Antipyretic Effect and H2O2
1. H2O2 is the essential reason of hyperthermia
ROS can be produced in various pathways, including enzymatic reaction, autoxidation of reductive molecules, and mitochondrial electron transfer chain, participate in cell growth and proliferation, development and differentiation, aging and apoptosis, as well as many physiological and pathological processes. ROS can be reciprocal transformated . In the antioxidant enzyme system, the O2·- produced by organism firstly generates H2O2 through the action of SOD, and then decomposes by CAT and POD. Among many ROS, the half-life of O•-2 is about 1 μs, the 1O2 is 3μs [42,43,44], with short life span but strong activity and great destructive power. But the activity of H2O2 is weak, with a long life span, high content, can run over long distances, and become the main ROS regulating cell metabolism [45,46].
Fever is closely related to H2O2. In order to clearly show the correlation between different data in the administration group and the model group, all the correlation data have been reflected in Table 1.Figures 3 and Figures 4 show that the correlation coefficient between body temperature and H2O2 content in model group is 0.9689, indicating that a large amount of H2O2 accumulated in various tissues of the body may be the essential reasons of fever reaction. Figure 5 shows that the correlation coefficient between POD activities and H2O2 contents is 0.8085 before giving the medicine to rats. This strong positive correlation is related to the characteristics of POD which is an inducible enzyme. Only when cells produce more H2O2 under exogenous stimulation, can POD activity be improved by feedback induction [48,49,50]. The mean value of serum POD activity before fever was 4.08U/mgprot, and that of serum POD after administration for 8h (i.e. 7.5h after modeling) was 4.51U/mgprot, with a increase of 10.54%, indicating that the POD activity had been activated and increased under the stimulation of H2O2 within 7.5h after being induced fever. That is, POD activity also increases along with the increase of H2O2 content. However, in the absence of electron donors, can POD eliminate H2O2 [51,52]. Without electron donor, H2O2 content must remains at a high level, which result in higher body temperature of rats. The rats were given cimicifugin, a electron donor was supplied, with a result of that the higher the POD activity, the lower the content of H2O2. The significant positive correlation was revered to a significant negative correlation (r = -0.5070), as be shown in Figure 5. Figure 2 has also showed that the body temperature decreased significantly after administration, indicating that the fever effect is closely related to the content of H2O2.
2 .POD is the main enzyme to eliminate H2O2 in rats
POD and CAT are the main antioxidant enzymes to eliminate H2O2. After administration, the correlation coefficient between the antipyretic effect of cimicifugin and POD was 0.5582, and CAT was 0.2335, shown in Figure and Figure 8, indicating that the antipyretic effect of cimicifugin was closely related to the activity of POD. Figure 9 shows that the optimum temperature of the POD is 39.5℃, further mantain high level at high teperature, but the CAT is 37.5℃, decrease sharply as increasing teperature, Fig-6 also shows that correlation between CAT and H2O2 was weak after fever, indicating that CAT is not the main enzyme to remove H2O2 at high temperature, while POD has high activity at 38 ℃~42 ℃, which is more than 2.5 times higher than that at normal temperature. The POD activity (U / mg prot) is be represented by the mass of catalytic substrate (1μg) per milligram of tissue protein (serum protein), and the amount of substance (1 μmol) of catalyzing substrate (H2O2) per milliliter of serum (or tissue protein) is taken as an activity unit (U / ml). With this, it concluded that the ratio of H2O2 consumption of POD and CAT of the same protein unit is about 1.47:1, H2O2 consumption ratio of POD and CAT of the same active unit at 37 ℃ is about 2.08:1, and that of POD and CAT with the same active unit at 39.5 ℃ is about 2.94:1, as shown in Figure 10. Under the fever condition, the consumption of H2O2 by the same POD is almost 3 times that of CAT. CAT is specific to H2O2 and can directly eliminate excessive H2O2, but it has poor thermal stability, and its activity is weak when the body temperature is high . POD is a glycoprotein with strong stability, which usually shows high activity under adverse conditions [48,49,50], indicating that POD is the main enzyme to eliminate H2O2, especially when the content of H2O2 is high and the body temperature is high.
Pain, Inflammation and H2O2
Radix Saposhnikoviae has antipyretic, analgesic and anti-inflammatory effects [38-39], which are interrelated and coexist.
Pain is also closely related to H2O2. It has been found that neuropathic pain and inflammatory pain are related to ROS [53,54,55]. ROS in the central nervous system mainly comes from the electron transport chain in the inner membrane of mitochondria. ROS, from Endoplasmic reticulum, induced by virus, can result in neuralgia[56,57], and oxidative scavengers can significantly eliminate mechanical abnormal pain . Figure 11 shows that the correlation coefficient between analgesic effect and POD is 0.6685, indicating that the analgesic effect of cimicifugin is mainly achieved by eliminating H2O2 in the body.
Inflammation is the host’s defensive immune response to foreign damage factors, and it is also closely related to ROS. When endothelial cells are injured, ROS, as a cell signal molecule, is also an inflammatory mediator . During the occurrence of lithiasis, the main components of the lithiasis can activate over-production of ROS and interact thioredoxin protein in a dose-dependent manner [60,61]. Figure 12 shows that the correlation coefficient between anti-inflammatory effect and POD is 0.4466, indicating that the anti-inflammatory effect of cimicifugin is mainly achieved by eliminating H2O2 in the body.
Mechanism of Action of Cimicifugin
The main effective component of Radix Saposhnikoviae is chromone. According to the Chinese Pharmacopoeia (Volume I of 2015 Edition), the total content of cimicifugoside and 5-O-methylviadine in Radix Saposhnikoviae should not be less than 0.24%. Because of the high content of cimicifugoside and 5-O-methylviadine, it is regarded as the standard component. The absorption of cimicifugin and 5-O-methylvisamminoside are very small, which can only be detected at high-dosing, while the main component in the blood is cimicifugin . Therefore, in order to reduce the impact of intestinal absorption process, intravenous administration was used in this study. The ability of flavonoids to eliminate ROS depends on the amount and location of -OH [64,65]. Cimicifugin lacks -OH, instead of -OCH3, with much lower activity than -OH. POD present in animal and plant cells widely and is an important enzyme to eliminate H2O2, but it needs polyphenols and other electronic donors to play a role [51,52]. The synergism of POD and Cimicifugin can effectively remove H2O2 in the cells, and exhibit antipyretic, analgesic, and anti-inflammatory effects.
The components of herbal medicine are very complex. Recent researches on drug action and mechanism mainly focused on the differential metabolites and metabolic pathways. In many cases, although there is correlation, it is not clear whether these metabolites are the upstream or downstream substances leading to the cause of disease, and it is difficult to clear whether it is causation or parallel relations. Darwin’s theory of evolution is the three major discoveries of Natural Science in the 19 th century, which greatly promoted the development of life science. According to this theory, the evolution of life has underwent from inorganic small molecules, to organic small molecules, to organic macromolecules, to independent systems, to primitive life. Animals and plants should have a common ancestor and the same basic life law. The ecological function of chromone of Saposhnikovia divaricata is to eliminate the ROS in cells, which is also an important factor of fever, pain and inflammation. The antipyretic, analgesic and anti-inflammatory effects of Radix Saposhnikoviae also eliminate ROS through the synergistic action of chromone and POD, which indicate that the ecological and pharmacological mechanisms of chromone of Radix saposhnikoviae are the same. At present, botany and zoology are two relatively independent research fields. The research results from plant ecology and herbal medicine pharmacology learning from each other can open up new ideas for the researches of plant pharmacology.