Application of ethyl acetate extraction fraction of Beaumontia grandiflora Wall. in the preparation of anti-inflammatory and analgesic drugs

Abstract

The present invention discloses the use of the ethyl acetate extract of Qingming flower in the preparation of anti-inflammatory and analgesic drugs: the stems of Qingming flower are crushed and extracted with ethanol, then extracted with petroleum ether and ethyl acetate, and the ethyl acetate extracts are combined and concentrated under reduced pressure to obtain the ethyl acetate extract of Qingming flower. The ethyl acetate extract of Qingming flower is safe, effective, and has no acute toxicity, and can increase the level of IL-10 in serum and inhibit the levels of IL-6 and IL-1 in serum. β and TNF‑ α The ethyl acetate extract of Qingming flower has anti-inflammatory and analgesic activities, with a daily dose of 9-80 g.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of an ethyl acetate extraction fraction of qingming flower in the preparation of anti-inflammatory and analgesic drugs. Background Art

[0002] Inflammation is an adaptive response triggered by physical factors (such as temperature, radioactive substances and mechanical damage), chemical factors (such as strong acids, strong alkalis and decomposition products of endogenous toxic substances) or biological factors (such as bacteria and toxins) under certain conditions, which manifests as redness, swelling, heat, pain and even dysfunction. Normally, inflammation can activate the body's immune system to resist various stimuli, thereby protecting the body from injury and infection. However, excessive and long-term inflammatory responses may cause cell damage or metabolic disorders, thereby triggering a series of diseases, including atherosclerosis, diabetes, myocardial infarction, cancer and neurodegenerative diseases. Therefore, the control and treatment of inflammation is particularly important, and inhibiting inflammatory responses is also considered to be one of the means to treat the above diseases.

[0003] Pain is one of the primary symptoms of inflammation, and the two are mutually causal and mutually influential, leading to the development of anti-inflammatory analgesics. Among the medications used clinically in my country, anti-inflammatory analgesics are the second largest class of drugs, second only to anti-infectives. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs) are the primary anti-inflammatory analgesics in clinical use, including aspirin, acetaminophen, indomethacin, naproxen, and diclofenac. The primary mechanism of action of these drugs is to inhibit cyclooxygenase-2 (COX-2), thereby reducing the production of prostaglandin E2 (PGE2). Because PGE2 has a protective effect on various tissues, including the gastric mucosa, NSAIDs, while inhibiting PGE2 production and thus exerting their anti-inflammatory and analgesic activity, also deprive other related tissues of PGE2's protection, leading to numerous adverse reactions, including gastrointestinal bleeding, peptic ulcers, liver and kidney damage, and cardiovascular disease. This has made the safety of NSAIDs a hot topic in the global pharmaceutical community. Therefore, the research and development of new anti-inflammatory and analgesic drugs has attracted widespread attention in the medical field and society.

[0004] Beaumontia grandiflora Wall. is a plant of the Apocynaceae family, mainly distributed in Yunnan and Guangxi, my country, as well as in areas such as Laos, Myanmar, Nepal, and Bhutan. Its roots and leaves are used as medicine, and it is pungent in the mouth and warm in nature. It has the effects of dispelling wind and dampness, promoting blood circulation, and relieving pain. It is mainly used to treat rheumatic arthralgia, lumbar muscle strain, traumatic injury, and bone fracture swelling and pain. It is an important Dai medicine resource in my country. There are currently no reports on its anti-inflammatory and analgesic effects and its ethyl acetate fraction being used for anti-inflammatory and analgesic purposes, which has restricted its medicinal development and clinical application to a certain extent. Summary of the Invention

[0005] The present invention aims to provide an application of an ethyl acetate extract of qingming flower in the preparation of an anti-inflammatory and analgesic drug, which is characterized by being prepared by the following steps:

[0006] (1) Ethanol extraction: 10 kg of Qingming flower stems were crushed and extracted four times in 90% ethanol at room temperature, 10 L each time, for 2 hours each time; the combined extracts were then removed by rotary evaporation under reduced pressure to obtain the total extract;

[0007] (2) Extraction and separation: The total extract was suspended in water, extracted with petroleum ether 5 times, and then extracted with ethyl acetate 5 times. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract of Qingming flower.

[0008] The ethyl acetate extract of Qingming flower upregulates the level of IL-10 in serum.

[0009] The ethyl acetate extract of Qingming flower inhibits the level of IL-6 in serum.

[0010] The ethyl acetate extract of Qingming flower inhibits the levels of IL-1β and TNF-α in serum.

[0011] The dosage of the ethyl acetate extract of Qingming flower is 9 to 80 g per day.

[0012] The dosage of the ethyl acetate extract of Qingming flower is 24g per day for a 60kg person.

[0013] Advantages of the present invention:

[0014] The advantages and positive effects of the present invention lie in providing a non-toxic and safe ethyl acetate extract of Qingming flower, which has the potential to be used in the preparation of anti-inflammatory and analgesic drugs. The application of Qingming flower in the preparation of anti-inflammatory and analgesic drugs has been developed. Qingming flower has the effects of dispelling wind and dampness, promoting blood circulation, and relieving pain, but its anti-inflammatory and analgesic activity has not been reported. The ethyl acetate extract of Qingming flower provided by the present invention has good anti-inflammatory and analgesic effects, and its anti-inflammatory mechanism may be related to upregulating IL-10 levels and downregulating IL-6, IL-1β, and TNF-α levels, and does not exhibit acute toxic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The organ indexes (%) of the total extract of Qingming flower and its four polar parts in male mice. A to F are the organ indexes of heart, kidney, liver, lung, spleen and thymus respectively. *** P < 0.001. Calculation formula: Organ index (%) = (organ mass / body mass) × 100. Vehicle: blank group; BEH: total extract group; BPE: petroleum ether group; BEA: ethyl acetate group; BNA: n-butanol group; BAE: water group (n = 5).

[0016] Figure 2 The organ indexes (%) of the total extract of Qingming flower and its four polar parts in female mice. A to F are the organ indexes of heart, kidney, liver, lung, spleen and thymus respectively. *** P < 0.001. Calculation formula: Organ index (%) = (organ mass / body mass) × 100. Vehicle: blank group; BEH: total extract group; BPE: petroleum ether group; BEA: ethyl acetate group; BNA: n-butanol group; BAE: water group (n = 5).

[0017] Figure 3 The effect of the total extract of Qingminghua flower and its four polar fractions on xylene-induced ear swelling in mice. Vehicle: blank group; BEH: total extract group; BPE: petroleum ether fraction group; BEA: ethyl acetate fraction group; BNA: n-butanol group; BAE: water fraction group; DXMS: dexamethasone group (n=6). Compared with the model group, the treatment group had a significantly higher ear swelling than the model group. * P<0.05, *** P<0.001.

[0018] Figure 4 The effects of the total extract of Qingminghua flower and its four polar fractions on the writhing reaction of mice induced by glacial acetic acid were studied. Vehicle: blank group; BEH: total extract group; BPE: petroleum ether fraction group; BEA: ethyl acetate fraction group; BNA: n-butanol group; BAE: water fraction group; ASP: aspirin group (n=6). Compared with the model group, the drug-treated group had a significantly higher score than the control group. * P<0.05, *** P<0.001.

[0019] Figure 5The effects of the total extract of Qingminghua flower and its four polar fractions on the hot plate reaction in mice. A to D are the hot plate pain reactions at 30, 60, 90, and 120 minutes, respectively. Vehicle: blank group; BEH: total extract group; BPE: petroleum ether fraction group; BEA: ethyl acetate fraction group; BNA: n-butanol group; BAE: water fraction group (n=6). Compared with the blank group, the drug-treated group * P<0.05, ** P<0.01, *** P<0.001.

[0020] Figure 6 The effect of ethyl acetate extract of Qingming flower on xylene-induced ear swelling in mice. Model: model group; BL: low-dose group of ethyl acetate extract of Qingming flower; BM: medium-dose group of ethyl acetate extract of Qingming flower; BH: high-dose group of ethyl acetate extract of Qingming flower; DXMS: dexamethasone group (n=6). ** P<0.01, *** P<0.001.

[0021] Figure 7 The effect of ethyl acetate extract of qingming flower on inflammatory cytokines in mouse serum. IL-10 (A), IL-6 (B), IL-1β (C) and TNF-α (D). Vehicle: blank group; Model: model group; BL: low-dose group of ethyl acetate extract of qingming flower; BM: medium-dose group of ethyl acetate extract of qingming flower; BH: high-dose group of ethyl acetate extract of qingming flower; DXMS: dexamethasone group (n=10). Compared with the blank group, the model group ### P<0.001; compared with the model group, ** P<0.01, *** P<0.001.

[0022] Figure 8 The effect of ethyl acetate extract of Qingming flower on writhing reaction induced by glacial acetic acid in mice. Model: model group; BL: low-dose ethyl acetate extract of Qingming flower group; BM: medium-dose ethyl acetate extract of Qingming flower group; BH: high-dose ethyl acetate extract of Qingming flower group; Asp: aspirin group (n=6). *** P<0.001.

[0023] Figure 9The effect of ethyl acetate extract of Qingming flower on the hot plate reaction of mice. A to D are the hot plate pain reactions at 30, 60, 90 and 120 min, respectively. Vehicle: blank group; YHZT: Yuanhu analgesic tablet group; BL, BM, BH are low, medium and high dose groups of ethyl acetate extract of Qingming flower (n=6). Compared with the blank group, the drug-treated groups * P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0024] The present invention provides an application of an ethyl acetate extract of qingming flower in preparing an anti-inflammatory and analgesic drug, which is characterized by being prepared by the following steps:

[0025] (1) Ethanol extraction: 10 kg of Qingming flower stems were crushed and extracted four times in 90% ethanol at room temperature, 10 L each time, for 2 hours each time; the combined extracts were then removed by rotary evaporation under reduced pressure to obtain the total extract;

[0026] (2) Extraction and separation: The total extract was suspended in water, extracted with petroleum ether 5 times, and then extracted with ethyl acetate 5 times. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract of Qingming flower.

[0027] The ethyl acetate extract of Qingming flower upregulates the level of IL-10 in serum.

[0028] The ethyl acetate extract of Qingming flower inhibits the level of IL-6 in serum.

[0029] The ethyl acetate extract of Qingming flower inhibits the levels of IL-1β and TNF-α in serum.

[0030] The dosage of the ethyl acetate extract of Qingming flower is 9 to 80 g per day.

[0031] The dosage of the ethyl acetate extract of Qingming flower is 24g per day for a 60kg person.

[0032] The present invention is further described below, but is not intended to limit the present invention in any way. Any changes made based on the present invention fall within the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of Qingming Flower Stem Extract

[0035] Stem and leaf samples of Beaumontia grandiflora were collected from Jinghong City, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, in May 2020. The specimen (No. B G20200517001) was authenticated by Associate Researcher Liu Jian of the Kunming Institute of Botany, Chinese Academy of Sciences, and is preserved in the Key Laboratory of Dai and Yi Medicine of Yunnan Province, Yunnan University of Chinese Medicine.

[0036] The collected Qingming flower stems (10 kg) were crushed and extracted with 90% ethanol at room temperature for 4 times, 10 L each time, and 2 hours each time; the extracts were combined and the solvent was removed under reduced pressure on a rotary evaporator to obtain a total extract (BEH). The total extract was suspended in water and extracted with petroleum ether, ethyl acetate and n-butanol in sequence, 5 times each time, and the extracts were combined and concentrated under reduced pressure to obtain petroleum ether (BPE), ethyl acetate (BEA), n-butanol (BNA) and water 4 polar parts (BAE). Among them, the ethyl acetate (BEA) part is the Qingming flower ethyl acetate extraction part described in the present invention.

[0037] Example 2

[0038] 1 Materials and Methods

[0039] 1.1 Experimental Materials

[0040] 1.1.1 Experimental instruments

[0041] Table 1 Main instruments used in the experiment

[0042]

[0043] 1.1.2 Drugs and reagents

[0044] Table-2 Manufacturers and product numbers of the main drugs and reagents used in the experiment

[0045]

[0046] 1.1.3 Medicinal material samples

[0047] Prepared by Example 1.

[0048] 1.1.4 Experimental animals

[0049] ICR mice, 3 weeks old and weighing 18–22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (Production License No.: SCXK(Beijing)2019-0010). Mice were acclimated for 7 days at standard laboratory room temperature (22 ± 2°C) with a 12-h light / dark cycle, with free access to food and water. The animal husbandry and experimental methods involved in the experiment were approved by the Animal Experimentation Ethics Review Committee of Yunnan University of Traditional Chinese Medicine (R-062019S004).

[0050] 1.2 Experimental methods

[0051] 1.2.1 Acute toxicity evaluation

[0052] 110 ICR mice, half male and half female, were randomly divided into 22 groups, with 5 mice in each group. Except for the blank group (equal volume of normal saline), two dosage groups of 1000 and 2000 mg / kg were set for total extract, petroleum ether, ethyl acetate, n-butanol and water, respectively. If 100% of the mice died at both the 1000 and 2000 mg / kg doses in each dosing group, the dose of the dosing group in this category will be reset to 500 mg / kg and continued observation will be carried out. The experiment was administered by gavage and lasted for 14 days, during which the death and behavior of the animals were recorded. The acute toxicity was evaluated using organ indexes of the heart, kidney, liver, lung, spleen and thymus.

[0053] 1.2.2 Screening of effective parts

[0054] Evaluation of anti-inflammatory activity of the total extract of qingming flower and its four polar fractions

[0055] The anti-inflammatory activity of the total extract and each polar fraction was studied using a xylene-induced mouse ear swelling model. Forty-two ICR mice were randomly divided into seven groups, with six mice in each group. Except for the model group (an equal volume of normal saline) and the positive control drug (dexamethasone, 5 mg / kg), the remaining treatment groups were given the total extract and four polar fractions (all at 400 mg / kg). Drugs were administered by gavage for 5 consecutive days. One hour after the last dose, 30 μL of xylene was applied to the right ear surface of each treatment group, while an equal volume of normal saline was applied to the left ear. One hour after application, the mice were sacrificed, both ears were clipped, and ear pieces were punched at the same location with an 8 mm ear punch and weighed. The ear swelling and inhibition rate were calculated using the following formula.

[0056] (1) Ear swelling (%) = (right ear mass – left ear mass) / left ear mass × 100%

[0057] (2) Inhibition rate (%) = (average swelling degree of model group - average swelling degree of drug-treated group) / average swelling degree of model group × 100%

[0058] Evaluation of analgesic activity of the total extract of qingming flower and its four polar fractions

[0059] The analgesic activity of the total extract of Qingming Flower and its polar parts was evaluated using glacial acetic acid to induce writhing reaction in mice. Seven groups were set up, with 6 mice in each group. The model group was given an equal volume of normal saline, and the other drug-treated groups were the positive drug control group (aspirin, 400 mg / kg) and the total extract of Qingming Flower group and its four polar parts group (all 400 mg / kg). The drug was administered continuously by gavage for 5 days. One hour after the last administration, each mouse was intraperitoneally injected with 0.2 mL of 6% glacial acetic acid, and the number of writhing reactions of mice in each group was recorded within 30 minutes. The inhibition rate was calculated using the following formula.

[0060] Inhibition rate (%) = (average number of writhing times in the model group – average number of writhing times in the drug-treated group) / average number of writhing times in the model group × 100%

[0061] The hot plate method was used to screen the analgesic activity of the total extract of Qingming Flower and its polar parts. Female ICR mice were divided into 7 groups. The blank group was given an equal volume of normal saline, and the other dosing groups were given 400 mg / kg (oral administration). The mice were placed in an intelligent hot plate instrument, and the hot plate temperature was set to 55±0.5℃. The hot plate pain threshold of the mice was observed and measured. If the test mouse still did not lick the hind paw for more than 60 seconds, the experiment was terminated to avoid scalding the animal. At the same time, mice that jumped during the test were eliminated. Mice with a pain threshold of 5s to 30s were selected for subsequent experiments. The pain threshold of each group of test mice before drug administration was determined as the pain threshold before drug administration. Each group of mice was placed in an intelligent hot plate instrument 30, 60, 90 and 120 minutes after drug administration, and the reaction time of licking the hind paw or jumping was recorded, and the inhibition rate was calculated using the following formula.

[0062] Inhibition rate (%) = (average pain reaction time of model group - average pain reaction time of drug-treated group) / average pain reaction time of model group × 100%

[0063] 1.2.3 Study on the anti-inflammatory activity of effective fractions

[0064] The anti-inflammatory activity of the active ingredients was studied using the xylene-induced mouse ear swelling model (see 1.2.2 for methods) and the carrageenan-induced paw swelling model. The carrageenan-induced paw swelling model was tested as follows.

[0065] Sixty ICR mice were randomly divided into six groups. Normal and model groups were given equal volumes of saline, while the positive control group received aspirin (400 mg / kg). Experimental groups included low, medium, and high doses (200, 400, and 800 mg / kg) of the ethyl acetate extract of Qingminghua. All mice were gavaged with 1 mL / 100 g of the extract for 5 consecutive days. Except for the blank group, all other groups received a 25 μL subcutaneous injection of a 1% carrageenan aqueous solution into the left hind paw 1 hour after the last dose to induce inflammation. The blank group received an equal volume of saline at the same site. The left hind paw volume of the mice was measured using a PV-200 paw volume meter at 0, 1, 2, 3, 4, 5, and 6 hours after inflammation. The degree of paw swelling and inhibition rate were calculated using the following formulas.

[0066] Foot swelling (%) = (V n –V0) / V0×100%

[0067] V0: volume of the forefoot after carrageenan injection;

[0068] V n : Paw volume inhibition rate at each time point after carrageenan injection (%) = (average paw swelling degree of model group - average paw swelling degree of drug-treated group) / average paw swelling degree of model group × 100%

[0069] Six hours after carrageenan injection, blood was collected from the eyeballs of mice in each group under anesthesia. After 30 minutes of rest, the serum was centrifuged and stored at -80°C. Enzyme-linked immunosorbent (ELISA) was used to measure the levels of cytokines IL-10, IL-6, IL-1β, and TNF-α in the serum of mice in each group. Cytokine levels were measured according to the kit instructions.

[0070] 1.2.4 Study on analgesic activity of effective fractions

[0071] The analgesic activity of the effective parts was studied using the acetic acid-induced writhing method and the hot plate method. The methods were the same as 1.2.2. A positive control drug group (Yuanhu analgesic tablets, 550 mg / kg) was set up for the hot plate method.

[0072] 1.2.5 Statistical analysis

[0073] GraphPad 9.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation. Comparisons between two groups were performed using the Student's t test, and comparisons between three or more groups were performed using one-way ANOVA. P < 0.05 indicated statistical significance.

[0074] 2 Results

[0075] 2.1 Acute toxicity evaluation

[0076] The results showed that, except for the n-butanol fraction, the total extract and the other three polar fractions of Qingming flower did not show any acute toxicity to the experimental animals. After administration of 1000 and 2000 mg / kg of the n-butanol fraction, mice died rapidly. Therefore, the dose of the n-butanol fraction was adjusted to 500 mg / kg and continued for 14 days. This fraction did not show any acute toxicity to the experimental animals. Figure 1 and Figure 2 .

[0077] 2.2 Screening of effective parts

[0078] 2.2.1 Evaluation of the anti-inflammatory activity of the total extract of Qingming flower and its four polar fractions

[0079] The results showed that compared with the model group, the total extract and ethyl acetate fraction of Qingming flower could significantly inhibit xylene-induced ear swelling in mice (P<0.05, P<0.001, respectively), with inhibition rates of 34.30% and 44.73%, respectively. Figure 3 , Table-3).

[0080] Table-3 Inhibitory effect of the total extract of qingming flower and its four polar fractions on xylene-induced ear swelling in mice

[0081]

[0082] Model: model group; BEH: total extract group; BPE: petroleum ether fraction group; BEA: ethyl acetate fraction group; BNA: n-butanol group; BAE: water fraction group; DXMS: dexamethasone group (n=6).

[0083] 2.2.2 Evaluation of analgesic activity of the total extract of Qingming flower and its four polar fractions

[0084] Glacial acetic acid-induced writhing reaction in mice

[0085] The results showed that compared with the model group, the total extract group, ethyl acetate group and water group could significantly reduce the number of writhing reactions induced by glacial acetic acid in mice (P<0.001), and the inhibition rate of the ethyl acetate group (64.34%) was higher than that of the total extract group and water group (63.95% and 35.27%, respectively). Figure 4 , Table-4).

[0086] Table-4 Inhibitory effect of the total extract of Qingming flower and its four polar fractions on the writhing reaction of mice induced by glacial acetic acid

[0087]

[0088]

[0089] Model: model group; BEH: total extract group; BPE: petroleum ether fraction group; BEA: ethyl acetate fraction group; BNA: n-butanol group; BAE: water fraction group; Asp: aspirin group (n=6).

[0090] Hot plate analgesia experiment

[0091] like Figure 5 As shown in the results, compared with the blank group, the total extract, ethyl acetate fraction and water fraction of Qingming flower could significantly prolong the pain reaction time at 5 observation time points.

[0092] 2.3 Study on the anti-inflammatory activity of effective fractions

[0093] 2.3.1 Effect of the active fraction on xylene-induced foot swelling in mice

[0094] like Figure 6 The results showed that compared with the model group, the medium and high dose groups of the ethyl acetate extract of Qingming flower could significantly inhibit xylene-induced ear swelling in mice (respectively: P < 0.01, P < 0.001), among which the inhibition rate of the high dose group (52.65%) was higher than that of the positive control drug (37.2%) (Table 5).

[0095] Table-5 Inhibition rate of xylene-induced ear swelling in mice by ethyl acetate extract of Qingming flower

[0096]

[0097] Model: model group; BL: low-dose group of ethyl acetate extraction of qingming flower; BM: medium-dose group of ethyl acetate extraction of qingming flower; BH: high-dose group of ethyl acetate extraction of qingming flower; DXMS: dexamethasone group (n=6).

[0098] 2.3.1 Effect of ethyl acetate extract of Qingming flower on carrageenan-induced foot swelling in mice

[0099] The results showed that compared with the blank group, the model group had significantly increased paw swelling at all observation time points after carrageenan injection (P < 0.001). Compared with the model group, the high-dose group of the ethyl acetate extract of Qingming flower significantly reduced paw volume in mice 4, 5, and 6 hours after carrageenan injection (P < 0.001). See Table 6.

[0100] Table-6 Effects of ethyl acetate extract of Qingming flower on carrageenan-induced foot swelling in mice ( n=6)

[0101]

[0102]

[0103] Model: model group; BL: low-dose group of ethyl acetate extract of Qingming flower; BM: medium-dose group of ethyl acetate extract of Qingming flower; BH: high-dose group of ethyl acetate extract of Qingming flower; DXMS: dexamethasone group. Compared with the blank group: ### P<0.001; compared with the model group: *** P<0.001.

[0104] The results of ELISA detection of related cytokine levels in mouse serum showed that ( Figure 7 Compared with the blank group, the IL-10 level in the model group was significantly decreased (P<0.001), while the IL-6, IL-1β, and TNF-α cytokine levels were significantly increased (P<0.001). Compared with the model group, all doses of the ethyl acetate extract of Qingming flower increased the IL-10 level (P<0.001), while the medium and high dose groups significantly inhibited the IL-6 level (P<0.05), and the high dose group significantly inhibited the IL-1β (P<0.001) and TNF-α (P<0.05) levels.

[0105] 2.3.2 Study on analgesic activity of effective fractions

[0106] Effects of active fractions on writhing reaction induced by glacial acetic acid in mice

[0107] The results showed that compared with the model group, the low, medium and high dose groups of ethyl acetate extract of Qingming flower could significantly reduce the number of writhing reactions induced by glacial acetic acid in mice ( Figure 8 ), and showed a dose-dependent inhibition rate of 29.25%, 47.00% and 65.00%, respectively (Table 7).

[0108] Table-7 Inhibition rate of ethyl acetate extract of Qingming flower on acetic acid-induced writhing reaction in mice

[0109]

[0110] Model: model group; BL: low-dose group of ethyl acetate extraction of qingming flower; BM: medium-dose group of ethyl acetate extraction of qingming flower; BH: high-dose group of ethyl acetate extraction of qingming flower; Asp: aspirin group (n=6).

[0111] Compared with the model group, *** P<0.001.

[0112] Study on the analgesic activity of effective parts by hot plate method

[0113] Compared with the blank group, the positive control drug (P<0.001) and the high-dose group of the ethyl acetate extract of Qingming flower significantly prolonged the pain reaction time of mice at 30 (P<0.05), 60 (P<0.001), 90 (P<0.001) and 120 (P<0.001) minutes after administration. The medium-dose group significantly prolonged the pain reaction time of mice at 90 (P<0.001) and 120 (P<0.001) minutes after administration. Figure 9 .

[0114] 3 Conclusion and Significance

[0115] 3.1 Conclusion

[0116] Compared with the model group, the total extract and ethyl acetate fraction of Qingming flower could significantly inhibit xylene-induced ear swelling in mice (P<0.05, P<0.001), and the total extract group, ethyl acetate fraction group and water fraction group could significantly reduce the number of writhing reactions in mice induced by glacial acetic acid (P<0.001), and significantly prolong the pain reaction time in the hot plate test, suggesting that the ethyl acetate fraction is the anti-inflammatory and analgesic active fraction of Qingming flower. Further studies showed that, compared with the model group, the ethyl acetate extract of Qingminghua significantly inhibited xylene-induced ear swelling in mice (P<0.01, P<0.001) in the medium and high dose groups. The high dose significantly reduced carrageenan-induced paw swelling in mice. All dose groups increased IL-10 levels (P<0.001), while the medium and high dose groups significantly inhibited IL-6 levels (P<0.05). The high dose group significantly inhibited IL-1β (P<0.001) and TNF-α (P<0.05). All dose groups significantly reduced the number of writhing reactions induced by glacial acetic acid in mice in a dose-dependent manner. The medium and high dose groups exhibited analgesic effects in the hot plate test. Polar toxicity tests showed that, with the exception of the n-butanol extract, the total extract, petroleum ether extract, ethyl acetate extract, and water extract did not produce acute toxic reactions in experimental animals at a dose of 2000 mg / kg. The dose of n-butanol was adjusted to 500 mg / kg and continued for 14 days. No acute toxic reaction was observed in the experimental animals.

[0117] In summary, the ethyl acetate extract of Qingming flower may be its main active part for anti-inflammatory and analgesic effects, and it has good anti-inflammatory and analgesic effects. Its anti-inflammatory mechanism may be related to the upregulation of IL-10 levels and the downregulation of IL-6, IL-1β and TNF-α levels. The ethyl acetate extract did not show acute toxic reactions.

[0118] 3.2 Dosage

[0119] The ethyl acetate extract of Qingming flower has good anti-inflammatory and analgesic effects at doses of 200, 400, and 800 mg / kg. For a person weighing 60 kg, the daily dose is 12, 24, and 48 g. For a person weighing 45 kg, the dose is 9, 18, and 36 g. For a person weighing 100 kg, the dose is 20, 40, and 80 g. The optimal dose is 24 g per person per day.

Claims

1. A method for preparing an anti-inflammatory and analgesic extract of Qingming flower, characterized in that: Prepared by the following steps: (1) Ethanol extraction: 10 kg of Qingming flower stems were crushed and extracted in 90% ethanol at room temperature four times, 10 L each time, for 2 hours each time; the combined extracts were removed by decompression on a rotary evaporator to obtain the total extract; (2) Extraction and separation: The total extract was suspended in water, extracted with petroleum ether 5 times, and then extracted with ethyl acetate 5 times. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain.

2. An anti-inflammatory and analgesic extract of Qingming flower prepared by the preparation method according to claim 1.

3. Use of the anti-inflammatory and analgesic extract of chrysanthemum morifolium according to claim 2 in the preparation of anti-inflammatory and analgesic drugs.

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