Abstract
Purpose
To evaluate the function of cinnabar in Shangke Jiegu tablet (SKJGT) via pharmacodynamics and toxicity investigations to determine whether cinnabar should be removed from SKJGT.
Materials and methods
The pharmacodynamic differences between SKJGT and cinnabar-free Shangke Jiegu tablet (CFSKJGT) were systematically compared in five animal models. Anti-inflammatory effects were assessed on ear swelling and paw edema by measuring the degree of swelling in each. Then, the acetic acid-induced writhing reaction and hot-water tail-flick were also evaluated by counting pain reactions. The pharmacodynamic effects on soft tissue contusions were identified through histopathological observation. Chemical markers of fracture healing, including osteocytes and the blood calcium and phosphorus level, were determined via radiographic examination and biochemical assay, respectively. In addition, the maximum dosages of SKJGT and CFSKJGT were tested in mice to compare their toxicities.
Results
SKJGT and CFSKJGT showed anti-inflammation effects (swelling inhibition ratios of 40.8% and 44.0%, respectively), analgesia (pain threshold ratios of 48.2% and 44.1%, respectively, at 60 min in the hot-water tail-flick test), and soft tissue contusion repair compared with the control (p < 0.05), and the degree of swelling inhibition and the number of pain reactions were dose-dependent. SKJGT and CFSKJGT both significantly improved the bone healing in the rat fracture model, as indicated by the increased osteocyte size during weeks 1–6 and elevated blood calcium and blood phosphorus levels (reaching maximum concentrations of 7.5 mmol/L and 6.8 mmol/L, respectively) during weeks 1–2. The maximum doses for the SKJGT and CFSKJGT groups were 9.0 g/kg in the acute toxicity experiment. The seizure rate of the SKJGT group (25.0%) was lower than that of the CFSKJGT group (50.0%) when the toxicity was observed after administration.
Conclusion
This is the first report to investigate the pharmacodynamics and acute toxicity of cinnabar in SKJGT. Broadly, this study offers novel, valuable insights into the efficacy of cinnabar in prescribed SKJGT.
Keywords: Shangke Jiegu tablet, Anti-inflammatory, Analgesia, Soft tissue contusion, Bone fracture, Acute toxicity
Graphical abstract
Shangke Jiegu tablet; Anti-inflammatory; Analgesia; Soft tissue contusion; Bone fracture; Acute toxicity.
1. Introduction
Shangke Jiegu tablet (SKJGT) is a famous Chinese patent medicine for treating bone fractures and joint injury by promoting blood circulation, relieving pain, reducing swelling, relaxing sinew, and strengthening bone, recorded in the 2020 edition of the Chinese Pharmacopoeia. It includes twelve Chinese medicines, including Semen Strychni (the mature seed from Strychnos nux-vomica L.), cinnabar (a sulfide-like mineral medicine), Carthami flos (the petals from Carthamus tinctorius L.), Eupolyphaga seu Steleophaga (the dry body from Eupolyphaga sinensis Walker), myrrh (the resin from Commiphora myrrha Engl.), Notoginseng radix et rhizoma (the dried root and rhizome from Panax notoginseng (Burk. F. H. Chen), starfish (the dry body of Craspidaster hesperus (Muller et Troschel), chicken bone (bones from Gallus gallus domesticus Brisson), borneolum syntheticum (extracts from Cinnamomum camphora (L.) Presl), calcined pyritum (a sulfide mineral in the pyrite group), olibanum (the resin from Boswellia carterii Birdw.) and Melo Semen (the mature seed from Cucumis melo L.). The efficacy of SKJGT was reported to exceed some well-known orthopedic medicines, such as Dieda Qili pills and Jianbu Huqian pills, which are recorded in the National Prescription volume of Traditional Chinese Medicine, a book compiled according to the representative Chinese patent medicine in 25 large and medium-sized cities in China (Fu, 2005; Zhu, 2014).
Historically, cinnabar was used in China as a tranquilizing mineral medicine and was first recorded in Shennong Bencao Jing during the Han Dynasty. It was used to treat hyperpyretic convulsions, insomnia, etc. as a raw powder (Zhang, 2016). Almost 5% of Traditional Chinese Medicine (TCM) formulas have included cinnabar, and nearly 16.1% of them are pediatric medicines (Kang et al., 2019). However, cinnabar in prescription medicines aroused public concerns because it contains HgS, which has the potential to induce mercury poisoning (Li, 2007; Xu and Feng, 2008).
As a mineral medicine, 99% of cinnabar was excreted (Liang and Shang, 2005) after oral administration, so its toxicity was long considered limited. However, modern pharmacological studies showed that cinnabar could induce accumulative mercury toxicity, including brain damage, hepatotoxicity, etc., and the trace amounts of released mercury from cinnabar in the brain would take 240 days to be metabolized (Ding, 2010), so the debate about whether cinnabar is necessary in TCM continues. Some have appealed to omit cinnabar from TCM prescriptions to ensure their safety, making the re-evaluation of cinnabar-containing Chinese patent medicines urgent (Qi et al., 2010).
To investigate cinnabar's function in SKJGT, we compared its efficacy and acute toxicity with cinnabar-free Shangke Jiegu tablet (CFSKJGT). This will significantly improve our understanding of the effects of cinnabar in SKJGT and measure the effect of adding cinnabar to TCMs.
2. Materials and methods
2.1. Materials
Semen Strychni, cinnabar, Carthami flos, Eupolyphaga seu Steleophaga, myrrh, Notoginseng Radix et Rhizoma, starfish, chicken bone, borneolum syntheticum, calcined pyritum, olibanum, and Melo Semen were obtained from Merro Pharmaceutical Co., Ltd. (Liaoning, China), which is the manufacturer of SKJGT. Voucher specimens (Semen Strychni, MQZ-SPU-001; cinnabar, ZS-SPU-002; Carthami flos, HH-SPU-003; Eupolyphaga seu Steleophaga, TBC-SPU-004; myrrh, MY-SPU-005; Notoginseng Radix et Rhizoma, SQ-SPU-006; starfish, HX-SPU-007; chicken bone, ZJG-SPU-008; borneolum syntheticum, BP-SPU-009; calcined pyritum, DZRT-SPU-010; olibanum, ZRX-SPU-011; and Melo Semen, TGZ-SPU-012) were deposited in the School of Pharmacy, Shenyang Pharmaceutical University. Shang-Ke-Jie-Gu tablet (SKJGT, batch number: 20130110) and cinnabar-free Shang-Ke-Jie-Gu tablet (CFSKJGT, batch number: 20120901) were prepared by mixing the powders of each ingredient according to the protocol recorded in the 2020 Chinese Pharmacopoeia. The preparation process and quality control for the SKJGT and CFSKJGT are given in the Supplemental Material.
Xylene and carrageenan were purchased from Sigma Corporation (St. Louis, USA). Acetic acid, sodium sulfide, talcum powder, soap powder, and corn starch were purchased from Laibo Chemical Industries, Ltd. (Shenyang, China). A post-surgery x-ray instrument (Number: PLX5200, Nanjing Perlove Medical Equipment Co., Ltd., China) was used in the fracture study. All other reagents used in the experiment are commercially available from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China).
2.2. Animals
This study was approved by the Institutional Animal Ethical Committee of Shenyang Pharmaceutical University (approval ID: SYPU-IACUC-C2019-48209). Experimental animals (equal numbers of male and female pathogen-free Sprague-Dawley rats, 220–250 g, and Kunming mice, 18–22 g) were kindly supplied by the Experimental Animal Center of Shenyang Pharmaceutical University. Rats and mice were housed in an air-conditioned animal center (humidity, 50% ± 10%; temperature, 22 ± 2 °C) with a 12 h light/dark cycle. Before the experiment, all animals were allowed ad libitum access to drinking water and standard rat chow and provided with a 7-day acclimation period.
2.3. Anti-inflammatory potential of SKJGT and CFSKJGT
2.3.1. Xylene-induced ear swelling in mice
Xylene-induced mouse ear swelling is a classic experimental model for acute inflammation (Xu and Bian, 2002). The 60 Kunming (KM) mice used in this experiment, 30 male and 30 female, were randomly assigned to five groups and treated according to the standard procedure (control group, 0.5% CMC-Na; SKJGT group (positive group), 0.52 g/kg/day; CFSKJGT-L group, 0.26 g/kg/day; CFSKJGT-M group, 0.52 g/kg/day; and CFSKJGT-H group, 1.04 g/kg/day) for 7 days. Then, 30 min after the last administration, the left ear of each mouse was wiped with 0.03 ml xylene. The round part of the ears (8 mm in diameter) was excised, and the wet weight was measured 2 h later. The difference in mass between the left and right ears was used to calculate the degree of swelling.
2.3.2. Carrageenan-induced paw edema in rats
Carrageenan-induced rat paw edema is an appropriate animal model to assess the anti-edematous activity of natural products (Vinegar et al., 1969). The right ankle circumference of each rat was measured as the reference before carrageenan administration. For this experiment, 60 Sprague-Dawley (SD) rats, 30 male and 30 female, were randomly divided into five groups. Animals in the control group were wiped with 0.5% CMC-Na, while treated groups were administered 0.18 g/kg/day SKJGT (SKJGT group), 0.09 g/kg/day CFSKJGT (CFSKJGT-L group), 0.18 g/kg/day CFSKJGT (CFSKJGT-M group), or 0.36 g/kg/day CFSKJGT (CFSKJGT-H group), for 7 days. Then, 30 min after the last administration, 0.05 ml of 1% suspension of carrageenan was injected into the sub-plantar area of the right paw (Xu and Bian, 2002). The ankle circumferences of each rat were measured at 1, 2, 3, 4, 5, and 6 h after injection. The mass difference between the left and right ankles was used to calculate the degree of edema.
2.4. Analgesic potential of SKJGT and CFSKJGT
2.4.1. Hot-water tail-flick test
The hot-water tail-flick test is a classic, reliable screening method to study analgesics. The hot-water tail-flick test measures the body surface pain caused by heat stimulation and is suitable for evaluating central analgesics. A total of 60 kM mice (30 male and 30 female) were randomly divided into five groups. Animals in the control group were wiped with 0.5% CMC-Na, while treated groups were administered with 0.52 g/kg/day SKJGT (SKJGT group), 0.26 g/kg/day CFSKJGT (CFSKJGT-L group), 0.52 g/kg/day CFSKJGT (CFSKJGT-M group), or 1.04 g/kg/day CFSKJGT (CFSKJGT-H group).
The hot-water tail-flick latencies of the test animals were recorded at 30 min, 60 min, and 120 min after administration, and the increased rate of the pain threshold was calculated to evaluate drug efficacy. The animals’ tail-skin temperature was manipulated by immersing the tails in water at a temperature of 47 °C.
2.4.2. Acetic acid-induced writhing reaction
The acetic acid-induced writhing reaction is a reliable animal model for evaluating analgesics. A total of 60 kM mice (30 male and 30 female) were randomly divided into five groups and treated according to the previous description (control group, 0.5% CMC-Na; SKJGT group, 0.52 g/kg/day SKJGT; CFSKJGT-L group, 0.26 g/kg/day CFSKJGT; CFSKJGT-M group, 0.52 g/kg/day CFSKJGT; and CFSKJGT-H group, 1.04 g/kg/day CFSKJGT). Then, 120 min after the last administration, the number of writhes was counted for 30 min immediately after the injection of 0.6% acetic acid (i.p.) at a rate of 0.1 mL/10 g body weight. The number and incubation period of the writhes was counted for 15 min after injection.
2.5. Soft-tissue contusion in mice
Mice are good soft-tissue contusion models for evaluating drugs intended to promote soft-tissue repair (Cheng et al., 2007; Zhou et al., 1991). In this study, 60 kM mice (30 male and 30 female) were shaved on the left side of the posterior 24 h before hair removal agent administration. The hair removal agent was composed of 2.5 g sodium sulfide, 3.5 g talc, 0.5 g soap powder, and the necessary amounts of water and corn starch to make the paste (Xiao et al., 2013). The soft-tissue contusion device was made according to the method described in the literature (Zhou et al., 1991). After pilot trials, the experimental condition was established as 50 g of balance weight, lifting to 7 cm high, and hitting the shaved skin 7 consecutive times.
Successfully contused mice were randomly divided into 5 groups: animals in the control group were administered 0.5% CMC-Na; those in the SKJGT group were given 0.52 g/kg/day SKJGT; the CFSKJGT-L group, 0.26 g/kg CFSKJGT; the CFSKJGT-M group, 0.52 g/kg CFSKJGT; and the CFSKJGT-H group, 1.04 g/kg CFSKJGT once per day for 7 days. On the eighth day, the animals were sacrificed, their swollen parts were dissected, tissue repair was observed, and a visual evaluation score was given. The histopathological section was microscopically applied to the histological evaluation score. Vision and histology scoring have changed since 1972 (Ingram and Grasso, 1975; Landsdown, 1972; Lashmar et al., 1989). A scoring system was prepared from the selected method and is shown in Table 1. The appearance of each mouse's visual or histological features was scored in a system where the absence of a trait was marked as 0.
Table 1.
Scoring system of the mouse soft-tissue contusion experiment.
| Histological appearance | Score | Visual appearance | Score |
|---|---|---|---|
| Ecchymosis | 2 | Ecchymosis | 2 |
| Muscle fiber fracture and variation | 2 | Bruise | 1 |
| Inflammatory cell infiltration | 1 | Angiogenesis | 1 |
| Tissue edema | 1 |
2.6. External force-induced fracture in rats
A total of 200 SD rats (100 male and 100 female) were randomly divided into five groups of 20 each, including one control group (0.5% CMC-Na) and four treatment groups (SKJGT group, 0.18 g/kg/day SKJGT; CFSKJGT-L group, 0.09 g/kg/day CFSKJGT; CFSKJGT-M group, 0.18 g/kg/day CFSKJGT; CFSKJGT-H group, 0.36 g/kg CFSKJGT). The treatments were administered daily for 6 weeks. The fractured legs of the control group were treated with 0.5% CMC-Na on the same schedule.
Anesthesia was induced and maintained with ether throughout the surgery and post-surgery x-ray. The fracture surgery was performed as previously described (Smitham et al., 2014; Zwingenberger et al., 2013); briefly, a 3-mm incision was made on the right radius and blunt dissection was performed to visualize the radius. The radius nerve was also visualized and avoided. A plastic external fixator was fixed to the anterolateral side of the radius with four titanium pins. The fixator and pins combined weigh 0.20 ± 0.01 g. A wire saw was passed underneath the radius, and the transverse osteotomy (0.4 mm) was conducted between the intermediate pins through the total thickness of the bone. Postoperative x-rays of all animals with external fixators were obtained during surgery to confirm the correct placement of the fixator and pin.
Half of the animals in each of the five treatment groups were sacrificed on the 28th day of treatment and the remaining half on the 42nd day of treatment according to the dosing protocol described above. Each radius was dissected, washed with chilled saline, weighed, and an ulna-radius syndesmosis was obtained for the histopathology study. For the biochemical assay, blood samples were collected from the animals at 7, 14, 21, 28, and 42 days after oral SKJGT and CFSKJGT administration.
2.7. Acute toxicity observation of SKJGT and CFSKJGT in mice
For the acute toxicity study, 48 kM mice (24 male and 24 female) were randomly divided into two groups of 12: one SKJGT and one CFSKJGT group. The dosing protocol was 9.0 g/kg/day SKJGT for the SKJGT group; 9.0 g/kg/day CFSKJGT for the CFSKJGT group. The rate of seizures was measured 30 min after SKJGT and CFSKJGT administration.
2.8. Data analysis
All of the results are expressed as mean ± S.E.M. values. The differences between groups were evaluated via one-way analysis of variance (ANOVA) followed by Tukey's comparison test. The level of significance was p < 0.05. For the hot-water tail-flick test and external force-induced fracture experiment, the differences between the SKJGT group and CFSKJGT groups were analyzed via two-way ANOVA followed by Tukey's comparison test. The level of significance was p < 0.05. SPSS software version 20 was used for statistical analysis.
3. Results
3.1. Results regarding the anti-inflammatory potential of SKJGT and CFSKJGT
3.1.1. Anti-inflammatory effects on xylene-induced ear swelling in mice
After the xylene wipe, ear swelling was observed at 30 min. SKJGT and CFSKJGT treatments inhibited swelling differently. As seen in Figure 1A, 120 min after administration, the ear swelling caused by xylene was significantly inhibited in each treated group (p < 0.05) compared with the control. Additionally, the inhibition rates were different—SKJGT (40.8% inhibition) and CFSKJGT-M (44.0% inhibition)—but this difference was not significant when treatment groups were compared with each other (p > 0.05). Therefore, SKJGT and CFSKJGT similarly and significantly suppressed xylene-induced ear swelling in rats.
Figure 1.
Statistical results of ear swelling (A) and paw edema (B) experiments. Note: Control: control group; SKJGT: original formula group; CFSKJGT-L: low-dose cinnabar-free group; CFSKJGT-M: medium-dose cinnabar-free group; and CFSKJGT-H: high-dose cinnabar-free group. The SKJGT and CFSKJGT groups were compared with the control group. ∗ indicates p < 0.05 and ∗∗ indicates p < 0.01.
3.1.2. Anti-inflammatory effects on carrageenan-induced paw edema in rats
In this experiment, the rats in each group developed paw edema 1 h after carrageenan injection, and the average ankle circumference increased from 0.8 cm to 1.2 cm, indicating that the model was successfully established. As shown in Figure 1B, the ankle circumference of the control group increased rapidly within 1 h and reached its highest point (1.2 cm) at 2 h, while the ankle circumferences of the treatment groups decreased within 6 h. Around 4 h, the ankle circumference of all treatment groups had significantly decreased (p < 0.05) compared with the control: SKJGT (37.5% inhibition rate), CFSKJGT-M (40.0% inhibition rate), and CFSKJGT-H (46.3% inhibition rate). However, there was no significant difference among the treated groups (p < 0.05). The results suggest that SKJGT and CFSKJGT both significantly suppressed carrageenan-induced rat paw edema. Within 5–6 h, edema increased to similar levels in all groups, indicating that carrageenan-induced inflammation in rats lasted for 5 h.
3.2. Results regarding the analgesic potential of SKJGT and CFSKJGT
3.2.1. Hot-water tail-flick experiment results
Tail-flick latency inhibition was different among different CFSKJGT treatment groups. As shown in Figure 2 and 1 h after administration, tail flicking was significantly inhibited in all treatment groups (p < 0.05) compared with the control, and the inhibition rates of the four preparations were different: the SKJGT group showed a 48.2% pain threshold rate; the CFSKJGT-L group, a 29.7% pain threshold rate; the CFSKJGT-M group, a 44.1% pain threshold rate; and the CFSKJGT-H group, a 50.4% pain threshold rate. The pain threshold rate of the CFSKJGT-L group was significantly lower than that of the SKJGT group (p < 0.05). The results indicate that SKJGT and CFSKJGT could both significantly increase the pain threshold rate for tail-flicking, and their therapeutic effects were similar. At 1 h after administration, the pain threshold rate of the CFSKJGT-M group was significantly lower than that of the SKJGT group (p < 0.05). The results indicate that SKJGT and CFSKJGT at various dosages could noticeably increase the tail-flicking threshold rate.
Figure 2.
Statistical results of mouse hot-water tail-flick experiment. Note: Treatment groups compared with the control group, ∗ indicates p < 0.05 and ∗∗ indicates p < 0.01; CFSKJGT groups compared with the SKJGT group, # indicates p < 0.05.
3.2.2. Analgesic effect on acetic acid-stimulated writhing in mice
The writhing test measures visceral pain induced by chemical stimulants and is used to evaluate weak central and peripheral analgesics. The number of writhes reached a peak at 5–10 min and almost disappeared 15 min after the injection of acetic acid. The number of writhes was reduced by pretreatment with CFSKJGT and SKJGT, depending on the dose. As seen in Figure 3 and 15 mins after the injection of acetic acid, all of the treated groups showed significantly inhibited acetic acid-induced writhes (p < 0.05) compared with the control. There was no significant difference between the CFSKJGT and SKJGT groups.
Figure 3.
Statistical results of acetic acid-induced writhing in mice. Note: The SKGJT group and the CFSKJGT groups were compared with the control group. ∗ indicates p < 0.05 and ∗∗ indicates p < 0.01.
3.3. Promoting soft-tissue contusion repair in mice
After hair removal, no visual changes were observed on the shaved skin of each mouse, indicating that our hair-removal agent (Xiao et al., 2013) was acceptable. The skin was observed on the left side of the mice's rears immediately after blows were struck with the contusion device and a clear bruise appeared after 1 h, indicating successful modeling. After SKJGT and CFSKJGT administration for 7 days, extensive ecchymosis, bruising, and angiogenesis were observed in the control group. In the treatment groups, incidental lesions and angiogenesis were occasionally observed, but the bruise disappeared. Therefore, SKJGT and CFSKJGT reduced soft-tissue contusion. The scores of the treatment groups were significantly lower than that of the control group (p < 0.05), as shown in Table 2. Histological examination showed ecchymosis, muscle fiber destruction, and inflammatory cell infiltration in the muscle tissue of the control group (Figure 4A). In the treatment groups, ecchymosis and muscle fiber fracture disappeared, although inflammatory cell infiltration remained (Figure 4B, C, D, E).
Table 2.
Statistical data for the mouse soft-tissue contusion experiment.
| Group | Dose (g/kg) | Histological score | Visual score |
|---|---|---|---|
| Control | - | 1.38 ± 0.74 | 2.38 ± 0.74 |
| SKJGT | 0.52 | 0.50 ± 0.76 ∗ | 1.13 ± 0.99 ∗ |
| CFSKJGT-L | 0.26 | 0.50 ± 0.93 ∗ | 1.13 ± 0.64 ∗ |
| CFSKJGT-M | 0.52 | 0.50 ± 0.76 ∗ | 1.00 ± 0.93 ∗ |
| CFSKJGT-H | 1.04 | 0.50 ± 0.93 ∗ | 0.63 ± 0.92 ∗ |
Note: The SKJGT group and the CFSKJGT groups were each compared with the control group and ∗ indicates p < 0.05.
Figure 4.
Pathological sections of contused mice on the seventh day after treatment with 0.5% CMC-Na or different formulations of SKJGT or CFSKJGT. (A) Control group: the arrow indicates inflammatory cell infiltration, muscle fiber fracture, and congestion of blood vessels, confirming that the modeling method induced soft-tissue contusion. (B) SKJGT group: the arrow indicates slight inflammatory cell infiltration; muscle cells are separated by red blood cells. (C) CFSKJGT-L: the arrow indicates inflammatory cell infiltration, slight intermuscular hemorrhage, and congestion of blood vessels. (D) CFSKJGT-M group: the arrow indicates slight inflammatory cell infiltration, intermuscular hemorrhage, and congestion of blood vessels. (E) CFSKJGT-H group: the arrow indicates congestion of blood vessels.
The SKJGT, CFSKJGT-L, CFSKJGT-M, and CFSKJGT-H groups showed remarkable therapeutic effects on the soft-tissue contusion. However, the differences between these four groups were not significant (p > 0.05). Therefore, SKJGT and CFSKJGT both offered good therapeutic effects.
3.4. Effects of SKJGT and CFSKJGT on osteotylus formation after fracture in rats
We used Nilsson's histological grading standard (Table 3) to score the histological observations of each specimen, with bone healing properties, osteotylus formation, and appearance of bone marrow as the analysis indicators. Osteotylus formation was observed after radiographic examination in all treatment groups (Figure 5A). Osteotylus was not observed in all groups 1 week after fracture induction. In the control group, an observable osteotylus had formed at week 4, followed by gradual compaction over the experimental period. In contrast, in the treatment groups, the osteotylus increased steadily in size from weeks 4–6. In addition, the rate of osteotylus compaction was significantly greater in the treatment groups, suggesting that SKJGT and CFSKJGT promoted the remodeling of the soft osteotylus into a hard osteotylus during the fracture-healing process.
Table 3.
Histomorphometric grading of radial fracture repair.
| Class | Degree | Score |
|---|---|---|
| Union | No sign | 0 |
| Fibrous | 1 | |
| Fibrocartilaginous | 2 | |
| Bony | 3 | |
| Complete, with trabecular and cortical bone | 4 | |
| Osteotylus | None | 0 |
| Small amount | 1 | |
| Moderate amount | 2 | |
| Profuse | 3 | |
| Bridging | 4 | |
| Bone marrow | None in resected area | 0 |
| Beginning to appear | 1 | |
| Present in more than half of the defect | 2 | |
| Complete colonization by red marrow | 3 | |
| Mature fatty marrow | 4 |
Figure 5.
The effects of SKJGT and CFSKJGT on fracture healing in rats. A: Results of radiographic examination, typical X-ray images of the broken bones: (i) immediately after the fracture, (ii) SKJGT group with osteotylus, week 4; (iii) CFSKJGT-L group with small osteotylus, week 4; (iv) CFSKJGT-M group with osteotylus, week 4; (v) CFSKJGT-H group with small osteotylus, week 4. B: Results of histopathological section: (i) immediately after the fracture, (ii) SKJGT group with many osteoblasts and bone marrow, week 4; (iii) CFSKJGT-L group with fewer osteoblasts and bone marrow, week 4; (iv) CFSKJGT-M group with many osteoblasts and bone marrow, week 4; (v) CFSKJGT-H group with many osteoblasts and bone marrow, week 4. C: histopathological scoring.
Osteoblasts and bone marrow were observed in all treated animals, and mature bone tissue was observed in the CFSKLGT-H group at week 4 (Figure 5B & C). After two weeks, a few osteoblasts were observed in all treated animals. Compared with the control group, observable bone marrow and osteoblasts were formed in the SKJGT and CFSKJGT groups, followed by a gradual increase throughout the experimental period. Meanwhile, the bone tissue size in the SKJGT and CFSKJGT treatment groups increased similarly from weeks 1–6, suggesting that both SKJGT and CFSKJGT promoted the remodeling of the soft osteotylus into hard osteotylus during the fracture-healing process.
3.5. Effects of SKJGT and CFSKJGT on plasma bone-specific alkaline phosphatase activity and the blood calcium and phosphorus level
Bone-specific alkaline phosphatase (BALP) is involved in bone formation, stable in serum, and a marker of osteoblast maturation and activity. The proliferation, differentiation, and maturation of BALP are closely related to normal bone growth and development. BALP is synthesized in the mature stage of the bone matrix and is closely related to bone-matrix mineralization. When bone mineralization is blocked, osteoblast cells produce substantial alkaline phosphatase, significantly increasing serum BALP.
As shown in Figure 6A, similar levels of plasma BALP activity were observed in all groups before fracture induction. After fracture induction, the BALP activity in all groups increased gradually from weeks 1–2 in response to the fracture. This elevation did not persist but decreased until week 6 in all groups, reaching pre-fracture levels. However, 1 week after fracture induction, there was no noticeable BALP change in any treatment group compared with the control group (p > 0.05). BALP activity in the SKJGT group was slightly higher than in the CFSKJGT groups, but no significant differences between treatment groups were observed (p > 0.05). SKJGT and CFSKJGT exhibit little variance in promoting fracture healing.
Figure 6.
Effects of SKJGT and CFSKJGT on alkaline phosphatase (A) and blood calcium and blood phosphorus (B). Note: Control: control group; SKJGT: original formula group; CFSKJGT-L: low-dose cinnabar-free group; CFSKJGT-M: middle-dose cinnabar-free group, CFSKJGT-H: high-dose cinnabar-free group. Treatment groups compared with the control group, ∗ indicates p < 0.05; CFSKJGT groups compared with the SKJGT group, # indicates p < 0.05.
Calcium and phosphorus in the blood mainly exist in the form of Ca2+ and HPO42− and are maintained in a dynamic balance. When the calcium and phosphorus levels are too high, they are deposited in bone tissues in the form of bone salts. Therefore, increased blood calcium and phosphorus is conducive to the deposition of calcium salts and promotes fracture healing.
As shown in Figure 6B, compared with the control group, noticeable blood calcium and blood phosphorus level changes were observed in the SKJGT and CFSKJGT-M groups 1 week after fracture induction (increased by 18.1 ± 9.8% and 13.5 ± 5.8%, respectively; p < 0.05) and 2 weeks after (increased by 14.4 ± 5.1% and 10.4 ± 7.8%, respectively; p < 0.05). After fracture induction, the blood calcium and blood phosphorus in all groups increased gradually during weeks 1–2 in response to the fracture. This elevation of blood calcium and blood phosphorus did not persist but decreased until week 6, reaching pre-fracture levels. These results indicate that the first two weeks were the critical period for fracture healing and that SKJGT was significantly more successful at promoting fracture healing than CFSKJGT.
3.6. Toxicological results for SKJGT and CFSKJGT
In the SKJGT and CFSKJGT groups, Semen Strychni toxicity symptoms occurred about 12 min after administration. Mice that were not dead within 1 day after administration were continuously observed for 14 days, and no further deaths occurred. In the SKJGT group, toxicity symptoms occurred approximately 20 min after administration, and the rate of seizures reached 25.0%, as shown in Table 4. In the CFSKJGT group, toxicity symptoms occurred approximately 12 min after administration, and the rate of seizures reached 50.0%. These results indicate that SKJGT's reduction of Semen Strychni neurotoxicity was significantly better than that of CFSKJGT. Cinnabar may be crucial to SKJGT's reduction of Semen Strychni neurotoxicity.
Table 4.
Acute toxicity test results for SKJGT and CFSKJGT.
| Groups | Rate of seizures (%) |
|---|---|
| SKJGT (9.0 g/kg) | 25.0 |
| CFSKJGT (9.0 g/kg) | 50.0 |
4. Discussion
Mineral medicines are essential in TCM and have been documented in many ancient codes and records, having been used in China for thousands of years. However, research into mineral medicines’ efficacy and safety has been limited, so people have questioned the necessity of these medicines. Cinnabar is a controversial ingredient of compound medicines as it may induce mercury poisoning. SKJGT allowed us to explore whether cinnabar is necessary in TCM prescriptions.
In this study, pharmacological models corresponding to its clinical indications were designed to systematically evaluate cinnabar's possible role in SKJGT. Both SKJGT and CFSKJGT offer significant anti-inflammatory and analgesic effects, as indicated by the relevant animal models. There was no significant difference in the effects of SKJGT and CFSKJGT preparations on most of the tested animal models. However, blood calcium and blood phosphorus and the increasing rate of pain threshold in the hot-water tail-flick test indicate that SKJGT is more effective than CFSKJGT.
Cinnabar may play a crucial role in SKJGT's ability to increase the rate of pain threshold in the hot-water tail-flick experiment. It detoxifies, calms nerves, clears heart fire, and relieves convulsions (Tian et al., 2019). Modern pharmacological studies have shown that cinnabar has anxiolytic effects and is associated with decreased serotonin 5-hydroxytryptamine (5-HT) levels in the brain (Wang et al., 2007). This may explain why cinnabar exerts a sedative and analgesic effect.
The metabolism of serum calcium and phosphorus is an important component of bone metabolism. Compared with simple serum calcium and phosphorus, the product of calcium and phosphorus is more stable, directly affecting calcium salt deposition (Broggini et al., 2015; Cho et al., 2013).
Under normal conditions, bone salts are constantly dissolved, releasing calcium into the blood, and bone constantly removes calcium from the blood to calcify bone cells. After fracture occurs, an observable callus of osteocytes forms near the fracture, followed by gradual compaction with osteocytes to ossify. Large amounts of calcium salts are formed and deposited during the early fracture period. This may be why the blood calcium and phosphorus content increased significantly in the treatment and control groups during the first two weeks after fracture induction. At the same time, the results of this experiment show that cinnabar can promote blood calcium and blood phosphorus in rat serum to a certain extent in weeks 1–2. Cinnabar's promotion of blood calcium and blood phosphorus has never been reported, and we thought this might be an indirect effect derived from its calming effect, which stabilizes the body for recovery from a bone fracture.
Another interesting finding was that cinnabar could alleviate the neurotoxicity induced by Semen Strychni in SKJGT. Semen Strychni, the mature seed of Strychnos nux-vomica L., has been used in TCM for thousands of years to treat facial nerve paralysis, rheumatoid arthritis, and bone fractures (Chen et al., 2012; Commission, 2020). Although Semen Strychni shows good curative efficacy, its application has been limited due to the neurotoxicity of its alkaloids (Yange et al., 2014). The alkaloids strychnine and brucine, accounting for 70% of total Semen Strychni alkaloids (Wu et al., 2007), have been recognized as the main components that are responsible for its pharmaceutical effects and its neurotoxicity (Nagasawa et al., 1996). Strychnine is a well-known, potent antagonist of glycine receptors in the vertebrate CNS and a powerful blocker of nicotinic acetylcholine receptors in various muscle and neuron cells (Li et al., 2018; Yin et al., 2003). Strychnine poisoning is characterized by a short prodromal phase, after which there are unusual seizures with intact sensoria.
In our research, CFSKJGT displayed obvious neurotoxicity caused by Semen Strychni, which was evaluated via the convulsion rate of treated mice. However, cinnabar appeared to significantly alleviate Semen Strychni-induced convulsions in the SKJGT group. According to TCM, cinnabar is sweet, slightly cold, and calms and detoxifies. Cinnabar is widely used in TCM to treat palpitations, epilepsy, and convulsions in children. Semen Strychni is bitter, warm, and relieves pain, disperses knots, and reduces swelling. In Traditional Chinese Medicine, Semen Strychni is mainly used for fall injuries, fractures, swelling, and pain.
Semen Strychni's strong neurotoxicity manifests as ankylosing convulsions and epileptic symptoms. Cinnabar and Semen Strychni have opposing properties; cinnabar's calming and detoxifying effects can be used clinically to treat epilepsy. Therefore, cinnabar combined with Semen Strychni reduces the toxicity of Semen Strychni, according to TCM theoretical guidance.
5. Conclusion
In summary, our study indicated that the analgesic and fracture-healing promotion effects of SKJGT were significantly stronger than those of CFSKJGT according to the hot-water tail-flick test and blood calcium and blood phosphorus levels. Cinnabar also appears to reduce the toxicity of Semen Strychni in rats and is, thus, potentially safe and effective when used according to TCM principles. The findings of these pharmacodynamic and acute toxicity studies confirmed that cinnabar plays a vital role in prescribed SKJGT.
Declarations
Author contribution statement
Taotao Wang; Na Han; Jun Yin: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; analysis tools or data; Wrote the paper.
Qiao Li; Ming Yang: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.
Haoying Xi; Zhihui Liu: Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data.
Ruimao Feng: Analyzed and interpreted the data.
Funding statement
Jun Yin was supported by Innovative Research Group Project of the National Natural Science Foundation of China [81573628 & 81872982], Natural Science Foundation of Liaoning Province [[2022] No. 47].
Data availability statement
Data will be made available on request.
Declaration of interest’s statement
The authors declare no conflict of interest.
Additional information
Supplementary content related to this article has been published online at [URL].
Appendix A. Supplementary data
The following is the supplementary data related to this article:
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Data Availability Statement
Data will be made available on request.