Mechanism of Pharmacological Action of Chromones in Radix Saposhnikoviae Based on Biological Signicance of Secondary Metabolites

According to origin of species, both animals and plants come from a common ancestor, so they all possess similar/same metabolic activities. Plants adapt to stress conditions by eliminating reactive oxygen species (ROS) partly with secondary metabolites, which are often the medicinal ingredients. With this, the mechanism of pharmacological action and biological action of secondary metabolites may be the same. Radix saposhnikoviae the root of Saposhnikovia divaricata, has antipyretic, analgesic and anti-inammatory effects. It was analyzed that the correlation between fever, analgesia and inammation and the activities of POD, CAT, the H 2 O 2 contents in serum before and after intravenous administration for cimicifugin, an active component. For the antipyretic, the correlation coecient between the body temperature and the H 2 O 2 contents was 0.9689 in the model group and 0.5221 in the treatment group, indicating that fever was closely related to the H 2 O 2 . In the presence of electron donor, POD can eliminate ROS, the correlation coecient between POD activities and H 2 O 2 content before and after administration were 0.8085 and -0.5070, respectively, indicating that cimicifugin can eliminate ROS through POD. At 39.5 ℃ , POD activity was about 1.47 times that of normal body temperature, and the scavenging eciency of POD was 2.94 times that of CAT, which indicated that cimicifugin enhanced the elimination of H 2 O 2 mainly through POD. For the analgesic and anti-inammatory, the correlation coecients of POD activity with analgesic and anti-inammatory effects were 0.6685 and 0.4466, respectively, indicating that they were closely related to the H 2 O 2 . In this paper, we found the ROS is an important factor of fever, pain and inammation, the pharmacological actions of Radix Saposhnikoviae also is elimination of ROS through the synergistic action of chromone and POD, the mechanism of pharmacological action of Chromones being the same as the biological action of secondary metabolites.


Introduction
Radix saposhnikoviae, the dried root of Saposhnikovia divaricata (Turcz.) Schischk, with antipyretic, analgesic and anti-in ammatory effects [1] , and is the most common herbal medicine in many Asian countries.
Drought, coldness, high salinity, metal toxicity, ultraviolet radiation and pathogen invasion are common stress for plants [2,3] . Under stress conditions, the equilibrium of xed CO 2 and O 2 in chloroplast of plant cells is collapsed, which result in the accumulated O 2 being reduced to O 2 ·-, and then transformed into other Reactive oxygen species (ROS) [4,5] , an oxygen-containing free radicals or superoxide which form free radicals easily, such as superoxide anion (O 2 ·-), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (·OH), etc., with an extremely high activity [6,7] . Due to trong oxidation ability of ROS, the secondary and tertiary structures of proteins (including enzymes) are usually constructed by the -S-S-groups between two nonadjacent amino acids, so ROS have the ability to regulate the activities of enzymes [8,9] , further, regulate various metabolism. However, too much of ROS can also do damage to organic macromolecules such as enzymes, interfere with normal metabolism, and affect the normal growth of plants [10] . Antioxidant enzymes, exist in animals, plants, fungi cells, and other organisms [12] , mainly include superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), etc. [11] , reponsible to eliminate excessive ROSs [13] .
However, antioxidant enzymes are also protein, can be destroyed by excessive ROS [14] . It has been proved that excessive ROS under severe stress conditions can reduce the activities of SOD, CAT, and POD [15] , further increase the destructive force of ROS, and result in damage to cell membrane, DNA, and protein [16] , even making cells to death. Animals have the ability to avoid over-production of ROS by dodging harsh environment, the survival of individuals mainly depends on the food competition among species, not harsh environment, but plants cannot move, it is certain that more ROS are produced because of more stress [17] , with this, it is di cult to eliminate too much ROS only by antioxidant enzymes, therefore, plants evolve additional secondary metabolism system to reduce production or the harm of ROS besids antioxidant enzymes [18,19,20] .  [21] , and can scavenge ROS and inhibit lipid peroxidation [22] . Their antioxidant activities depend on the number and position of hydroxyl groups (-OH or methoxy group (-OCH 3 ) substituents. The -OH at C5, C6 and C7 positions on A-ring and C3 ', C4' and C5' positions on B ring are the main sites in uencing the activity [23,24] . The activities are also signi cantly enhanced when hydroxyl group is introduced into B ring and double bond is introduced into C ring [25] . The high-quality product areas of Radix saposhnikoviae are semi-arid grasslands. Cimifugin, Prim-O-glucosylcimifugin and 4 -O-β-D-glucosyl -5-O-methylvisamminol are the main secondary metabolites of Saposhnikovia divaricata. However, chromones from Saposhnikovia divaricata contain methoxy -substituted (-OCH 3 ), instead of -OH, while the activity of -OCH 3 is far lower than -OH. In this case, only presence of POD can chromones get the best out of them. POD is a enzyme related to adversity, only under stress condition can POD exhibite higher activity. POD eliminating H 2 O 2 must have a electron donor, and chromones exactly is what POD need [26] . It has been proved that POD activity of Saposhnikovia divaricata increased under the stress conditions [26] . The structures of chromones from Radix Saposhnikovia is shown in Figure 1.
According to origin of species, animals and plants have a common ancestor [27] , so they all possess similar/same cell structure, biological enzyme, genetic material, and metabolism, et al.. But in the face of different environments, they have evolved their own strategies over hundreds of millions of years [28,29] .
Plants adapt to stress conditions by eliminating excessive ROS with secondary metabolites [30,31] , which are often the medicinal ingredients [32,33] . It has been proved that animals will also produce much of ROS when they are exposed to radiation, hyperbaric oxygen, cigarette smoke, air pollution, heavy metals, pesticides, pathogens and other external factors [34,35] . All kinds of diseases of animals have an inseparable relationship with ROS. For example, ROS is involved in the pathological process of cancer, diabetes, in ammation, pain, hypertension, atherosclerosis, diabetic angiopathy, and ventricular remodeling after myocardial infarction [36,37] . Radix saposhnikoviae has de nite antipyretic, analgesic and anti-in ammatory effects [38,39] . The biological mechanism of the ecological action and pharmacological action of chromone of Radix saposhnikoviae may be the same.

Materials And Methods
Instruments

Antipyretic Effect of Cimifugin
Animal Screening and Modeling. The basic body temperature of SPF male SD rats was measured after 3 days of adaptive feeding. The rats whose body temperature was more than 39.0 ℃ or whose twice body temperature uctuations were more than 0.5℃ were eliminated. Finally, 20 SD rats were selected as quali ed experimental animals for modeling.
Temperature monitoring of rats. The quali ed experimental animals were divided into experimental group (15 animals) and model group (5 animals). The rats were forbidden to feed but not drink for 12 h before the experiment, and the average temperature was measured at an interval of 15 minutes for three times as the normal temperature before modeling, and the temperature was monitored at an interval of 1 hour from 0 h to 22 h after administration.
Determination of antipyretic effect of Cimifugin. The experimental groups were injected with 0.216mg/kg of Cimifugin solution by tail vein at 0 h and 6 hours, respectively. The model group was given the same volume of normal saline. 20% dry yeast suspension was injected subcutaneously into the back after the rst injecting Cimifugin solution half an hour later. The antipyretic effect (△t) of Cimifugin was calculated with change of the body temperature for 6 h and 8 h after administration.
The antipyretic effect △T ℃ = T 8h -T 6h The serum of rats was collected before administrate drug (0 h) and at 8 h after administrate drug for testing POD and CAT activities.

The Relationship between Fever and H 2 O 2 in Rats
Five rats of the model group were selected to measure the contents of H 2 O 2 and body temperature at 0h, 8h and 22h. Hydrogen peroxide (H 2 O 2 ) content was determined with hydrogen peroxide (H 2 O 2 ) kit.
Determination of CAT and POD activity CAT activity was detected by Catalase (CAT) kit. The amount of substance (1 μmol) decomposing substrate (H 2 O 2 ) per ml of serum was used as one activity unit (U / ml). POD activity was measured by POD kit, and the amount of substance (1 μg) per mg of tissue protein (serum protein) was used as one activity unit (U / mgprot).
Determination of Optimum Temperature of POD and CAT Blood was collected from 5 rats, and the serum was mixed equally, the activities of POD and CAT were measured at 37.0 ℃, 37.

Analgesic Effect of Cimifugin
Screening of experimental animals and determination ofaverage pain threshold. Before the experiment, 30 KM female mice were screened. The pain threshold was determined by hot plate method. The temperature of hot plate was set at 55 ℃. Fifteen mice with pain threshold from 5s to 30s were selected as quali ed experimental animals. The average pain threshold (s) was measured before the experiment.
Determination of POD activity and analgesic effect. The serum of 15 mice was collected before administration for the determination of POD activities. The pain threshold was measured 1 hour after 0.5mg/kg dosage of cimifugin solution was administrated through tail vein. Each mouse was measured for more than 30s. The average value of three times was used as the indication of the analgesic effect Analgesic effect (s) = average pain threshold after administration (s) -average pain threshold before administration (s).

Anti-in ammatory Effect of Cimifugin
Serum samples of 15 KM mice, half male and half female, were collected before administration for determination of POD activities. Cimicifugin was given by tail vein at the dose of 0.5mg/kg. After 30 minutes, both sides of the left ear of each mouse was coated 0.2ml xylene, and the right ear was used as control. One hour later, the mice were killed by cervical dislocation. Two ears were cut along the base line of the auricle respectively. Round ear pieces were made on the same part of the left and right ears with an 8mm diameter punch and were weighed. The weight difference between the two ear pieces of the same mouse was taken as the indication of the auricle swelling degree (mg).

Results
Relationship between fever and H2O2 in rats. The body temperature of model group reached 41.4 ℃ after 8 h of modeling, and then decreased, while the temperature of treatment group was 38.9℃ after 8 h and then decreased. The body temperature of the treatment group was signi cantly lower than that of the model group, indicating that cimicifugin has antipyretic effect, as shown in Figure 2. The basal body temperature of rats was 36.5 ℃, and the contents of H2O2 in was 47.97~54.49 mmol/L. After 8 hours, the body temperature reached the highest value of 39.9~41.5℃. The H2O2 contents increased to 149.95~162.53 mmol/L. After 22 hours, the body temperature dropped to 37.3~37.7 ℃, and the H2O2 content returned to the normal level of 50.76~61.47mmol/L. The correlation coe cient between body temperatures and H2O2 contents in model group was 0.9689 based on above the numerical data, indicating that there was a close relationship between fever and H2O2 in rats, as shown in Figure. 3. The correlation coe cient between body temperatures and H2O2 contents in the treatment group was 0.5221, indicating that body temperature was also closely related to serum H2O2, as shown in Figure 4.
Relationship between the antipyretic and the POD, CAT. POD activities were extremely signi cantly positive correlated with the contents of H2O2 in serum, with a correlation coe cient of 0.8085. However, after administration for cimicifugin, the relationship between the them was reversed, from a signi cant positive correlation to a signi cant negative correlation, with a correlation coe cient of -0.5070, indicating that cimicifugin signi cantly reduced the contents of H2O2, as shown in Figure 5. The activities of POD and CAT increased with the increase of body temperature. The activity of CAT was the highest at 37.5 ℃, and then decreased sharply. The correlation coe cients between CAT activity and H2O2 content in serum before and after administration were 0.3002 and 0.0361, respectively, as shown in Figure 6.The correlation coe cient between the CAT and temperature rise amplitude were 0.5470 and 0.2335 before and after administration, respectively, as shown in Figure. 7 and Figure. 8. But the optimum temperature of the POD and CAT are different. The optimum temperature of the POD is 39.5℃, further still mantain high activity at higher teperature, but the CAT is 37.5℃, decrease sharply as increasing teperature, indicating that CAT played an important role in eliminating H2O2 at normal temperature. While POD activity increased rapidly above 38.0 ℃ and 39.5 ℃, with a 1.47 times higher than that of normal temperature, indicating that POD plays an important role in eliminating H2O2 under fever condition. At 37.0 ℃, the elimination e ciency of POD is 2.07 times that of CAT, and at 39.5 ℃, 2.94 times that of CAT, indicating that POD is the main way to eliminate H2O2 under fever condition, as shown in Figure.  processes. ROS can be reciprocal transformated [40] . In the antioxidant enzyme system, the O 2 ·produced by organism rstly generates H 2 O 2 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 [41] , the 1 O 2 is 3μs [42,43,44] , with short life span but strong activity and great destructive power. But the activity of H 2 O 2 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 H 2 O 2 [47] . In order to clearly show the correlation between different data in the administration group and the model group, all the correlation data have been re ected in Table 1. Figures  3 and Figures 4 show that the correlation coe cient between body temperature and H 2 O 2 content in model group is 0.9689, indicating that a large amount of H 2 O 2 accumulated in various tissues of the body may be the essential reasons of fever reaction. Figure 5 shows that the correlation coe cient between POD activities and H 2 O 2 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 H 2 O 2 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 H 2 O 2 within 7.5h after being induced fever. That is, POD activity also increases along with the increase of H 2 O 2 content. However, in the absence of electron donors, can POD eliminate H 2 O 2 [51,52] . Without electron donor, H 2 O 2 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 H 2 O 2 . The signi cant positive correlation was revered to a signi cant negative correlation (r = -0.5070), as be shown in Figure 5. Figure 2 has also showed that the body temperature decreased signi cantly after administration, indicating that the fever effect is closely related to the content of H 2 O 2 .
2 .POD is the main enzyme to eliminate H 2 O 2 in rats POD and CAT are the main antioxidant enzymes to eliminate H 2 O 2 . After administration, the correlation coe cient between the antipyretic effect of cimicifugin and POD was 0.5582, and CAT was 0.2335, shown in Figure and 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 H 2 O 2 was weak after fever, indicating that CAT is not the main enzyme to remove H 2 O 2 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 (H 2 O 2 ) per milliliter of serum (or tissue protein) is taken as an activity unit (U / ml). With this, it concluded that the ratio of H 2 O 2 consumption of POD and CAT of the same protein unit is about 1.47:1, H 2 O 2 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 H 2 O 2 by the same POD is almost 3 times that of CAT. CAT is speci c to H 2 O 2 and can directly eliminate excessive H 2 O 2 , but it has poor thermal stability, and its activity is weak when the body temperature is high [53] . 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 H 2 O 2 , especially when the content of H 2 O 2 is high and the body temperature is high.
Pain is also closely related to H 2 O 2 . It has been found that neuropathic pain and in ammatory 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 signi cantly eliminate mechanical abnormal pain [58] . Figure 11 shows that the correlation coe cient between analgesic effect and POD is 0.6685, indicating that the analgesic effect of cimicifugin is mainly achieved by eliminating H 2 O 2 in the body.
In ammation 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 in ammatory mediator [59] . During the occurrence of lithiasis, the main components of the lithiasis can activate overproduction of ROS and interact thioredoxin protein in a dose-dependent manner [60,61] . Figure 12 shows that the correlation coe cient between anti-in ammatory effect and POD is 0.4466, indicating that the anti-in ammatory effect of cimicifugin is mainly achieved by eliminating H 2 O 2 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-Omethylvisamminoside are very small, which can only be detected at high-dosing [62] , while the main component in the blood is cimicifugin [63] . Therefore, in order to reduce the impact of intestinal absorption process, intravenous administration was used in this study. The ability of avonoids to eliminate ROS depends on the amount and location of -OH [64,65] . Cimicifugin lacks -OH, instead of -OCH 3 , with much lower activity than -OH. POD present in animal and plant cells widely and is an important enzyme to eliminate H 2 O 2 , but it needs polyphenols and other electronic donors to play a role [51,52] . The synergism of POD and Cimicifugin can effectively remove H 2 O 2 in the cells, and exhibit antipyretic, analgesic, and anti-in ammatory 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 di cult 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 in ammation. The antipyretic, analgesic and antiin ammatory 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 elds.   Changes of fever temperature in rats