Anti-Aging Effects of Lycium ruthenicum Murr. Granules in Caenorhabditis elegans

Ju Zhang1,2, Dong-Sheng Jia3, Shi-Hao Zhao1,2, Xiao-Liang Xie3, Hong Chen4 and Hai-Feng Li4,*

1College of Biology Science and Engineering, Hebei University of Economics and Business, Shijiazhuang 050072, China

2Hebei Poultry Meat Deep Processing Technology Innovation Center, Shijiazhuang 050072, China

3Institute of Cash Crop, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050061, China

4School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China

ABSTRACT

In order to investigate the anti-aging effects of Lycium ruthenicum Murr. granules (HGQ) and potential underlying mechanism of actions, Caenorhabditis elegans was exploited as model organisms. The anti-aging capacity of HGQ was determined by lifespan and lipofuscin fluorescence assays in wild-type C. elegans N2. Calorie restriction pathway was detected by pharyngeal pumping rate assay and lifespan examination in sir-2.1 mutant nematodes. The antioxidant activity of HGQ was investigated by determining reactive oxygen species (ROS) level, paraquat-stress survival rate, malondialdehyde (MDA) content as well as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. We observed that HGQ exerted little effect on the growth of wild-type N2 nematodes at concentration < 0.6 mg/ml, and 0.05 mg/ml of the HGQ significantly prolonged the lifespan and reduced the lipofuscin level in N2 nematodes. However, the HGQ failed to influence the pharyngeal pumping rate in N2 nematodes or to affect the lifespan of sir-2.1 mutant nematode VC199, indicating the anti-aging effect of HGQ is independent of calorie restriction pathway. Further experiments showed that the HGQ decreased ROS and MDA contents and increased the survival rate and SOD (not CAT or GPx) activities in paraquat-stressed N2 nematodes. Our results suggest that HGQ can effectively delay the senescence of C. elegans, and this effect may be related with its significant antioxidant activity.

Article Information

Received 29 July 2019

Revised 23 October 2019

Accepted 21 December 2019

Available online 06 March 2020

Authors’ Contribution

ZJ, LHF, XXL and JDS designed the study. ZJ, ZSH, CH and LHF performed experimental work and analyzed the data. ZJ and LHF wrote the article.

Key words

Lycium ruthenicum Murr., Caenorhabditis elegans, Aging, Oxidative stress, Reactive oxygen species, Antioxidant enzyme.

DOI: https://dx.doi.org/10.17582/journal.pjz/20191016031055

* Corresponding author: [email protected]

0030-9923/2020/0003-1061 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

With improving living standards and health awareness around the world, delaying the senescence has recently gained enormous attentions in China. The agents that claim to prolong lifespan include traditional medicinal, alternative medicine and food items (Breckenridge, 2013). Among them, Lycium ruthenicum Murr. has been recorded in the pharmaceutical classics such as Uyghur Medicine, The Four Medical Tantras, and Jing Zhu Materia Medica. This has been reported to enhance immune function, enrich yin and nourish kidney, and delay senescence (Liu, 1999). Recent studies have shown that Lycium ruthenicum Murr. contains a variety of bioactive components (Zhao et al., 2018), and its mature berries are rich in polyphenols such as anthocyanin and flavone, which have strong pharmacological activities such as anti-oxidation (Wu et al., 2016; Chen et al., 2018) anti-fatigue (Ni et al., 2013), anti-cancer (Zhang et al., 2019), and immunity enhancing (Peng et al., 2014) properties.

At present, people often extract Lycium ruthenicum Murr. in water, and then concentrate and dry to formulate Lycium ruthenicum Murr. granules (HGQ). These are then used in tea to enhance human immunity and delay aging. However, there is no systematic research and reports on its anti-aging activity and any underlying molecular mechanisms, and thus warrant studies.

A large number of studies have shown that natural onset of aging is closely related to the generation of excessive free radicals in vivo. Therefore, enhancing the body’s antioxidant capacity and scavenging excessive free radicals can effectively delay aging (Si et al., 2014; Simioni et al., 2018). In addition, previous investigations have shown that “calorie restriction” also can delay aging and prolong lifespan through enhancing the activity of longevity genes such as sir-2 (Testa et al., 2014; Al-Regaiey, 2016). Polyphenols, including anthocyanin and flavonoid, can exert anti-aging biological activities through their strong anti-oxidation and free radicals scavenging effects (Zilić et al., 2012; Loffredo et al., 2017; Cirillo et al., 2016), and regulations of calorie restriction signaling pathways (Kulkarni and Cantó, 2015; Timmers et al., 2011). The study conducted by Shang et al. (2017) showed that Lycium ruthenicum Murr. is rich in active polyphenol components content (up to 4%), and the average content of anthocyanin can reach to 2.3%. These results provide a foundation to further investigate the anti-aging activity of Lycium ruthenicum Murr. granules (HGQ).

In this study, we applied a model organism Caenorhabditis elegans to assess the anti-aging impacts of HGQ. The Caenorhabditis elegans was adapted mainly due to its relatively short life cycle, simple experimental operation, rich genetic resources, and key-signaling pathway similarities with human. Different approaches were used to assess the lifespan, anti-oxidation and calorie restriction pathways. Collectively, the data proposed the anti-aging activity and provided novel insights into the molecular mechanisms in anti-aging effects of HGQ.

 

Materials and methods

Strains and materials

Wild-type C. elegans N2, mutant strain VC199 [sir2.1 (ok434)] and Escherichia coli (OP50 and NA22) were obtained from the Caenorhabditis Genetics Center (University of Minnesota) and maintained under standard conditions.

Lycium ruthenicum Murr. granules were purchased from Hebei Kangping Health Food Co., Ltd. The granules were prepared by concentrating and drying the water extract of Lycium ruthenicum Murr.

Reagents and instruments

Agar, tryptone, yeast extract, ampicillin, and 2’-deoxy-5-floxuridine were purchased from Sigma Company, USA. Pierce BCA Protein Quantitative Analysis Kit was purchased from ThermoScientific, USA; Reactive Oxygen (ROS) Assay Kit, Total Superoxide Dismutase (SOD) Activity Assay Kit, Lipid Peroxidation MDA Assay Kit, Catalase (CAT) Assay Kit and Total Glutathione Peroxidase (GPx) Assay Kit were purchased from Beyotime Biotechnology Company, Ltd., China.

Full-wavelength microplate reader (BioTek Co., Vermont, MA, USA); Fluoroskan Ascent FL microplate reader (Thermo Electron Co., Waltham, MA, USA) and grinding machine (Shanghai Jingxin, JX-24) were applied to record diverse assays.

Sample preparation

Appropriate amount of HGQ was taken and dissolved in the liquid S. Medium of C. elegans to make 5 mg/ml HGQ sample solution. The solution was filtered through 0.22 μm sterile millipore membrane, and was saved aside as standby until use.

Food clearance assay

The food clearance assay was carried out to determine the range of HGQ concentrations as described (Zhang et al., 2016). Synchronized L1 nematodes N2 (10-15/well), E. coli NA22 (OD570≈0.70), and sample solution (final concentration 0.01-1.0 mg/ml) were added to a 96 wells plate (final volume 100 μl/well) and incubated at 20°C. The control group was added with S. Medium, and 8 parallel wells were set for each sample concentration. The absorbance of the culture was measured at 570 nm every day from day 1-7 using a full-wavelength microplate reader (BioTek Co., Vermont, MA, USA). The day HGQ added was designed as day 1.

Lifespan assay

The lifespan assay for C.elegans was performed in liquid culture at 20°C as described previously (Zhang et al., 2012). Briefly, synchronized L1 larvae nematodes were incubated with E. coli NA22 in S. Medium for about 42h to L4 larvae and then treated with 100 μg/ml ampicillin, and 75 μg/mL 5-fluoro-2’-deoxyuridine to prevent progeny growth for another 24h until young adult larvae. Then animals were transferred into 96 wells plate (10-15 nematodes/well) and treated with the HGQ (10 wells/concentration). The day of HGQ addition was designated as day 0 during the analysis. The alive animals were counted microscopically every two days based on their movement until all were dead.

Lipofuscin level assay

The lipofuscin assay was performed as described previously (Gerstbrein et al., 2005). Nematodes were prepared as described in lifespan assay but black-96 wells plates were used instead, and the fluorescence intensity of lipofuscin was determined by Fluoroskan Ascent FL microplate reader (Thermo Electron Co., Waltham, MA, USA) with 355 nm excitation and 460 nm emission every 3 days from day 1 to day 10.

Pharyngeal pumping rate assay

Pharyngeal pumping rate assay was carried out as previously described (Wang et al., 2016). Nematodes were prepared as the method of lifespan assay with a modification of 24-well plate with about 500 nematodes per well. After incubation at 20°C for 4 days, nematodes were collected and transferred on the NGM plate coated with E. coli. OP50. After an adaption of another 30 min at 20°C, the pharyngeal pumping rate was microscopically counted for about 50 nematodes per concentration treatment, and the data was expressed as mean ± SEM of three independent experiments.

Determination of ROS level

The ROS level was determined using the 2’-7’-dichloro-fluorescein diacetate as has previously been described (Wu et al., 2006). Briefly, synchronized young adult nematodes were prepared as shown in lifespan assay and then added into a 24-well plate with or without HGQ treatments in three parallel wells for each concentration. After incubation for 2 days at 20°C, 10 μl of paraquat (ROS induction reagent, final concentration of 10 mM) was added, and then the nematodes were cultured for another 2 days at 20°C. Approximately 1500 nematodes per treatment were collected and washed three times with S. Medium and then homogenized in PBST buffer (1*PBS with 0.1%Tween 20) using a grinder at 4°C for 6min (grinding for 45 s, suspending 15 s). The supernatant collected through centrifugation (4°C, 12000 rpm, 10 min) was used as nematode lysate. Pierce BCA Protein Quantitative Analysis Kit (Beyotime, Nanjing, China) and ROS Assay Kit (Beyotime, Nanjing, China) were used to determine the protein concentration and ROS level of the lysate, respectively. The results were normalized by protein content.

Determination of MDA content

The MDA content assay was performed as previously described (Xiao et al., 2014). The treatment of nematodes, the preparation of lysate and the quantification of protein content were same as ROS detection. The MDA content was measured using Lipid Peroxidation MDA Assay Kit (Beyotime, Nanjing, China) and results were normalized by protein contents.

Paraquat survival assay

The oxidative survival assay was performed as described previously using paraquat (Wang et al., 2014). The preparation of C. elegans was performed as shown in lifespan assay. Synchronized young adult nematodes were transferred to 96 wells plates along with HGQ treatment. After incubation for another 24 h, the nematodes were exposed to 60 mM paraquat. The time of HGQ addition was designated as hour 0 and the number of live or dead nematodes were scored microscopically every 12 h based on their movement until all were found dead.

Determination of antioxidant enzyme activity

Antioxidant indices were determined as previously described (Xiang et al., 2017; Wang et al., 2016). The nematodes and lysate were prepared the same as ROS detection and the protein content of lysate was determined using BCA Assay. The SOD, CAT and GPx activities were measured using respective Assay Kits (Beyotime, Nanjing, China), combined with a full-wavelength microplate reader, and the results were normalized by protein contents.

Statistical analysis

The statistical analysis was performed primarily using GraphPad Prism 5.01 (GraphPad Software, San Diego, CA). All experiments were performed more than three times independently. The C. elegans lifespan and survival data were analyzed by the Logrank (Mantel-Cox test) method and the representative Kaplan–Meier survival curves were shown. The other experimental data were expressed as mean ± SEM or mean ± SD. One-way analysis of variance (ANOVA) and Dunnett’s post-test and un-paired t-test were performed to analyse the difference between the experimental and control groups. p<0.05 was considered to be significantly different.

 

Results

Concentration of HGQ administration

The food clearance rate can indirectly reflect the influence of compounds on the growth and reproduction of C. elegans at different concentrations (Voisine et al., 2007). In this study, the initial concentration of HGQ was estimated by food clearance assay in wild-type N2 nematodes. The results shown in Figure 1 indicated that the concentration of < 0.5 mg/ml had no significant influence on the food clearance compared with control, but the concentration of ≥ 0.6 mg/ml prominently decreased the food clearance rate, suggesting that the higher concentration can affect the normal growth of C. elegans. Therefore, we considered a concentration range of 0.05 ~ 0.5 mg/ml for subsequent experiments.

Senescence of C. elegans

As lifespan is regarded as an unequivocal anti-aging index (Wang et al., 2014), we applied the lifespan assay to assess the anti-aging activity of HGQ in N2 nematodes. The median lifetime of nematodes treated by HGQ at the concentration of 0.05 mg/ml was significantly extended by 7% from 26 days to 28 days (p < 0.05) compared with control group (Fig. 2A), while the other concentrations had no obvious influence on the lifespan of N2, suggesting a certain activity of anti-aging effect of HGQ at lower concentrations.

Studies have shown that age-related autofluorescent lipofuscin accumulated in the intestine over time is regarded as a biomarker of aging, and their accumalation is shown to be inversely correlated with longevity (Terman and Brunk, 2004). The model organism of C. elegans has transparent body, and the changes in the accumulation of lipofuscin in vivo can be quantitatively analyzed based on its optical properties using a fluorescent microplate reader. Therefore, we further studied the anti-aging activity of HGQ using lipofuscin content test. As shown in Figure 2B, compared with the control group, the relative fluorescence intensity of the 0.05 mg/ml HGQ treatment group was significantly decreased on day 4, 7, and 10, indicating that HGQ could significantly reduce lipofuscin content in the body of nematodes at this concentration, and thereby inhibit the aging of nematodes, showing a certain anti-aging activity, while the HGQ sample with a concentration of 0.1-0.5 mg/ml had no significant effect on lipofuscin content.

Calorie restriction pathway of C. elegans

A large number of studies have confirmed that limiting calorie intake has certain anti-aging biological effects and can delay the occurrence of aging-related diseases (Sutphin and Kaeberlein, 2008; Timmers et al., 2011). In order to confirm whether the anti-aging activity of HGQ is related to the calorie restriction of C. elegans, we detected the pharyngeal pumping rate in N2 nematodes to investigate the influence of HGQ on the food swallowing ability. Compared with the control group, no obvious changes of pharyngeal pumping rate were observed in the nematodes treated by HGQ at the tested concentration, indicating that it had little influence on the feeding and calorie intake (Fig. 3A).

Studies have also shown that the anti-aging effects of calorie restriction pathways is related to the mechanisms such as enhancing the activity of longevity genes such as sir-2.1 and reversing the expression of longevity genes (Testa et al., 2014; Al-Regaiey, 2016). In order to further verify the relationship between the anti-aging activity of HGQ and the calorie restriction pathway, we examined the influence of HGQ on the lifespan of C. elegans VC199, which has the mutants defective in the sirtuin-homologue SIR-2.1. The results in Figure 3B revealed that the lifespan of VC199 was also prolonged by HGQ at the concentrations of 0.05, 0.1 and 0.3 mg/ml, among which the extensive effect at 0.05 mg/ml was the most significant, similarly as in N2 nematode, suggesting that the anti-aging activity of HGQ was not related to the regulation of gene sir-2.1.

 

 

 

Oxidative stress level in C. elegans

According to the free radical theory of aging, the occurrence of aging is directly related to the formation of excess free radicals in vivo and the resulting oxidative stress environment (Finkel and Holbrook, 2000). In order to study whether the anti-aging activity of HGQ is related to the reduction of oxidative stress level, we took the paraquat, a herbicide capable of generating superoxide radicals, to induce oxidative stress in nematodes and then examined the influence of HGQ on the ROS level and malonaldehyde (MDA, ROS-mediated membrane lipid oxidation product) content. Compared with the control group, the 0.05 mg/ml HGQ treatment could significantly reduce the ROS level in paraquat-stressed N2 nematodes, while 0.1 mg/ml treatment exhibited a lowering and non-significant trend (Fig. 4A). The high-concentrations of 0.5 and 0.3mg/ml administration showed a little effect on ROS levels in nematodes.

The MDA content measurement (Fig. 4B) revealed that compared with the control group, the low-concentration of 0.05 mg/ml HGQ treatment significantly reduced the MDA content in paraquat-stressed N2 nematodes. It suggested that, HGQ treatment could effectively reduce the in vivo oxidative stress level and exerts certain anti-oxidation capacity in C. elegans at low concentrations. Thse results were consistent with the anti-aging activity of HGQ at low concentration treatment.

Antioxidant capacity in C. elegans

In order to further verify the antioxidant capacity of HGQ, we simultaneously determined the nematodes survival rate under 60 mM paraquat oxidative stress. The results shown in Figure 5 indicate that the survival time of N2 nematodes exposed to paraquat was obviously extended after the treatment of HGQ at the concentration of 0.05, 0.1 and 0.3 mg/ml, especially the 0.05 mg/ml treatment exhibiting stable significant difference (p < 0.05) compared with control among 3 independent experiments, while no influence observed at the high concentration of 0.5 mg/ml. These results confirmed that the HGQ was capable of reducing oxidative damages in C. elegans, especially at the low concentration treatment.

Activity of antioxidant enzymes in C. elegans

Antioxidant enzymes are key substances in the cellular antioxidant system. Enhancing the activity of antioxidant enzymes helps to clear excess ROS and alleviate oxidative stress (Finkel and Holbrook, 2000). In order to explore the mechanism of HGQ’s antioxidant activity, we detected the influence of HGQ on the activity of SOD, CAT and GPx in C. elegans. Compared with the control group, HGQ treatment at concentrations of 0.05 and 0.3 mg/ml could significantly enhance the SOD activity (Fig. 6A), however the effects on the enzyme activities of CAT and GPX were non-significant (Fig. 6B, C). These results indicate that the free radical scavenging activity of HGQ should be mainly related to the increase of SOD activity in nematodes.

 

 

Discussion

Due to the rich polyphenolic active substances, Lycium ruthenicum Murr. has been extracted and dried to make Lycium ruthenicum Murr. granules (HGQ) in tea to be exploited as an anti-aging agent. However, studies were still lacking to provide systematic scientific verification on its biological effects. The model organism C. elegans has the advantages at overall animal level such as short life cycle, intact organ system, and simple and economical operation. Thus it has been widely used in the fields of biomedicine and health food in recent years. For example, Wang et al. (2019) and Zhang et al. (2019) have used the model organism C. elegans to study the antioxidant and anti-aging activities of abalone protein hydrolysate and rose tea polyphenol. Therefore, we studied the activity effect and the mechanism of HGQ using the model C. elegans.

Our results revealed that HGQ could significantly extend the median lifespan and reduce the accumulation of lipofuscin in the wild-type C. elegans N2 at a low concentration of 0.05 mg/ml treatment, showing a good anti-aging activity. The pharyngeal pumping rate assay and the lifespan examination of sir-2.1 mutant nematode showed that HGQ not only could not affect the food and energy intake of nematodes, but also exhibited an irrelevance between the life-extension and sir-2.1 gene, indicating the anti-aging effect of HGQ is independent of calorie restriction pathway. This result is consistent with Zhang et al. (2018) finding, which have revealed that polysaccharides from Dendrobium could effectively delay the aging and increase the anti-stress ability of C. elegans N2, but it also did not affect the nematode’s pharyngeal pumping rate and energy intake.

Further studies have found that HGQ can effectively reduce the ROS level and MDA content in vivo, increase the survival rate and enhance the activity of SOD in C. elegans N2 under paraquat oxidative stress, showing a good antioxidant capacity. Interestingly, this activity also showed a drug concentration-dependence with better effect at 0.05 mg/ml treatment, similarly with the HGQ’s anti-aging activity, which suggesting a considerable contribution of the antioxidant to the anti-aging effect of HGQ. The researches of Chen et al. (2016) and Wang et al. (2014) have also shown the similar results that both Hongshan flower Chinese cabbage extract and Angelica protolysate can delay the aging of C. elegans N2 through its good antioxidant activity in vivo and in vitro. So, our results suggest that HGQ can effectively delay the senescence of C. elegans, and this effect may be related with its significant antioxidant activity.

Previous findings (Song et al., 2014; Chen et al.,2018; Liu et al., 2013) have shown that the crude methanol extract, polyphenols and polysaccharide components from the Lycium ruthenicum Murr. exhibit free radical scavenging and antioxidant activities. Therefore, we reasoned that the anti-aging activity of HGQ should be related to its rich antioxidant active components. However, a study conducted by Shang et al. (2017) have revealed that there were great differences in the chemical composition of Lycium ruthenicum Murr. from different producing areas. For example, the free radical scavenging ability of Lycium ruthenicum Murr. from Alatanaobao, Alxa Right Banner, Inner Mongolia is the strongest, while the reducing ability of Lycium ruthenicum Murr. from Soumber, Mongolia is the strongest. Therefore, further studies will combine the raw materials of different producing areas to analyze the active function, thus providing a more optimized and sufficient theoretical basis for the deep development of Lycium ruthenicum Murr. granules.

 

Conclusions

In summary, data presented in this study indicate that Lycium ruthenicum Murr. granules can effectively delay the aging of C. elegans, and the anti-aging activity may be related to its significant antioxidant effect, but not related to the calorie restriction pathway. These finding provide foundational benefits of the HGQ in human healthcare and highlight the need of future studies in this domain.

 

Acknowledgements

This work was supported by the Science and Technology Research Project of Hebei Higher Education (No. ZD2018013); Key Research Project of Research Fund of Hebei University of Economics and Business (No. 2017KYZ05); Research Project Entrusted by Hebei Academy of Agriculture and Forestry Sciences (No. F18C02); Project Supported by China Agriculture Research System (No. CARS-21) and Natural Science Foundation of Guangdong Province (No. 2017A030313864).

 

Statement of conflict of interest

We declare no conflicts of interest in this study.

 

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