30.20 ± 0.01** |
| D-gal model | 93.86 ± 3.17 | 2.27 ± 0.08 | 3.91 ± 0.04 |
| Vit E | 79.55 ± 2.12* | 8.46 ± 0.29** | 11.90 ± 0.03** |
| Higher-dose AAP | 74.32 ± 1.72**# | 9.38 ± 0.23** | 16.21 ± 0.11**# |
| Lower-dose AAP | 75.53 ± 2.55** | 10.81 ± 0.16** | 13.66 ± 0.07**& |
Notes: *Compared with aging model group, P < 0.05; **Compared with aging model group, P < 0.01; #Compared with positive control group, P < 0.05; &Compared with AAP high-dose group, P < 0.05.
Discussion
Traditional Chinese herbal medicines have played a crucial part in the healthcare for centuries and made unique contributions to the medical and health services development. In this paper, enzyme-assisted extraction technique was performed to obtain the higher AAP yield from angelica sinensis and astragalus membranaceus through L9(3) [4] orthogonal test. Under the optimal extraction conditions, the AAP yield was 13.87% while the concentration was 82.82%, which were significantly higher than AAP obtained using conventional extraction method (7.83 and 63.41%, respectively). In addition, our previous paper indicated that AAP-2 was purified in turn through chromatography on DEAE-52 column and Sephadex G-100 column. AAP-2A is a kind of neutral and homogeneous polysaccharides without protein. MW, RMS and MW/Mn of AAP-2A was 2.252, 2.252 × 103 kda, 28.4 nm and 1.038, respectively. Moreover, AAP-2A was composed of Rha, Gal, Ara and Glc, which were linked by eight connection types, i.e. 1,3-connected rhap, 1,3-connected GALP, 1,3-connected Araf, 1,5-connected Araf, 1,3,5-connected Araf, terminal GLCP, 1,4-connected GLCP and 1,4,6-connected GLCP. Lastly, the surface morphology of AAP-2A was curly, loose and flaky surface and an amorphous solid under the scanning electron microscopy. The acquisition of AAP provided reliable materials for the subsequent anti-aging research.
The subacute aging model rat induced by the well-known D-gal is widely used to evaluate the anti-aging drugs. In this study, treatment with D-gal at a dose of 150 mg/kg for 6 weeks caused a significant damage in rat brain, heart and kidney tissues, indicating that senescence model was successfully induced by D-gal. The dosage of AAP was chosen from our previous toxicology research results, which among two doses were used in this study: the high-dose AAP group (2 g/kg) and the low-dose AAP group (0.5 g/kg). Subsequently, we evaluated the anti-aging effect mechanisms of AAP on aging rats, which including telomerase activity, p16 protein expression of the brain, oxidative damage and energy metabolism of the brain mitochondria, ROS damage of the kidney tissue, heart mtDNA concentrations, histopathological changes, peripheral blood and bone marrow hematopoietic function and BMNC cell cycle.
Current theories suggest that aging is triggered by cell intrinsic and cell extrinsic changes. During cell intrinsic changes, the cell undergoes a transformation that is caused by mitochondrial dysfunction, loss of protein homeostasis (proteostasis) and DNA damage [5]. Interestingly, these processes may be interrelated, indicating that cellular aging is not the result of a single cellular malfunction but rather a combination of molecular changes. Ultimately, these molecular changes result in cellular aging, which severely affects cell and tissue function.
One of the major causes of aging is mitochondrial oxidative stress [34]. Mitochondria produce energy for cells and may increase the level of free oxygen radicals, thereby damage the proteins, lipids and DNA [35]. These damages may lead to further mitochondrial dysfunction and contribute to senescence. SOD is a major active enzyme protecting the mitochondria against oxidative damages and attacks of O2−. MDA is a specific product of lipid peroxidation [36]. With aging, the level of MDA increases and the activity of SOD decreases. Thus, both SOD and MDA are important indicators of senescence. MAO is an enzyme, which mainly exists in the mitochondrial outer membrane in the liver, kidneys and brain [37]. SDH, which plays an important role in Krebs cycle, is a marker enzyme in the mitochondrial inner membrane [38], while MDH is an oxidation-reduction enzyme which participates directly in the tricarboxylic acid cycle as mitochondrial dehydrogenases. Therefore, MAO, SDH and MDH are evaluators of the mitochondrial function, which influences the aging process. Our results found that AAP significantly increased the activity of SOD, SDH and MDH while decreased the level of MDA and MAO activity of brain mitochondria in D-gal-induced aging rats. These results indicated that AAP could conserve the normal metabolism of the mitochondria by reducing the free radical damage to the brain mitochondria. To further investigate the underlying the anti-aging mechanism of AAP, telomerase activity and p16 protein expression were determined through ELISA. Telomerase maintains regular telomere length [39, 40], which prevents cellular senescence [41], which is associated with the decrease in telomerase activity. The p16-pRb pathway is a common indicator of cellular senescence. Our results demonstrated that AAP increased telomerase activity and decreased p16 protein expression. These changes may contribute to the anti-aging effects of polysaccharides.
The mtDNA is not wrapped around histones, which makes it more vulnerable to ROS. The mtDNA mutations and deletions could lead to mitochondrial dysfunction. Numerous studies showed that senescence increases mtDNA concentrations, which is similar to the change of mtDNA during natural senescence [42]. Increased mtDNA concentrations indicate enhanced mtDNA activity, which may lead to mtDNA mutations and accumulation. Three hypothesis explaining the mechanisms of increased mtDNA concentrations are the compensatory feedback regulation, replicative advantage and modulation disorder of mtDNA deletions. Nevertheless, polysaccharides could protect the mtDNA against the damage induced by ROS [43]. We found that AAP reduced the concentrations of mtDNA, which might slow the aging process in humans.
Histopathological examination displayed that AAP ameliorated the cardiac lesions in D-gal-induced aging rats. These results demonstrated that AAP exhibited obvious protective effects against the D-gal-induced heart damage. The free radical theory points out that the decline or disappearance of the antioxidant defenses with aging is a key factor affecting the aging process [44]. SOD and GSH-PX are important antioxidant enzyme. MDA is the stable metabolite of lipid peroxidation [45]. PC, the product of protein peroxidation, is regarded as an important index of aging. GSH contains sulfhydryl groups and is related to the antioxidant capacity of the body. Our results discovered that AAP increased SOD and GSH-PX activities, as well as GSH content, whereas it decreased PC, and MDA levels in the kidneys of D-gal-induced aging rats. So, we concluded that AAP decreased the ROS damage and played an important role in delaying senility of the kidneys.
Hematopoietic microenvironment plays an extremely important role in regulating the proliferation and differentiation of hematopoietic stem cells. BMNCs, which are the core components of bone marrow hematopoietic induction microenvironment, play an important role in maintain hematopoietic function of body. Various indexes of aging rats were tested by blood routine, and the effect of AAP on hematopoiesis in aging rats was studied. And we found the whole blood cells and BMNCs increased after administration of AAP in aging rats. Generally, with aging occurs, cells will appear G0/G1 phase arrest. In this study, the cell cycle of BMNCs was analyzed by flow cytometry to explore the effect of AAP on the cell cycle. The results displayed that AAP accelerated G1 phase cell to enter S phase in cell cycle in aging rats, which also indirectly reflected the effective hematopoietic function of AAP.
In conclusion, we optimized the conditions of enzyme-assisted extraction of CAAP, which were the concentration of cellulase of 1.5%, optimal temperature for enzymatic action of 50°C, extraction pH of 5 and extraction time of 180 min. When the optimum conditions were employed, the maximum CAAP yield was 13.87% while polysaccharide content was 82.82%. More importantly, our study found that AAP significantly increased telomerase activity and decreased p16 protein expression of the brain in D-gal-induced aging rats. In addition, it reduced the contents of MAO and MDA and increased the activities of SOD, SDH and MDH of brain mitochondria in D-gal-induced aging rats. Furthermore, it lessened the concentrations of mtDNA in the heart and prevented the heart tissues from damage. At the same time, it markedly enhanced SOD, GSH and GSH-PX activities and decreased PC and MDA levels of the kidneys in D-gal-induced aging rats. In addition, AAP could restore the number of cells in the peripheral blood and BMNC by inhibiting the decline of the cells of each line. As well as, AAP accelerated G1 phase cell to enter S phase in BMNC cycle.
Taken above all, our results provide solid evidence that AAP exert protective effects against senescence through multiple actions on D-gal-induced aging rats. Therefore, we have reason to believe AAP from angelica and astragalus could be explored as a food supplement or novel natural senility-delaying medicine in food and pharmaceutical field. Above all, AAP may offer some obviously advantages, such as simplified process, low cost, high-usage, adequate water solubility and minimal side effects compared with western medicines.
Acknowledgement
This work was supported by the National Natural Science Foundation of China [grant number 81860257], the Fundamental Research Funds for Key Laboratory of Drug Screening and Deep Processing for Traditional Chinese and Tibetan Medicine of Gansu Province (No. 20180808).
Author Contributions
X.Y.P. and A.M.L. designed research; H.S., K.L.Z., C.Y.T. and B.C. performed research and analyzed the data; H.S. and X.Y.P. wrote the paper. All authors read and approved the final manuscript.
Conflict of interest statement
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
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