Unlocking the Antioxidant Potential of White Tea and Osmanthus Flower: A Novel Polyphenol Liquid Preparation and Its Impact on KM Mice and Their Offspring

doi:10.21203/rs.3.rs-3100776/v1

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Unlocking the Antioxidant Potential of White Tea and Osmanthus Flower: A Novel Polyphenol Liquid Preparation and Its Impact on KM Mice and Their Offspring

https://doi.org/10.21203/rs.3.rs-3100776/v1

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Scope: White tea rich in polyphenols has excellent antioxidant effect, but there is still some space for research and development of its functional application. In this study, we formulated a liquid polyphenol preparation derived from white tea and osmanthus flower(wtofLPP), which exhibited rich antioxidant content and desirable rheological properties.

Methods and results: The average particle size of wtofLPP was determined using a nanoparticle analyzer to assess the rheological properties of wtofLPP. The antioxidant performances in vitro were evaluated by in vitro antioxidant method. Notably, the wtofLPP substantially enhanced superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzyme activities in KM mice and their offspring. Furthermore, it reduced malondialdehyde (MDA) levels, extended swimming time, and significantly increased small molecules such as glutathione (GSH), ceruloplasmin, and Vitamin E, which are known to exert antioxidant activities. The preparation also elevated liver glycogen levels, while decreasing lactic acid and urea nitrogen concentrations. Oxidative damage poses significant health risks, leading to alterations in physiological markers and potentially causing severe epigenetic effects on offspring.

Conclusion: Collectively, these findings indicated that the liquid polyphenol formulation(wtofLPP) protected against fatigue-induced oxidative damage, offering potential health benefits for both the parent generation and their progeny.

liquid preparation

polyphenol

anti-fatigue

oxidative stress

This research paper aimed to evaluate the effect of a liquid polyphenol preparation made from white tea and osmanthus flower on antioxidant indices in KM mice and their offspring. Specifically, the impact of the wtofLPP on enzyme activities of SOD, Gsh-PX, and CAT, MDA level, swimming time, GSH, cupruloplasmin, Vitamin E, liver glycogen level, lactic acid, and urea nitrogen was determined. The paper also explores the potential of functional liquid preparations, such as tea preparation, as a convenient and ready-to-drink product that provides health benefits by providing nutrients or other substances, such as flavonoids, specific amino acids, and antioxidants. The findings of this research could provide insights into the potential use of polyphenols as a natural antioxidant to prevent oxidative stress-related diseases.

Oxidative stress is a well-known physiological response induced by various types of exercises, including prolonged endurance, resistance, high-intensity anaerobic, and centrifugal exercise. This response results in an increase of oxidation biomarkers in skeletal muscles and blood (Bloomer & Goldfarb, 2004; Groussard et al., 2003; Scheffer et al., 2012) .Excessive exercise leads to the accumulation of nitrogen radicals (RNOS) in the body, causing oxidative stress and damaging biomacromolecules(Bai et al., 2014; Zhang et al., 2011).

Polyphenols are compounds with benzene ring and phenolic hydroxyl structures that exhibit potent antioxidant capacity (6–7 times of vitamin E) and hypoglycemic, anti-aging functions and are widely present in plants(Nan, Meng, Di-Zhe, & Xiao-Yuan, 2016). The polyphenol structure can remove free radicals, thereby improving the activity of antioxidant enzymes and achieving antioxidant effects (Perron & Brumaghim, 2009). Researchers extract polyphenols from a variety of natural substances, which are abundant active components with strong antioxidant activity and(Qiang & Jingwen, 2019) can remove a large number of ROS and act as antioxidants against damage (Yahfoufi, Alsadi, Jambi, & Matar, 2018).

Functional liquid preparations are becoming increasingly popular among consumers due to their health benefits and convenience. Among various functional preparations, tea preparations are popular due to their long history and cultural significance. However, traditional tea soaking methods may not cater to modern consumer needs, leading to the emergence of liquid tea preparations. At present, green tea, black tea and oolong tea liquid preparations are prevalent, while liquid preparations of yellow tea, white tea are relatively scarce. The development of white tea liquid preparations has been limited due to its slow growth rate. However, white tea contains polyphenols and caffeine that can effectively eliminate excessive radicals in the body, delay aging, and enhance antioxidant capacity while promoting endocrine organ activity and reducing fatigue. Polyphenols, one of the active substances in tea, have remarkable antioxidant capacity. Its hydroxyl scavenging ability is stronger than that of vitamin C, increasing with dose, making it an excellent candidate for application in functional liquid preparations. Functional tea preparations, due to their inherent antioxidant performance, have the potential to promote digestion, prevent colds, enhance immunity, and provide other health benefits. Therefore, developing white tea liquid preparations is vital for promoting the rapid growth of the whole tea preparation market.(Jie, Xin-yang, Ying, Xu-jie, & Xiao-ping, 2018; Si-meng, Zheng-shan, Hui-hui, Xue-li, & Xin, 2020).

2.1. Performance of wtofLPP

In Fig. 1 (a), it is evident that the particle size of the wtofLPP was 842 ± 46 nm, with a PDI of 0.435. These values indicate a homogeneous and well-dispersed formulation with a small particle size. The smaller the particle size, the higher the stability of the wtofLPP and the less prone it is to agglomerate into clusters.

As depicted in Fig. 1 (b), the viscosity of wtofLPP decreased significantly with increasing shear rate from 0.1s^− 1 to 100s^− 1. The elevated shear rate disrupted the structure of wtofLPP particles, resulting in decreased binding between molecules and a rapid decline in viscosity(Wei-du, 2019). The most substantial reduction in viscosity was observed when the shear rate was 0.1s^− 1 ~ 10s^− 1. When the shear rate exceeded 10 s^− 1, the viscosity stabilized, and the shear force destroyed the flocculation between particles, resulting in a decrease in viscosity. Ultimately, the molecular structure was almost destroyed, exhibiting pseudoplastic fluid characteristics. This property is beneficial for pipeline transportation during production and reduces energy consumption.

The changes in the energy storage and energy dissipation modulus of the wtofLPP is illustrated in Fig. 1 (c). Both the energy storage modulus and energy dissipation modulus exhibit a gradual increase within the frequency range of 0.1Hz ~ 100Hz, indicating that wtofLPP exists in a viscoelastic state. Notably, the G' values are higher than the G" values in the system, which implies that the wtofLPP is predominantly elastic, with higher elastic behavior than viscous behavior.

2.2. antioxidant activity in vitro

As shown in Fig. 2 (a), the scavenging ability of each component on ABTS free radicals continued to increase in the concentration range of 0-1.2 mg/mL, even reached 66.73% when the concentration was 1.2mg/mL, and the scavenging rate was higher than that of ternary compound. The IC₅₀ value of ABTS free radical scavenging of wtofLPP was calculated, and the CI index was found to be 0.94 ± 0.01, indicating that the ternary complex of wtofLPP has synergistic antioxidant effect with taurine.

In Fig. 2 (b), the IC₅₀ value of wtofLPP was 10.08, and the CI index was 0.94 ± 0.02, which was higher than that of other groups, indicating a synergistic effect among the components. The scavenging activity of wtofLPP on superoxide free radicals reached 68.77% when the concentration reached 12 mg/mL.

As shown in Fig. 2 (c), the CI index of wtofLPP was 0.97 ± 0.01, indicating a synergistic effect among the components but it was not significant. At a 12 mg/mL concentration, the hydroxyl radical scavenging rate of fruit juice was 72.11%, that of white tea soup was 63.02%, and that of osmanthus extract was 32.27%. The hydroxyl radical scavenging activity of wtofLPP reached 67.61%.

In Fig. 2 (d), the absorbance was found to be stronger with greater antioxidant capacity, and it gradually increased with the concentration. At a concentration of 12 mg/mL, the absorbance of wtofLPP was 0.801, which was higher than that of ternary compound (0.683), juice (0.787), tea soup (0.462) and osmanthus extract (0.372). This indicates that a synergistic antioxidant effect could be achieved among the components.

2.3. Effects on weight bearing swimming and body weight in mice

The influence of wtofLPP on body weight of mice is shown in Fig. 3 (a). After four-week intragastric administration, body weight of mice in all groups, except for the blank group. increased gradually Meanwhile, the body weight of swimming-trained mice was lower than that of blank group. By the 4th week of the experiment, the body weight of the mice had stabilized, with the medium and low dose groups exhibiting the slowest rates of increase. The water intake of the mice in each group was normal, and there were no instances of mortality during the study.

As shown in Fig. 3 (b), Single-factor variance analysis revealed that the mice's exhaustion time in each administration group was significantly extended compared to the model group. Furthermore, within the dose range examined, the exhaustion time increased with higher doses of the polyphenol preparation, indicating that the wtofLPP can significantly improve the exercise endurance of mice.

2.4. Changes in antioxidant enzyme indexes in mice

As shown in Fig. 4 (a), the SOD value in the model group was significantly lower than that of the normal group (P < 0.01), indicating that fatigue leads to redox imbalance and oxidative stress. In contrast, the SOD value of medium and high dose groups was significantly higher than that of model group (P < 0.01), suggesting that the wtofLPP can effectively enhance SOD activity in mice.

As shown in Fig. 4 (b), the GSH-Px activity in model group was significantly lower than that of blank group (P < 0.01). Notably, the GSH-Px activity of each wtofLPP group was higher than that of model group, and the difference was significant (P < 0.01), which indicates that wtofLPP can effectively increase the GSH-Px activity in mice in a dose-dependent manner.

As shown in Fig. 4 (c), the CAT activity in the model group was significantly lower than that of the blank group (P < 0.01). The CAT activity in high-dose group of the wtofLPP was significantly increased compared with that in model group (P < 0.01), suggesting that the wtofLPP can effectively enhance CAT activity in mice.

As shown in Fig. 4 (d), MDA content in model group was significantly higher than that of blank group (P < 0.01), indicating that fatigue-induced oxidative stress increased lipid peroxidation in mice. Notably, the MDA content of wtofLPP groups was significantly lower than that of model group (P < 0.01), indicating that wtofLPPs can reduce the production of lipid peroxidation and protect against oxidative stress in mice(Huang, Lin, & Deng, 2015).

2.5. Changes of nonenzymatic antioxidant indices in mice

The effect of wtofLPP on GSH synthesis in mice was shown in Fig. 5 (a). The GSH level in model group was significantly lower than in blank group (P < 0.01), indicating its depletion in combating free radicals(Trevisan et al., 2001). Compared to the model group, the GSH level in the wtofLPP groups increased significantly (P < 0.01) in a dose-dependent manner(Quliang & Wutong, 2001).

The effect of wtofLPP on the synthesis ability of ceruloplasmin in mice was shown in Fig. 5 (b). The ceruloplasmin production in model group was significantly lower than in the blank group (P < 0.01). In contrast, the production of ceruloplasmin in the 00000wtofLPP groups was significantly higher than in the model group (P < 0.01).

Vitamin E can inhibit lipid peroxide production, and reduce oxidative damage in the body(Johnston, McCance, Holmes, Young, & McGinty, 2016; Zhu et al., 2016). The effects of wtofLPP on the synthesis ability of Vitamin E in mice were shown in Fig. 5 (c). The Vitamin E level in model group was significantly lower than that in blank group (P < 0.01), while the high-dose wtofLPP group exhibited a significantly higher vitamin E level compared to the model group (P < 0.01).

2.6. Changes in biochemical parameters related to fatigue

Blood urea nitrogen (BUN) is an important indicator of protein degradation(Wei et al., 2016). As shown in Fig. 6 (a), BUN level of the model group exhibited a significantly higher BUN level than the normal group, reaching the highest BUN level, suggesting that the body was experiencing an energy deficiency, leading to the production of a considerable amount of protein and amino acid metabolites (J. Wang et al., 2014). The BUN levels in the wtofLPP groups were significantly lower than those in the model group (P < 0.01), displaying dose dependency. The wtofLPP was involved in the mice’s energy metabolism and significantly reduced their BUN levels.

Excessive LA can decrease exercise performance (Bao et al., 2016). The effect of the wtofLPP on LA levels inmice was demonstrated in Fig. 6 (b). The LA content in model group was considerably higher than in blank group (P < 0.01). Compared with the model group, the LA content in the medium and high dose wtofLPP groups and positive control group were significantly decreased (P < 0.01), showing that polyphenols can significantly reduce lactic acid content, providing an anti-fatigue effect(Yun, 2012).

The effect of wtofLPP on LDH level of mice was shown in Fig. 6 (c) The serum LDH value in the model group was significantly decreased (P < 0.01) In contrast, the LDH levels in the wtofLPP groups were significantly higher than those in the model group (P < 0.01), indicating increased LDH level following intragastric administration.

As shown in Fig. 6 (d), the content of muscle glycogen in model group was significantly lower than that in blank group, and the content of muscle glycogen in wtofLPP at medium and high doses was significantly higher than that in model group (P < 0.01), indicating that wtofLPP can delay fatigue.

2.7. Histopathology

Hematoxylin-eosin (HE) staining is a widely employed technique for pathological examination. Figure 7 (a) demonstrates that the lung tissue in the blank group exhibited normal morphology, distinct cytoplasmic nucleolar staining, no fibrous hyperplasia in the lung interstitium, and normal bronchial mucosa at all levels. In contrast, the model group showed local alveoli dilation, mucous membrane shedding, the presence of blood cells in the lumen, inflammatory cell aggregation around the trachea, and an abundance of red blood cells in the alveolar epithelium. The lung tissue in the wtofLPP groups exhibited varying degrees of improvement.

Figure 7 (b) reveals that the spleen tissue of the control group contained no macrophages, and displayed a distinct boundary between white and red pulp without hyperplasia. In the model group, severe white pulp shrinkage and the presence of macrophages were observed, consistent with previously reported findings(Xiu-xiu, Shang-li, Bo, Qi, & Li-jia, 2018). The high-dose group showed more significant improvement compared to the model group, and the treatment effect was dose-dependent.

As illustrated in Fig. 7 (c), the myocardial fibers (blank group) were closely arranged with clear nuclei, no shrinkage, and an absence of blood cells. In the model group, the cardiac fiber arrangement exhibited widened gaps, numerous blood cells and scattered inflammatory cells in the nucleus, which was consistent with literature reports(Xiu-xiu et al., 2018). The myocardial pathological changes were reduced in all dose groups, with the most substantial improvement observed in high-dose group.

Figure 7 (d) indicates that the liver cell plates in the blank group were neatly and radially arranged, with distinct cytoplasmic and nuclear boundaries. Conversely, the model group displayed irregular arrangements, cytoplasmic fusion, swelling, and nuclear contraction. The liver tissue pathological changes were mitigated in all dose groups, with high-dose group exhibiting the most significant improvement.

Figure 7 (e) shows that the kidney tissue of the blank group appeared relatively normal, without obvious edema or inflammatory cell infiltration. In the model group, glomerular atrophy, epithelial exfoliation, conspicuous vascular congestion in the interstitium, and localized interstitial hyperplasia were observed. The pathological changes were improved across all dose groups.

As shown in Fig. 7 (f), muscle tissues in the blank group were closely arranged, with evenly stained cytoplasmic nuclei, and no edema. The model group exhibited irregular muscle tissue arrangement, prominent edema, and nucleolar shrinkage, which was consistent with previously published findings(Li, 2020). The improvement of high-dose wtofLPP group demonstrated better improvement compared to low and medium-dose groups.

2.8. Effects on mice fertility and offspring weight

The average number of offspring produced by each mouse in each group is depicted in Fig. 8 (a). No significant difference in offspring numbers was observed among the wtofLPP groups (LP-LP, N-F, N-M) when compared to the normal group. The fertility rate of the model group, however, exhibited a significant decrease, indicating that the wtofLPP could protect against fatigue-induced parental fertility decline. The fertility rates of the N-F and M-F groups were lower than those of the M-M and N-M groups, implying that oxidative damage caused by fatigue in female mice had a more pronounced impact on fertility.

As shown in Fig. 8 (b). In general, the weight of male offspring mice in each group was higher than that of female mice. When compared to the normal group (male 18.62 ± 1.23 g, female 16.86 ± 0.91 g), the weight of male offspring mice in other groups decreased, with the model group (male 16.42 ± 1.30 g, female 15.34 ± 1.59 g) experiencing the most significant weight loss, while the other groups had more moderate weight reductions.

2.9. Effects on antioxidant of mouse offspring

Figure 9 (a) illustrates that SOD activity in each experimental group was significantly lower than that in the normal group (P < 0.01), with the lowest SOD activity observed in the model group. This indicates a considerable impact of fatigue on SOD activity. Notably, the activity of SOD in N-F group was significantly higher than in the model group (P < 0.05).

According to Fig. 9 (b), CAT activity in the model group was significantly reduced compared to the normal group. In contrast, CAT activity significantly increased in the M-M and N-F groups treated with the wtofLPP (P < 0.05).

As depicted in Fig. 9 (c), MDA content in all groups increased compared to the normal group, suggesting that fatigue induced oxidative damage affected the offspring. Compared to the model group, MDA content significantly decreased in the M-M, N-M, LP-LP and N-F groups treated with the wtofLPP (P < 0.01). Compared to the M-F group, The MDA content of offspring in the M-M and N-F groups demonstrated less difference than in lower than in the normal group (P < 0.01). In contrast, vitamin E levels in the N-F group treated with wtofLPP significantly increased (P < 0.01), with M-M and LP-LP groups also showing a significant increase (P < 0.05). The vitamin E levels of offspring in the M-M group were less different from the normal group when compared to the M-F group. Furthermore, vitamin E levels of offspring in the N-F group were closer to the normal group than in the N-M group, indicating that the oxidative damage in male mice had lesser impact on offspring than in fatigue damaged female mice.

As shown in Fig. 9 (e), ceruloplasmin levels in the model group were significantly lower than those in the normal group (P < 0.01). The ceruloplasmin levels in the N-F and M-M groups treated with the wtofLPP significantly increased (P < 0.01), and the levels of ceruloplasmin in the N-M and LP-LP groups also significantly increased (P < 0.05). Compared to the M-F group, the ceruloplasmin levels of the offspring in the M-M and N-F groups were less different from the normal group, indicating that fatigue-induced oxidative damage had a lesser effect on offspring in male mice than in the normal group.

Lastly, Fig. 9 (f) demonstrates that the GSH synthesis ability in the model group was significantly lower than in the normal group (P < 0.01). The synthesis ability of GSH in N-F and M-M groups was significantly higher than in model group (P < 0.01), and their GSH synthesis ability improved to some extent.

2.10. Effects on fatigue resistance of mouse offspring

As shown in Fig. 10 (a), the BUN level in the model group was significantly higher compared to the normal group, indicating increased fatigue. Conversely, the BUN levels in the N-F, M-M and N-M groups treated with wtofLPP was significantly lower than those in the model group (P < 0.01). The LP-LP group also exhibited a substantially reduced BUN level compared to the model group (P < 0.05). These results suggest that the wtofLPP can reduce exercise-induced protein catabolism and nitrogen excretion in offspring.

Figure 10 (b) shows that the LA level in the model group was notably higher than that in the normal group (P < 0.01). The LA level of wtofLPP. The N-F group treated with polyphenol liquid preparation displayed a significantly lower LA level than the model group (P < 0.05), while no significant differences were observed in other groups (P < 0.05). These findings suggest that the experimental administration groups can enhance lactic acid aerobic metabolism, alleviating fatigue (Hsu, Ho, Lin, Su, & Hsu, 2005). The M-M and N-F groups exhibited a smaller difference in offspring LA levels compared to the M-F and N-M groups, indicating that fatigue-induced oxidative damage in female mice had a more pronounced impact on offspring than that in male mice.

As depicted in Fig. 10 (c), liver glycogen content was substantially reduced in the model group, while the content of liver glycogen in LP-LP, N-F, M-M and N-M groups treated with wtofLPPs displayed significantly higher liver glycogen levels than the model group (P < 0.01). This suggests that the wtofLPPs can mitigate liver glycogen consumption in vivo. Exercise-induced physical exhaustion occurs concurrently with liver glycogen depletion, as liver glycogen is progressively broken down to maintain blood glucose balance and support exercise, leading to a decline in liver glycogen levels.

Lastly, Fig. 10 (d) reveals that muscle glycogen content in the model group was significantly reduced (P < 0.01). The M-M group treated with wtofLPP exhibited a substantially higher muscle glycogen content compared to the model group (P < 0.01), while the N-M group showed a significant increase (P < 0.05). No significant differences were observed among the other groups. These results indicate that increased glycogen reserves can delay the onset of fatigue (Wilson et al., 2019).

The rheological analysis of the wtofLPP investigated in this study (Figure. 1) demonstrated desirable characteristics.

In the study of antioxidant properties of wtofLPPs in vitro, a comparative analysis was conducted by SPSS software contrapose the IC₅₀ values. In addition to this, the combined action index was also calculated. The result showed that the combination of each component of wtofLPPs and taurine had synergistic effect (Figure. 2).

In vivo experiments with KM mice revealed that wtofLPP offered protection against oxidative stress in parent mice, effectively enhancing the activities of GSH-Px, SOD, and CAT in mice and their offspring. It also reduced MDA levels, improved the synthesis capabilities of Vitamin E, GSH and ceruloplasmin, decreased inflammatory response, and mitigated the detrimental effects of oxidative damage. These results indicated that wtofLPPs can maintain the stability of antioxidant system in mice and their progeny (Figure. 4–6).

Intragastric administration of wtofLPP effectively decreased serum LA and BUN concentrations and increased glycogen levels in offspring, but the difference was not significant. Moreover, parental administration of the preparation promoted offspring generation and increased the fertility rate of fatigue-damaged mice, with a more substantial effect on female mice. However, no significant differences were observed in the sex ratio and body weight of offspring (Figure. 8–10).

Exhaustive swimming resulted in changes to the physiological and biochemical indexes of the mice. Compared to the model group, the exhaustive time of mice in each administration group was prolonged, while BUN and LA levels decreased and glycogen contents increased. Morphological observation showed that wtofLPPs ameliorated the pathological changes in the heart, lung, liver, kidney, muscle and spleen to varying degrees (Figure. 7).

In conclusion, this study indicates that wtofLPPs exhibit favorable taste and stability, providing a theoretical foundation for developing and utilizing functional liquid preparation products. WtofLPP can eliminate the accumulation of fatigue-generated metabolites, enhance the synthesis ability of antioxidant enzymes and non-enzymatic antioxidant substances within the body, protect against damage induced by fatigue stress, and delay fatigue onset. These findings lay a solid theoretical foundation for the development and applicability of novelanti-oxidative stress food products.

Ethics Statement

All animal experimental procedures conformed with the Guidelines of the Institutional Animal Care Use Committee, Heilongjiang province, China. The animal protocols were approved by the Animal Ethics Committee of Harbin Institute of Technology (IACUC-2020032).

Reagents and Materials

The primary ingredient for this experiment was premium Fuding white tea, aged for two years or less. This tea was procured from Ningde City, Fujian Province, while the osmanthus was sourced from Guangxi Zhuang Autonomous Region.Assay kits for the quantification of malondialdehyde (MDA), blood lactic acid (LA), blood urea nitrogen (BUN), superoxide dismutase (SOD), catalase (CAT), Vitamin E, ceruloplasmin, lactic dehydrogenase (GSH), glutathione (GSH), glutathione peroxidase (GSH-Px), and liver/muscle glycogen were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Taurine was purchased from Hubei Yuanda Life Science and Technology Co. (Huangshan, Hubei, China), while L-carnitine was procured from Liaoning Kesuo Nutrition Technology Co. (Dalian, Liaoning, China). Sodium carboxymethylcellulose (CMC), xanthan gum and sodium alginate were acquired from Shanghai Changguang Enterprise Development Co. (Shanghai, China), Shandong Gaofeng Fermentation Co. (Gaofeng, Shandong, China) and Qingdao Mingyue Seaweed Group Co. (Qingdao, Shandong, China), respectively.

Preparation of liquid formulations: Fresh Fuding white tea, no more than two years old, was selected for this study. The tea leaves were crushed using a grinder and subsequently extracted in an electric thermostatic water bath (HWS24, Shanghai Yiheng Technology Co., LTD., Shanghai Fengxian) at 80℃ for 45 minutes with a 1:80 (m:v) ratio. The extract was then cooled and set aside. Separately, osmanthus flowers were weighed and extracted in an electric thermostatic water bath (HWS24, Shanghai Yiheng Technology Co., LTD., Shanghai Fengxian) at 30℃ for 120 minutes using a 1:60 (m:v) ratio. The resulting extract was collected for further processing. Following the extraction process, the two extracts were combined and homogenized using a 30 Mpa high pressure homogenizer (Shandong Huaifang, Shandong Taihan Food Machinery Co., LTD.) for 5 minutes. The mixture was then sealed, sterilized sterilized at 63℃ for 30 minutes, and cooled at 4℃ for subsequent use.

The liquid formulations were optimized using the response surface methodology via Box-Behnken (4 factors and 3 levels) in Design-Expert 8.0.6 (Stat-Ease Inc, Minneapolis, MN, USA). The optimized formulation included a mixture of white tea soup, compound juice and osmanthus extract in an 8:2:1 ratio with 10% added water. Niacinamide (4 mg/100 mL) (Jiangsu Brothers Pharmaceutical Co., Suzhou, Jiangsu, China), 8% sugar, 0.2% compound stabilizer (xanthan gum: CMC: sodium alginate (2:3:1)) and taurine (50 mg/100 mL), L-carnitine (90 mg/100 mL), 0.025% potassium sorbate and 0.03% sodium dehydroacetate were also added. The mixture was then homogenized twice at 30 MPa and sterilized as described above.

Particle size and rheological performance: The average particle size of wtofLPP was determined using a nanoparticle analyzer. A 1 mL aliquot of the diluted sample was introduced into the preparation tank (medium: water) and maintained at room temperature. Subsequently, the sample was subjected to laser irradiation within the range of 0.3nm to 10.0µm (Shanmugam & Ashokkumar, 2015), and recorded at 25℃ across a frequency range of 0.1Hz to 100Hz and a shear rate range of 0.1 to100 s⁻¹.

Antioxidant performance in vitro:

The ABTS radical scavenging activity was determined by a previously reported method with slight modifications (Amensour, Sendra, Abrini, Perez-Alvarez, & Fernandez-Lopez, 2010). The absorbance A₁ was obtained after 6 minutes of light protection at 25℃. A parallel control experiment was conducted by replacing the wtofLPP with an equal volume of distilled water, yielding the reference absorbance (A₀). The ABTS radical scavenging activity was calculated using the following equation:

Clearance rate: M(%)=(A₀-A₁)/A₀×100%. (1)

The superoxide radical scavenging ability was determined using the pyrogallol autoxidation method as described (Kai, Ji-song, Fan-yu, & Peng, 2020). The wavelength was set at 320 nm, and the light absorption value A was measured every 30 seconds for 4 minutes. The absorption value or control absorbance A₀ was determined by Tris-Hcl solution instead of sample. The clearance rate (Y) was calculated as follows:

Clearance rate: Y(%)=(ΔA₀-ΔA)/ΔA₀×100%. (2)

The hydroxyl radical scavenging ability of the wtofLPP was assessed by following a modified protocol(Y. Wang et al., 2016). Absorbance was measured after reaction for 30 minutes.

Clearance rate: Z(%)=[A₀-(A_i-A_i0)]/A₀×100% (3)

The reducing power of the antioxidant wtofLPP was determined using a standard procedure(Du & Xu, 2014).

To evaluate the synergistic resistance effect of each component of the wtofLPP and taurine on fatigue-induced oxidative stress, the Chou-Talalay model was employed for calculating the Combination Index (CI). It can be expressed using the following equation:

Combination Index (CI): \(\text{CI=}\frac{{\text{D}}_{\text{1}}\text{+}{\text{D}}_{\text{2}}}{{\text{DX}}_{\text{1}}\text{+}{\text{DX}}_{\text{2}}}\) (4)

Here, DX₁ and DX₂ denote the doses corresponding to the clearance rate (expressed as X%) when only D1 or D2 are present, respectively. Conversely, D₁ and D₂ represent the respective doses required to achieve the same clearance rate (X%) when the two substances are combined.

Animal experiment:

Sixty specific-pathogen-free (SPF) KM mice (22 ± 2 g, Male, 4 weeks of age, SPF) were purchased from Liaoning Changsheng Biotechnology Co., LTD. (Shenyang, Liaoning, China). After a 7 days acclimatization period, the mice underwent a weight-bearing swimming experiment on day 8. They were randomly assigned to six groups: blank control (Normal), model control (Model), low-dose wtofLPP (LDG) (30 mL/kg), medium-dose wtofLPP(MDG) (60 mL/kg), high-dose wtofLPP (HDG)(90 mL/kg), and a positive control group(PCG)(China Beijing Red Bull Vitamin Beverage Co., LTD Red Bull Beverage 60 mL/kg). Following oral gavage, each group rested for 30 min. The mice were then subjected to a 30-minute weight-bearing swimming training session, with their tails bound with lead skin (5% body weight) and submerged in a water tank maintained at a temperature of 25 ± 1℃ and a depth of 30 cm. Treatment groups received low, medium, or high doses of wtofLPPs once daily, while the blank control and model control groups were administered distilled water on an empty stomach, for 28 consecutive days. During the exhaustive test, the exhaustive time was recorded when the mice were submerged at the bottom for more than 10 seconds.

Anti-fatigue in offspring mice: Sixty with an average weight of 22 ± 2 g KM mice (male and female) were selected. The mice were divided into three groups: a normal group, a model group (treated with distilled water), and a wtofLPP administration group (treated with a medium dose of wtofLPP). Following 28 days continuous gavage, mating was conducted in the pairs of figure (8). After a week, the pregnant female rats were housed individually and allowed to give birth to offspring. At 28 days postnatal, the offspring mice were subjected to 24 hours of food and water deprivation, after which blood samples were collected for analysis Commercial kits were utilized to measure serum biochemical parameters, including antioxidant enzyme system indexes (SOD, CAT, MDA) non-enzymatic antioxidant substance indexes (GSH, cupriplasmin, vitamin E) and anti-fatigue indexes (BUN, LA, LDH, liver glycogen and muscle glycogen).

Statistical analysis

Data analysis was executed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). All results were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed, followed by Tukey’s test and Duncan's multiple comparison tests to assess the statistical significance of differences between groups. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

BUN Blood urea nitrogen

CAT Catalase

GSH- Px Glutathione peroxidase

LA Lactic acid

MDA Malondialdehyde

SOD Superoxide dismutase

WtofLPP White tea and osmanthus flower liquid polyphenol preparation

Author Contributions

Hua Zhang and Zhenyu Wang are as the cooperation teachers of designing and supervising the research project; Yisen Wang mainly takes charge of writing and researching. Yisen wang and Jiaqi Xu provide some experimental instruments with their analysis. Xun Zhou analyzes the experimental data. Ziluan Fan and Hua Zhang provide important guidance and recommendations to the research proposal. Ziluan Fan makes a significant contribution to this paper revision. Hua Zhang provided financial support for the research and finished major revision and re-submission. All the authors reviewed the manuscript.

Conflict of Interest

The authors have declared no conflict of interest.

Funding Source Declaration：The National Science Foundation of China (31401483), the National Science Foundation of Heilongjiang Province, China (C2018034), the Initiation Fund for Postdoctoral research in Heilongjiang Province (LBHQ19104) and the Fundamental Research Funds for the Central Universities (hit.nsrif.2020024) financially supported this study. The authors thank these institutions for their contributions to this research.

Ethical approval: All animal experimental procedures conformed with the Guidelines of the Institutional Animal Care Use Committee, Heilongjiang province, China. The animal protocols were approved by the Animal Ethics Committee of Harbin Institute of Technology (IACUC-2020032).

Data Availability Statement：The data that support the findings of this study are available from the corresponding author, [author initials], upon reasonable request.

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No competing interests reported.

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Unlocking the Antioxidant Potential of White Tea and Osmanthus Flower: A Novel Polyphenol Liquid Preparation and Its Impact on KM Mice and Their Offspring

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Abstract

Figures

1. Introduction

2. Results

2.1. Performance of wtofLPP

2.2. antioxidant activity in vitro

2.3. Effects on weight bearing swimming and body weight in mice

2.4. Changes in antioxidant enzyme indexes in mice

2.5. Changes of nonenzymatic antioxidant indices in mice

2.6. Changes in biochemical parameters related to fatigue

2.7. Histopathology

2.8. Effects on mice fertility and offspring weight

2.9. Effects on antioxidant of mouse offspring

2.10. Effects on fatigue resistance of mouse offspring

3. Discussion

4. Experimental Section

Abbreviations

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