The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.
Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine. Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.
Decoction formula is the most commonly used dosage form of the traditional Chinese medicine and the liquid medicinal drugs1,2. The decoction formula of traditional Chinese medicine is processed by decocting the herbs in water after being soaked, followed by filtering to discard the dregs.
Because the decoction formula can be individualized based on clinical necessities, especially in line with the characteristics of evidence-based treatment in traditional Chinese medicine, this dosage form via trans-oral administration has been applied in clinical practice for thousands of years with specific and irreplaceable advantages3,4. The quality of the decoction formula is directly related to the clinical efficacy. Therefore, the decoction formula must be prepared based on the principles for the standardized decoction methods to ensure the active ingredients are maximally extracted and protected3. Meanwhile, decoction-ready medicines are the main form of raw Chinese herbs used in clinical practice; the quality standards and production methods of prescribed decoction-ready medicines must comply with the provisions of the Chinese Pharmacopoeia2. Moreover, the prescribed decoction-ready medicines must be soaked in cold drinking water for at least 30 min before being decocted in a special pharmaceutical pot by heating5. Studies have shown that most of the active ingredients can be extracted by decocting for two times, and the Code of Practice for the Administration of Decoction Rooms of Traditional Chinese Medicines in Medical Institutions also prescribes decoction to be done twice4,6,7. If special Chinese herbs, such as one with hard texture or tonics, are prescribed, the decoction time is recommended to be extended. The decoction utensils and storage containers that directly come in contact with the decoction formula, starting from the container in which the prescribed decoction-ready medicines are soaked, should be chemically stable and have lids, such as casseroles, ceramics, glass and stainless steel. However, aluminum and ordinary plastic products should not be used, while iron and other corrosive utensils should be prohibited to avoid possible chemical reactions, which will reduce therapeutic efficacy and even produce harmful effects4,6,7.
Due to the limitations of performing clinical experiments, small animals bearing the human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions including the decoction formula of traditional Chinese medicine8,9; however, unlike humans, the decoction formulas should be prepared every day, mixed together, and freshly administered 2x a day in clinical practice. Animals are unable to voluntarily take their medications on time with the required dosage, and most of the animal experiments related to traditional Chinese medicine are gavage-based oral administration of the decoction formulas at present. The decoction formulas used for in vivo animal experiments should be prepared in advance, aliquoted, stored at -20 °C and thawed just before use3,4,10. Moreover, substandard animal immobilization and gavage needle manipulation increase the risk of unnecessary injuries and negative emotions. Therefore, as an important method of trans-oral drug delivery, the skilled gavage technique is particularly important to avoid possible esophageal injury and drug spillage, thus ensuring that each model animal receives an equal amount of prescribed herbal medicine to achieve accurate experimental results. Here, we provide a gavage protocol for the decoction formula of traditional Chinese medicine.
This protocol describes the detailed gavage technique in osteosarcoma model mice by using the Yiqi Jiedu decoction as a sample. Four-week-old, male athymic mice (BALB/c nude) were used in this experiment and kept in the SPF-grade breeding room of Laboratory Animal Center in Shanghai University of Traditional Chinese Medicine. All animal experiments were approved by the Animal Management and Committee of Shanghai University of Traditional Chinese Medicine, complied with the Code of Ethics for Laboratory Animals (Animal Ethics Approval Number: PZSHUTCM2304060007), and conducted in strict accordance with the international requirements related to laboratory animals.
1. Materials
2. Dosage determination
3. Cell source and culture
4. Modeling methods
5. Preparation of traditional Chinese medicine decoction formula for gavage ( Figure 2)
6. Gavage administration in mice by using a gavage device
7. Course of treatment
8. Adverse reactions
The inhibitory effect of Yiqi Jiedu decoction on the growth of osteosarcoma in vivo was determined in an intra-tibial xenograft osteosarcoma mouse model, which was prepared by injecting the 143B osteosarcoma cells into the tibia of athymic mice. The Yiqi Jiedu decoction was administered to the mice of the Yiqi Jiedu decoction group by gavage for 28 days starting from the second day of the 143B injection. Physiological saline was given to the mice in the control group. All mice were euthanized after 28 days. The results showed that the tumor volume in the Yiqi Jiedu decoction group was significantly reduced compared with that of the control group (Figure 5).
Figure 1: Decoction consumables. The decoction-ready medicine consists of (A) Ginseng, (B) Hedyotis diffusa, (C) Barbed skullcap herb, (D) Graduated measuring cup, (E) Rubber bands, (F) Gauze, (G) Fixing three layers of gauze at the mouth of measuring cup with rubber bands. Please click here to view a larger version of this figure.
Figure 2: Preparation of decoction formula for traditional Chinese medicine. (A) The decoction-ready medicines except for Ginseng being soaked for 30 min. (B) Ginseng being soaked separately for 30 min. (C) Ginseng being decocted separately. (D) Filtering decoction formula of traditional Chinese medicine. (E) Storage of concentrated decoction formula of traditional Chinese medicine. Please click here to view a larger version of this figure.
Figure 3: Gavage consumables. Regular 1 mL syringe with original needle (left panel), and 1 mL syringe with original needle being replaced by a No. 12/55 mm elbow gastric perfusion needle to set a gavage device (right panel). Please click here to view a larger version of this figure.
Figure 4: Procedure of gavage administration in mouse. (A) Mouse on the cage surface. (B) Mouse tail being grasped with one hand. (C-D) Mouse being fixed with one hand. (E) Needle being inserted from the mouse mouth. (F) End of gavage administration. Please click here to view a larger version of this figure.
Figure 5: Inhibitory effect of the decoction in the osteosarcoma mouse model. (A) Osteosarcoma samples being collected from the control group and the Yiqi Jiedu decoction intervention group. Error bars stand for standard deviation (n=7) (B) Unpaired t test of tumor volume between control (Ctrl) group and Yiqi Jiedu decoction intervention group. Yiqi Jiedu decoction intervention for 28 days significantly inhibits the tumor volume (p<0.05). Error bars stand for standard deviation (n=7). (C) Body weight of mice in the Yiqi Jiedu decoction gavage group and the control group throughout the experimental period. Please click here to view a larger version of this figure.
The clinical efficacy of the prescribed herbs in traditional Chinese medicine is largely associated with the administered dose and dosage form; therefore, the establishment of a standardized decoction strategy and a skilled administration process are necessary in clinical practice, as well as in improving the reliability of experimental results.
In this study, we first provided a detailed strategy for the standardized decoction of Chinese herbs used in clinical practice of traditional Chinese medicine, which facilitates the guidance of people to scientifically decoct medicines, also practically improves the quality of decoction formula and clinical efficacy of herbal medicines; therefore, once the standardization is achieved, it will improve the accuracy and consistency of the herbal medicine efficacy in clinical practice.
The gavage strategy has been widely applied as an oral intake method in preclinical studies and is applicable to various experimental animals, including mice, rats, and rabbits, which offers some advantages, such as ease of administration, accurate dosage control, and ability to mimic the clinical route of drug administration13. However, the animals will struggle if they are immobilized in an incorrect position without anesthesia, which may threaten the health of the researchers and reduce the reliability of the experiments. Therefore, the establishment of a standardized gavage method is important to reduce these possibilities and to avoid decoction entering the esophagus by mistake or causing esophagus damage, which in turn causes animal death and experimental failure.
Here, we chose the gavage route to deliver the decoction formula into the osteosarcoma model mice, as it allowed us to precisely measure the dosage and warrant consistent drug delivery throughout the experiment. To prepare the decoction formula, we first calculated the quantity of decoction-ready medicines for the mouse based on a ratio of 20 times the quantity used for adults with a body weight of 60 kg; soaked, and decocted 2x by heating to boiling; filter concentrated, and stored the decoction in aliquots at -20 °C. To perform the gavage administration, load 200 µL of the concentrated decoction formula into a gavage device, insert the gavage needle into the mouth, gently push the gavage needle toward the stomach and inject the decoction formula into the stomach of the mouse. One of our findings is the significant inhibition of tumor growth observed in the group receiving the decoction formula of Yiqi Jiedu decoction via gavage. We observed an obvious reduction in tumor volume in the Yiqi Jiedu decoction group compared to the control group. These results indicate that the gavage strategy effectively delivers the active components of the decoction formula to the target site, resulting in potent anti-tumor effects. Furthermore, we evaluated the safety profile of the gavage strategy by monitoring the general condition and body weight of the mice throughout the experimental period. We found no significant differences in body weight changes or signs of toxicity between the Yiqi Jiedu decoction gavage group and the control group, indicating that the gavage administration of the decoction formula is well-tolerated and does not cause obvious adverse effects.
Although the gavage strategy proved effective in delivering the decoction formula to the experimental animals, it is important to realize the potential adverse reactions and certain limitations associated with this administration method. To avoid food-caused trachea obstruction, closely observe mouse status during and after gavage, such as difficulty breathing, shortness of breath, and cough. Once any of them appear, immediately stop the gavage and perform chest compressions upon a horizontal hard desktop; furthermore, immediate veterinary help is necessary for any serious condition. Moreover, we need to pay close attention to the following limitations. Firstly, gavage may induce stress in the animals, potentially affecting their overall physiology and response to treatment. However, we took actions to minimize stress by using a small feeding needle and handling the mice gently during the gavage procedure. Moreover, gavage could only be performed by a person who has been specifically trained and qualified to ensure that a standardized technique is used for avoiding the possible harm to the animals14. Secondly, the exact mechanism by which the active components of the decoction formula exert their anti-tumor effects remains unclear. It is possible that the gavage strategy may alter the pharmacokinetics and pharmacodynamics of the herbal compounds, potentially influencing their bioavailability and therapeutic efficacy. Future studies should focus on elucidating the precise mechanisms underlying the anti-tumor effects of the decoction formula delivered through gavage. Furthermore, studies have reported that isoflurane anesthesia application during long-term gavage reduces the incomplete ingestion of decoction and the animal death due to the unskilled gavage technique15. Meanwhile, adding cooking oil into the decoction reduces the gavage associated animal stress16.
In conclusion, we provided the standardized decoction process of Chinese herbs and the skilled gavage strategy for mice. These efforts not only avoid possible esophageal injuries and death of the experimental mice, but also avoid drug overflows ensuring that each experimental animal administers equal quantity of herbal medicines, therefore, improve the accuracy and consistency of the experimental results, also the therapeutic effects. Our study demonstrates the efficacy and safety of the gavage strategy for administration of the traditional Chinese medicine decoction formula in an osteosarcoma mouse model. These results highlight the potential of this delivery method in the treatment of osteosarcoma and lay the foundation for further research in optimizing the gavage strategy of traditional Chinese medicine administration. Additionally, future studies should investigate the exact action mechanisms of the active components in the decoction formula and explore potential synergistic effects with other treatment modalities to enhance the therapeutic outcomes in osteosarcoma patients. Therefore, these findings shed light on the potential benefits and limitations of this administration method and provide valuable insights into its application in osteosarcoma treatment.
The authors have nothing to disclose.
The current work is supported by grants from the (1) National Nature Science Foundation (81973877 and 82174408), (2) National Key R&D Program of China (2020YFE0201600), (3) Shanghai Top Priority Research Center construction project (2022ZZ01009), (4) Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation, and (5) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).
143B cell line | ATCC | CRL-8303 | osteosarcoma cell line |
Anesthesia machine | Shenzhen RWD Life Technology Co.,Ltd | R500IP | The Equipment of Anesthesia mice |
Automatic cell counter | Shanghai Simo Biological Technology Co., Ltd | IC1000 | Counting cells |
BALB/c athymic mice | Shanghai SLAC Laboratory Animal Co, Ltd. | Male | Animal |
Barbed skullcap herb | Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd. | 20230606 | Decoction-ready medicines |
Basement Membrane Matrix | Shanghai Uning BioscienceTechnology Co., Ltd | 356234, BD, Matrigel | re-suspende cells |
Centrifuge tube (15 mL) | Shanghai YueNian Biotechnology Co., Ltd | 430790, Corning | Centrifuge the cells |
Elbow gavage needle (12-gauge/ 55 mm) | RWD Life Science Co., Ltd. | C21014-12 | Component of gastric perfusion device |
Gauze | Haishi Hainuo Group Co., Ltd. | LC45 | Decoction filter |
Ginseng | Shanghai Wanshicheng Pharmaceutical Co., Ltd. | 20220704-2 | Decoction-ready medicines |
Hedyotis diffusa | Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd. | 2023071107 | Decoction-ready medicines |
isoflurane | Shenzhen RWD Life Technology Co., Ltd | VETEASY | Anesthesia mice |
Micro-volume syringe | Shanghai high pigeon industry and trade Co., Ltd | 0-50 μL | Inject precise cells into the tibia |
Phosphate-buffered saline | Beyotime Biotechnology | ST447 | wash the human osteosarcoma cells |
Rubber bands | Shanghai Hengfei Biotechnology Co., Ltd. | XPJ | Fixing gauze on the measuring cup |
Syringe (1 mL ) | Shanghai Mishawa Medical Industry Co., Ltd. | SBM0040 | Component of gastric perfusion device |
Trypsin (0.25%) | Shanghai YueNian Biotechnology Co., Ltd | 25200056, Gibco | trypsin treatment of cells |