Effects of Yiqi Fanggan Mixture on Anti-Stress Response and Immunity of Mice

To study the effect of Yiqi Fanggan Mixture on the anti-stress response and immunity of mice, a Chinese herbal compound (composed of some tonic Chinese medicine, including astragalus flavone, deep-fried atractylodes macrocephala koidz, fortune eupatorium herb, cyrtomium fortunei, white cardamom, windproof, dyers woad root, licorice, jobstears seed) was given to the mice for intragastric administration once a day for 2 weeks. Mice in each group are subjected to normal pressure hypoxia test, swimming test, cold resistance test and high temperature resistance test. Organ index and serum hemolysin content of mice were also monitored. The results indicated that the Yiqi Fanggan Mixture can improve the survival time of mice under the condition of normal pressure hypoxia; prolong the swimming time and high temperature resistance time of mice; reduce the death rate of mice under the low temperature condition; and improve the activity of serum hemolysin and organ index of mice. The results showed that Yiqi Fanggan Mixture could improve the ability of anti-stress response and immune ability in mice.


Introduction
Stress response is an adaptive response produced when the body is stimulated by changes of body's internal and external environment. During stress, a series of neuroendocrine reactions will appear, mainly manifested by the excitation of the sympathetic nervous system and the increased secretion of adrenocortical hormones by the hypothalamic-pituitary-adrenaline axis (HPA), which is a protective response. Stress ability is the ability of the body to adapt to various emergency situations. However, just like everything has two sides, persistent and excessively strong stress response will also cause obvious damage to the body [

Prescription and Preparation [5]
Preparation: Decoct Prescription: Astragalus flavone 150 g, deep-fried atractylodes macrocephala koidz 100 g, fortune eupatorium herb 100 g, cyrtomium fortune 50 g, white cardamom 100 g, windproof 100 g, dyers woad root 100 g, licorice 50 g, jobstears seed 100 g. Preparation: Decoct the above 9 herbs with water for three times. After 1.5 h of the first boiling, 1 h of the second boiling and 1 h of the third boiling, combine the decoction, centrifuge, filter, concentrate the filtrate to 850 ml, add 2 g potassium sorbate, mix, fill to 100 ml, and sterilize at 105˚C for 30 min.

Animal Grouping and Methods of Administration
Kunming male mice count 350, clean grade, weight (20 ± 2) g, provided by Yangtze University Medical Experimental Animal Center. Of these, 280 mice were first modeled and then divided into four groups at random: the large dose of the Yiqi Fanggan Mixture group (giving Yiqi Fanggan Mixture at the 5.0 ml/100g body weight), and the small dose group of the Yiqi Fanggan Mixture (giving Yiqi Fanggan Mixture at the 2.5 ml/100g body weight equivalent to concentrated crude drug solution), and blank control group (giving the same volume of physiological saline), and the positive control group (Andolin capsules, 0.64 g•kg −1 ). The remaining 70 mice were used as sham control group (giving the same volume of physiological saline). Each group is given intragastric administration, 1 time/d for 2 weeks.
The experimental plan has been approved by the Animal Experimental Ethics Committee of Yangtze University.

Test of Anti-Stress Ability of Mice
1) Normal pressure hypoxia test [6]. Taking the five groups of mice, each 2) Cold-resistant test [7]. Taking the five groups of mice, each group has 10 mice. Put the mice in −5˚C ± 1˚C refrigerator. The animals are removed after 1 h, counting the survival amount of mice in each group.
3) High temperature-resistant test [8]. Taking the five groups of mice, each group has 10 mice, and the mice were placed in a constant temperature oven at a temperature of 45˚C ± 1˚C. Observe the survival time of the mice.
4) Swimming test [8]. Taking the five groups of mice, each group has 10 mice,

Immune Testing of Mice
1) Determination of mouse organ index. The mice were sacrificed with cervical dislocation. Weigh the mouse first, then soak the mouse in 75% alcohol for 3 min, move the mouse to the ultra-clean workbench, take out the spleen and thymus, wipe the blood on the organ with filter paper, weigh the wet weight of the organ, and calculate the organ index of the mouse. Mouse organ index = organ weight/body weight (mg/g).
2) Determination of serum hemolytic pigment of mice. Starting from the 6 th day before the end of administration, the mice were intraperitoneally injected with 0.2 ml of 20% SSRBC every day. After 6 days, the eyeballs were removed for blood collection and placed in an anticoagulant centrifuge tube, centrifugated at 2000 r/min for 10 min at 4˚C. The serum was carefully absorbed and diluted 50 times with normal saline for testing. 1 ml of 50 times diluted mouse serum was in turn added to another 10 ml centrifugal tube, rehydrated (guinea pig serum) to 0.5 ml, added 20% SSRBC 0.5 ml, mixed and then placed in 37˚C constant temperature water bath for 10 min, and terminated the reaction by ice bath.
Blank control tube was filled with physiological saline, and the others were treated in the same way as example tubes. Read the absorbance value at 540 nm wavelength using a photometer.

Effects of Yiqi Fanggan Mixture on Anti-Stress Response of Mice
The effect of Yiqi Fanggan Mixture on the anti-stress ability of mice is shown in

Effect on Mouse Organ Index
The results of this test are shown in Table 2. Compared with the control group, the organ index of the administration group is improved to a certain extent, and the difference between the high-dose group and blank control group is statistically significant blank control group (P < 0.05), but the difference between the small dose group and blank control group is not statistically significant (P > 0.05).

Effect on Serum Hemolytic Content of Mice
The effect of Yiqi Fanggan Mixture on serum hemolytic pigment content in mice can be seen in Figure 1. Figure 1 shows that compared the high-dose and low-dose groups with blank control group, the content of serum hemolytic hemolyte in mice has a statistical difference (P < 0.05), indicating that the Yiqi Fanggan Mixture has a certain effect on improving the activity of serotonin.

Discussion
Stress is a nonspecific systemic reaction of the body when stimulated by various  Immunity is the ability of animals to protect themselves against external enemies, and the strength of immunity can measure the body's resistance to pathogenic microorganisms [11]. Serum hemolytic pigment is an important indicator of the immune function of animal bodies, and when hemolytic production content increases, the immunity of mice is also enhanced. The thymus and spleen,  [16]; Wind protection has anti-microbial, anti-inflammatory and immunomodulatory effects [17]; Volatile oils, endoester Mixtures, polysaccharides, glycosides and other components in white art can achieve anti-inflammatory effects by inhibiting the production of cytokines that mediate inflammatory reactions and regulating immunity [18]. Licorice has many pharmacological activities, such as antibacterial, anti-inflammatory, antiviral, immunomodulation effetcs, etc. [19]. The various components of radix isatidis have antibacterial, anti-endotoxin, antiviral and immunomodulatory effects, of which the anti-flu virus effect is particularly ob-  [21]. Therefore, this prescription has a pharmacological basis in improving positive gas, enhancing immunity and preventing new coronary pneumonia.

Conflicts of Interest
The author declares no conflicts of interest regarding the publication of this paper.