Herbal mixture extracts (HME) comprised of Semen Sojae Nigrum, Fructus Cnidii, Radix Glycyrrhizae, and Cornu Cervi. Herein, we employed three mouse models, including hot-plate test, acetic acid (AA)-induced writhing test and AA-induced vascular permeability test, to determine analgesic and anti-inflammatory effects of HME. Results revealed that HME exhibited analgesic effects in hot-plate test and in AA-induced writhing test, as evidenced by increasing the latency to lick paws and decreasing AA-induced writhing counts, respectively. HME also significantly and dose-dependently decreased AA-induced vascular permeability, indicating HME exhibited anti-inflammatory effects. Similar improvement can be observed in aspirin treatment that used as positive control in this study. Most of medicinal effects of Fructus Cnidii are considered to attribute to coumarins, such as osthol (OST) and imperatorin (IMP) with several pharmacological activities. We then used OST and IMP as bioactive components in HME. The content of OST and IMP in HME was 3.57 ± 0.10 mg/g and 1.20 ± 0.02 mg/g, respectively, from three independent batches. Only OST possessed inhibitory effects in three mouse models, suggesting that OST may partially involve in protective effects of HME. These results demonstrated that HME has a potential on anti-analgesic effects and anti-inflammatory actions.
Prevention and treatment of chronic diseases of herbal medicines may attribute to their synergistic actions [
These specific functions of each herbal medicine led us to hypothesize that these components when used in combinations could serve as an effective prescription with analgesic effects and anti-inflammation. In the present study, we employed three mouse models, including hot-plate test, acetic acid (AA)-induced writhing test, and AA-induced peritoneal permeability test, to investigate the protective effects of HME. Most of medicinal effects of Fructus Cnidii are considered to attribute to coumarins, such as osthol (OST) and imperatorin (IMP) with several pharmacological activities. In addition, we used osthol (OST) and imperatorin (IMP) as bioactive components in HME. The content of OST and IMP was investigated by HPLC from three independent batches to ensure the quality of manufactured HME. The protective effects of OST and IMP in three mouse models were also determined.
The prescription of HME was developed by Ko Da Pharmaceutical Co. Ltd. (Taoyuan, Taiwan). Semen Sojae Nigrum, Fructus Cnidii, Radix Glycyrrhizae, and Cornu Cervi were purchased from Taiwan or China herbal markets and identified in the R&D center of Ko Da Pharmaceutical Co. Ltd., where voucher specimens have been kept. 75 kg of Semen Sojae Nigrum and 25.2 kg of Fructus Cnidii were soaked in 10-fold (w/v) of 30% (v/v) ethanol for 12 h, and then extracted for 2 h followed by filtration through 40-sieve mesh. 75 kg of Radix Glycyrrhizae were extracted in 12-fold (w/v) of 95% ethanol for 1 h followed by filtration through 40-sieve mesh. All filtrates were collected and subjected to vacuum and reduced-pressure concentration to obtain 30 kg of extracts. 30 kg of Cornu Cervi were grinded to form powder and mixed with extracts at the ratio of 1:1 followed by dried in 60˚C oven for 12 h. The mixtures were granulated using granulator for 2 h, and then passed through 40-sieve mesh to obtain HME.
Male ICR mice (4-wk-old, 28 - 32 g) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). This study protocol was approved by the Institutional Animal Care and Use Committee of HungKuang University (approval No.: 10310). Mice were housed in cages with controlled temperature (25˚C ± 2˚C) and humidity (65% ± 5%) with 12 h-light/dark cycles and were accommodated for 1 wk. During the accommodation and experimental periods, mice were supplied water ad libitum and a standard rodent diet (Lab 5001, Purina Mills) containing 59.8% carbohydrate, 23.4% protein, 4.5% crude fat, as indicated by the supplier.
Mice were randomly divided into nine groups (n = 6 for each group) as follows: group 1, control; group 2, positive control; group 3, low dose of HME (77 mg/kg); group 4, medium dose of HME (154 mg/kg); group 5, high dose of HME (308 mg/kg); group 6, low dose of IMP (0.6 mg/kg); group 7, high dose of IMP (1.2 mg/kg); group 8, low dose of OST (2.2 mg/kg); group 9, high of OST (4.4 mg/kg). All groups, except positive control, were fed orally with normal saline or different doses of HME, IMP, and OST. Positive control mice were fed with aspirin (250 mg/kg) via i.p. injection. The temperature of the hot plate (Basile, Varese, Italy) was maintained at 55˚C ± 2˚C. Mice were placed on hot plate and determined the latency to licking paws for the first time within 30 s. The formula for calculating the difference of the latency to licking paws is the difference between before and after HME, OST, IMP or aspirin administration for 20 min.
Mice were randomly divided into nine groups (n = 5 for each group) as follows: group 1, control; group 2, positive control; group 3, low dose of HME (77 mg/kg); group 4, medium dose of HME (154 mg/kg); group 5, high dose of HME (308 mg/kg); group 6, low dose of IMP (0.6 mg/kg); group 7, high dose of IMP (1.2 mg/kg); group 8, low dose of OST (2.2 mg/kg); group 9, high of OST (4.4 mg/kg). All groups were fed orally with normal saline, aspirin (250 mg/kg), or different doses of HME, IMP, and OST. After administration for 30 min, mice were injected i.p. with 10 mL/kg of 0.6% AA solution. The number of writhes produced in mice was counted within 10 min.
Mice were randomly divided into ten groups (n = 5 for each group) as follows: group 1, control; group 2, AA; group 3, AA + positive control; group 4, AA + low dose of HME (77 mg/kg); group 5, AA + medium dose of HME (154 mg/kg); group 6, AA + high dose of HME (308 mg/kg); group 7, AA + low dose of IMP (0.6 mg/kg); group 8, AA + high dose of IMP (1.2 mg/kg); group 9, AA + low dose of OST (2.2 mg/kg); group 10, AA + high dose of OST (4.4 mg/kg). Mice were injected i.v. with 10 mL/kg of 1% Evans Blue dye solution (Sigma-Aldrich, St. Louis MO, USA) followed by i.p. injection of 10 mL/kg of 0.6% AA. Normal saline, aspirin (250 mg/kg), or different doses of HME, IMP, and OST were given orally 1 h prior to the injection of Evans Blue dye solution. Thirty minutes after injection of AA, mice were sacrificed by cervical dislocation. Peritoneal fluids were collected, and then washed with normal saline followed by centrifuged at 3000 rpm for 10 min. The supernatant was collected and determined the absorbance at 590 nm using a spectrophotometer (Perkin Elmer, Waltham, USA).
The test solution was prepared by mixing 1 g of HME with 50 mL of 70% (v/v) methanol under shaking for 1 h on orbital shaker (Deagle, New Taipei City, Taiwan) followed by filtration through a 0.45 μm filter. The standard solution was prepared by mixing OST and IMP (ChemFaces, Hubei, China) with methanol to obtain different concentrations through serial dilution. Analytical HPLC was performed on Hitachi D-7000 interface equipped with L-7100 pump, L-7420 UV detector and L-7200 autosampler (Tokyo, Japan). Chromatographic separation was carried out on a Mightysil RP-18 column (250 × 4.6 mm, 5 μm) using a gradient solvent system comprised of acetonitrile (A) and 0.1% (v/v) acetic acid (B). Gradient profile was set as follows at 0 - 5 min with the ratio of 40% A and 60% B; at 5 - 50 min with the ratio of 40% - 60% A and 60% - 40% B; at 50 - 65 min with the ratio of 60% - 40% A and 40% - 60% B. The UV wavelength, flow rate, and injection volume were set at 254 nm, 0.8 mL/min, and 10 μL, respectively.
Values are expressed as means ± SD and analyzed using one way ANOVA followed by Fisher’s protected least significant difference test for comparisons of group means, when the F value was significant (P < 0.05). All statistical analyses were performed using SPSS for Windows, version 10 (SPSS, Inc.); a P value < 0.05 is considered statistically significant.
Hot-plate test was used to evaluate the central analgesic effects [
Hot-plate test | AA-induced writhing test | |||
---|---|---|---|---|
Groups | Before Administration | After Administration | Difference | Number of writhing within 10 min |
The latency to licking paws (sec) | ||||
Control | 11.8 ± 4.7 | 12.3 ± 4.5 | 0.5 ± 0.6a | 42.5 ± 4.3a |
Aspirin | 11.3 ± 1.2 | 19.0 ± 2.8 | 7.7 ± 3.3c | 17.3 ± 4.1b |
HME 77 | 11.5 ± 3.9 | 12.8 ± 3.5 | 1.3 ± 0.8ab | 35.0 ± 2.4cf |
HME 154 | 12.0 ± 3.9 | 15.7 ± 4.5 | 3.7 ± 1.5ab | 31.0 ± 3.6cde |
HME 308 | 11.2 ± 2.7 | 21.8 ± 6.2 | 10.7 ± 4.4c | 27.2 ± 6.0e |
IMP 0.6 | 10.3 ± 1.2 | 14.0 ± 2.8 | 3.7 ± 2.2ab | 39.7 ± 3.6af |
IMP 1.2 | 10.0 ± 2.5 | 14.5 ± 2.9 | 4.5 ± 1.2b | 35.8 ± 2.6cf |
OST 2.2 | 9.8 ± 1.6 | 14.0 ± 2.6 | 4.2 ± 2.5b | 33.5 ± 6.4cd |
OST 4.4 | 10.3 ± 3.2 | 18.8 ± 5.2 | 8.2 ± 3.6c | 28.3 ± 4.1de |
Values are expressed as mean ± SD; mean in each column not sharing a letter differ statistically, P < 0.05. Abbreviation: HME, herbal mixture extract; IMP, imperatorin; OST, osthol.
with increasing from 0.5 s to 10.7 s, as compared to control group (
The writhing test induced by AA via i.p. injection is an experimental model used for determining the central and peripheral analgesic activity of drugs [
AA was shown to increase inflammatory mediators, including PGE2, histamine, and serotonin, in peritoneal fluids followed by induction of vascular permeability [
We stipulated that the recommended dietary allowance (RDA) of HME in humans is 1.5 g per day on the basis of HME as a commercial dietary supplement in Taiwan. In all mice models, a formula available for converting human equivalent dose to animal dose in mg/kg, i.e., multiply the human dose in mg/kg per day by 12.3 [
It should be noted that just one batch-specific formulation of HME was used to determine pharmacological effects in mice. We used OST and IMP as bioactive compounds to ensure the quality of manufactured HME and as in HME. The content of OST and IMP in HME was 3.57 ± 0.10 mg/g and 1.20 ± 0.02 mg/g, respectively, from three independent batches (
Most of medicinal effects of Fructus Cnidii are considered to attribute to coumarins, such as OST and IMP [
Groups | Value of OD590nm |
---|---|
Control | 0.03 ± 0.00a |
AA | 2.96 ± 0.11b |
AA + Aspirin | 2.12 ± 0.45cd |
AA + HME 77 | 2.19 ± 0.48c |
AA + HME 154 | 1.94 ± 0.42cd |
AA + HME 308 | 1.62 ± 0.33d |
AA + IMP 0.6 | 2.87 ± 0.16b |
AA + IMP 1.2 | 3.06 ± 0.03b |
AA + OST 2.2 | 2.06 ± 0.26cd |
AA + OST 4.4 | 1.89 ± 0.20cd |
Values are expressed as mean ± SD; mean in each column not sharing a letter differ statistically, P < 0.05. Abbreviation: AA, acetic acid; HME, herbal mixture extract; IMP, imperatorin; OST, osthol.
This research was supported by E10100034-204 from the Small Business Innovation Research (SBIR), Ministry of Economic Affairs, Taiwan.
Chien, M.-Y., Chuang, C.-H., Lin, Y.-T., Yang, C.-M. and Chen, C.-H. (2018) Analgesic and Anti-Inflammatory Effects of Herbal Mixture Extracts in Mice. Pharmacology & Pharmacy, 9, 1-9. https://doi.org/10.4236/pp.2018.91001