Aqueous root extract of Asparagus cochinchinensis (Lour.) Merr. Has antioxidant activity in D-galactose-induced aging mice

Background Extracts of plants have been considered as sources of natural antioxidant agents. In this study, we aimed to explore the antioxidant capacity of the aqueous root extract of Asparagus cochinchinensis (Lour.) Merr. Methods Using vitamin C (Vc) as a positive control, we analyzed the aqueous root extract of A. cochinchinensis free radical scavenging ability in vitro. We also established a mouse aging model using D-galactose and then treated it with aqueous root extract or Vc. The blood cell count and superoxide dismutase (SOD), catalase (CAT), and nitric oxide synthase (NOS) activities as well as malondialdehyde (MDA) and nitric oxide (NO) contents were measured; pathological examination of tissues was performed; and SOD, glutathione peroxidase (GPX), and NOS expression levels in the serum, liver, and brain tissues were investigated. Results In vitro, compared with the antioxidant Vc, the aqueous root extract showed similar 1,1-Diphenyl-2-picrylhydrazyl radical and 3-ethylbenzothiazoline-6-sulfonic·scavenging activities and even significantly increased superoxide anion (p < 0.05) and hydroxyl radical (OH) (p < 0.01) scavenging activities. The aqueous extract significantly increased the white blood cell count as well as enhanced SOD, CAT, and NOS activities (p < 0.01) in aging mice. In addition, the aqueous extract increased the NO content (p < 0.05) and reduced the MDA content (p < 0.05). Conclusions The aqueous root extract of A. cochinchinensis showed as strong antioxidant ability as Vc and might prevent aging by reducing radicals.


Background
Aging, which refers to a multidimensional process of physical and psychological changes, is closely related to most human diseases [1]. Aging is associated with reduced antioxidant enzyme, such as superoxide dismutase (SOD), activities, thereby attenuating the removal of oxygen radicals [2]. Currently, exogenous radical scavengers, such as butyl hydroxy toluene, butylated hydroxylanisole, and tert-butylhydroquinone, have been successfully used for resisting disease development [3]. However, the toxicity of artificial antioxidants can lead to risks of DNA damage and malignancy [4]. Thus, the safety of artificial antioxidants remains a subject of debate [5].
Natural antioxidants have attracted attention as replacements for artificial antioxidants, [6]. Okra leaf [7] and Rubus alceifolius Poir [8], which are traditional Chinese medicines, are deemed to be potential resources of natural antioxidants. Asparagus cochinchinensis (Lour.) Merr. (A. cochinchinensis), one of the medicinally important plants, has antibacterial, anti-inflammatory, anticancer, and antioxidant effects [9][10][11][12]. A methanol extract from A. cochinchinensis has a neuroprotective effect in cerebral infarction model animals [13]. Our recent study also demonstrated the antioxidant ability of shoot extract of A. cochinchinensis in mice with D-galactose-induced aging [14]. However, antioxidant effects of the tuberous root of A. cochinchinensis remain unclear.
A previous study revealed that an aging model can be induced using D-galactose [15]. In this study, Dgalactose-induced aging mouse model was established, and then the aging mice were treated with aqueous root extract or Vc. The blood cell count and superoxide dismutase (SOD), catalase (CAT), and nitric oxide synthase (NOS) activities as well as malondialdehyde (MDA) and nitric oxide (NO) contents were measured; pathological examination of tissues was performed; and SOD, glutathione peroxidase (GPX), and NOS expression levels in serum, liver, and brain tissues was investigated. In addition, we investigated the effect of the aqueous root extract of A. cochinchinensis on radical scavenging ability in vitro. We also aimed to explore the antioxidant mechanism and further application of the aqueous root extract of A. cochinchinensis.

Pharmaceutical preparation
The aqueous root extract was prepared as described previously [16]. A. cochinchinensis specimens were collected from Lewang Town, Wangmo County, Buyi and Miao Nationalities Autonomous Prefecture, Guizhou Province, and identified by Professor Wu Xian at Hunan Huaihua College. This material (voucher specimen number: HJH20150930013) was deposited in the Plant Herbarium, Institute of Biology, Guizhou Academy of Sciences. The roots of A. cochinchinensis were dried with hot air. After grinding, powdered roots (20 g) were dissolved in water (160 mL), and then boiled and extracted three times. The three extracts were combined, filtered, and concentrated using a rotating evaporator to obtain the aqueous extracts of A. cochinchinensis, in accordance with Chinese pharmacopoeia [17]. Theaqueous extracts were dissolved in distilled water to a stocking solution of 0.7 g/mL and frozen until use.

Measurement of radical scavenging ability in vitro
1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used for antioxidant assays. DPPH·and 3ethylbenzothiazoline-6-sulfonic (ABTS + ) scavenging activities was measured in accordance with the procedure described in our previous report [14]. In brief, 2 mL of 0.7 g/mL root extract solution was reacted with 2 mL of 1.25 × 10 −4 mol/L DPPH or 30 μL of 0.7 g/mL root extract solution was reacted with 3 mL of 7 mmol/L ABTS + at room temperature in the dark, after which the absorbance was detected at 517 or 734 nm, respectively. Negative and positive controls were ethanol (solvent) and vitamin C (Vc), respectively. Furthermore, the superoxide anion and hydroxyl radical (OH) levels were measured using commercial kits (Jiangcheng Bioengineering Institute, Nanjing, China). The absorption value was detected using a microplate reader (Thermo).

Animal models and drug treatment
Approval was obtained from the Animal Ethics Committee of the Animal Laboratory Center of Xiangya Medical School of Central South University prior to using the animals for the following experiments. In total, 80 healthy male KunMing (KM) mice, which weighed 20 ± 2 g and were aged 2 months old, were provided by the Xiangya Medical School of Central South University. Mice were randomly and equally assigned to four groups: negative control, aging model, Vc positive control, and extract treatment. Mice in the negative control group were subcutaneously injected with saline (100 mg/kg) daily. In the aging model group, aging was induced by D-galactose, in accordance with a previously described method [18]. Mice in the aging model, Vc positive control, and extract treatment groups received a subcutaneous injection of 500 mg/kg Dgalactose daily; meanwhile, mice in Vc positive control or extract treatment groups were treated with Vc or aqueous root extract (200 mg/kg) daily by intragastric administration for 30 consecutive days.

Preparation of blood and pathological tissue sample
After the final drug administration for 24 h, 20 μL blood samples were taken by eyeball extirpation and then centrifuged at 3000 r/min for 10 min. The supernatant was used for the following experiments.
The mice were killed and then their brains, hearts, kidneys, and livers were isolated. Tissues were pre-fixed in Bouin's solution for 24 h, followed by gradient ethanol dehydration, paraffin embedding, slicing (5-7 μm), and regular HE staining. Finally, a microscope (Motic Group Co., Ltd., Xiamen, China) was used to observe these sections.

Measurements of the NOS, SOD, and CAT activities and NO and MDA contents
Tissues (including livers, kidneys, hearts, and brains; 0.3-0.6 g) were ground. After centrifugation at 1000 r/min for 5 min, NOS, SOD, and CAT activities as well as NO and MDA contents in the supernatants were measured using commercial kits (Jiangcheng Bioengineering Institute).

Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR)
Total mRNA of the serum was extracted using an RNA extraction kit (Ambiogen Life Science Technology Ltd.). Then, cDNA was synthesized using a first-strand cDNA synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China). Primers used in the present study are shown in Table 1. PCR reaction was conducted in a 50 mm 3 system containing 5.0 mm 3 of 10× PCR buffer, 10 pmol forward and reverse primers, 0.3 mm 3 of 10 mM dNTPs, 2 U Taq (Ferments, USA), and 60 ng of the template under the following conditions: 5 min at 94°C, followed by 35 cycles of 60 s at 94°C, 50 s at 56°C or 67°C, and 50 s at 72°C and 10 min at 72°C. PCR reactions were terminated before the reaction reached platform, and amplicons were examined using gel electrophoresis.

Statistical analysis
Statistical evaluation was performed using SPSS 18.0 package (SPSS Inc., Chicago, IL, USA). All data are expressed as mean ± S.D. The t-test was used to compare differences between the two groups in the in vitro analysis. One-way analysis of variance was used

Radical scavenging ability in vitro
Compared with the Vc positive control group, 0.7 g/mL aqueous root extract of A. cochinchinensis had similar DPPH·and ABTS + ·scavenging activities, but significantly increased superoxide anion (p < 0.05) and OH scavenging activities (p < 0.01) (Fig. 1), which suggested strong radical scavenging ability of the aqueous root extract in vitro.

Antioxidation ability in vivo
As shown in Table 2, CAT, NOS, and SOD activities were clearly lower in the serum, kidney, heart, brain, and liver samples (p < 0.05) of the aging model group than in that of the negative control group. SOD, NOS, and CAT activities in the extract treatment group were elevated (p < 0.05) compared with those in the aging model group. No significant difference was observed in NOS, CAT, and SOD activities between the extract treatment group and Vc group. These results indicate that the aqueous root extract had similar activities for enzymes of the antioxidant system in comparison with Vc. Moreover, compared with the control group, decreased NO content and increased MDA content were observed in the aging model group (p < 0.05), whereas the extract of A. cochinchinensis significantly increased the NO content in aging mice and reduced their MDA content (Table 3). Meanwhile, NO and MDA contents were similar between extract treatment and Vc groups ( Table 3).
Effect of the aqueous root extract of A. cochinchinensis on the microstructure of mouse viscera HE results of tissues are shown in Fig. 2. In the control group, myocardial fiber cells were shuttle-shaped and arranged in parallel; had clear intercellular boundaries, close packing, clear visible band and intercalated disc; and distinct gradation in kidney, heart, brain, lung, and liver tissues. However, plump myocardial fiber cells, clear capillary vessels, and a widened interval were found in these tissues in the aging model group. After treatment with the aqueous root extract of A. cochinchinensis or Vc, a clear improvement in mouse viscera was observed. However, the aqueous root extract of A. cochinchinensis showed protective effects on the liver, brain, and kidney.

Transcriptional gene expression levels related to antioxidation
As shown in Fig. 3, compared with the control group, NOS, SOD, and GPX expression levels were significantly reduced in the aging group. However, compared with the aging model group, NOS, SOD, and GPX expression levels in the serum after treatment with root extract and Vc were elevated. NOS and GPX gene expression levels were increased in the extract treatment group compared with those in the negative control group. Moreover, NOS, SOD, and GPX gene expression levels in the liver of the negative control group was similar to those in the liver of the extract treatment group. In addition, NOS, SOD, and GPX gene expression levels in the kidney were similar among the control, Vc, and extract treatment groups.

Discussion
The present study investigated the mechanism of action of the natural antioxidant A. cochinchinensis in the aging process. The results revealed that the aqueous root extract of A. cochinchinensis showed strong radical scavenging ability in vitro and could also increase SOD and GPX expression levels and SOD and CAT activities in vivo. Moreover, the aqueous root extract of A. cochinchinensis reduced the MDA content, increased the NO content, and played important roles in pathological changes in the liver, kidney, and brain.  A previous study showed that radicals could cause all kinds of diseases related to oxidative damage, such as cancer, cardiovascular disease, immune system defects, and aging [19]. Natural substances have been considered to inhibit radical-caused damage [20] and radical production [21]. MDA, as a peroxide product of lipids, could reflect oxygen radical production [22]. SOD could reduce MDA production by inducing a disproportionation reaction of superoxide radicals [23]. CAT, as an antioxidant enzyme, was also reported to influence peroxide hydrogen production [24]. GPX was also reported to reduce oxidative stress [25]. This study found that the aqueous root extract of A. cochinchinensis showed radical scavenging ability in vitro and could significantly influence SOD, CAT, GPX, and MDA activities or contents in vivo, indicating that the radical scavenging ability of aqueous root extract might be mediated by antioxidant enzymes.
Moreover, we found that NOS gene expression and NO content were upregulated in aging mice after been treated with the aqueous root extract of A. cochinchinensis. NO is produced from L-arginine by NOS, and reduced NOS activity could result in aging [26]. Therefore, the aqueous root extract of A. cochinchinensis might promote antioxidant activity by enhancing NOS expression and NO content. Previous studies also indicated that NO was associated with a series of physiological processes and NO could enhance the antioxidant activity [27,28]. In addition, the overexpressed NO might cause aging by inducing toxicity of superoxide anions [29,30]. Thus, we speculated that the aqueous root extract of A. cochinchinensis might influence NO production and then delay the aging process by destroying homeostasis.

Conclusions
In conclusion, the antioxidant ability of the aqueous root extract of A. cochinchinensis was shown to occur through enhancement of antioxidant enzyme expression levels, increase in NOS, CAT, and SOD activities and the NO content, and reduction in the MDA content. However, chemical analysis of the extract is still required for identifying its active ingredient.