Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

The overall goal of this procedure is to prepare an er-xian decoction, a traditional Chinese herbal medicine for its in vivo and in vitro application. This method can help answer key questions in herbal medicine field about preparation and application of a traditional Chinese medicine. The main advantage of this technique is that the water extraction steps are similar to those used in a clinical setting.

Place the raw materials for a single er-xian decoction into a container with a lid, and add enough distilled water that the herbs are covered by about an inch. Put the lid on the container, and macerate the raw herbs for one hour. Then use a gas cooker on high power to heat the herbs until the water boils.

Turn the power down to a low simmer, and decoct the herbs for two hours. At the end of the first water decoction, cover a 500-milliliter glass beaker with gauze and cotton, and carefully pour the decoction liquid only into the beaker through the filter. Return any herbal residue on the filter paper to the container, and add enough fresh distilled water to cover the herbs with another inch of water.

After a second water decoction as just demonstrated, filter the decoction into the 1, 000-milliliter beaker, and place the beaker onto a gas cooker covered with asbestos-free wire gauze gas. Heat the decoction with a low simmer using a glass rod to slowly and continuously stir the solution. When the decoction reduces to 200 milliliters, transfer the solution to a 500-milliliter beaker, and reduce the volume to 100 milliliters as just demonstrated.

After confirming the appropriate level of sedation by toe pinch, use straight operating scissors to make a five-centimeter long, 0.5-centimeter deep incision in the skin and peritoneum of an er-xian decoction-treated animal one hour after the last decoction treatment from the abdomen to the bottom of the thorax. Use tissue paper to move the abdominal viscera, and remove the connective tissue of the abdominal aorta to expose the vessel. Next, use a 10-milliliter syringe equipped with a 22-gauge needle to slowly draw eight to 10 milliliters of blood from the abdominal aorta, and transfer the blood to a 15-milliliter sterile tube.

Clot the blood for 30 to 60 minutes at room temperature, then collect the cells by centrifugation. Carefully pool the serum from each animal into a single sterile 50-milliliter tube, and heat and activate the serum in a 56 degrees Celsius water bath for 30 minutes. Finally, use a syringe filter with a 0.22-micrometer pore size, and hydrophilic polyethersulfone membrane to filter the serum into a 50-milliliter conical tube for immediate use.

Hematoxylin and eosin staining of the lumbar vertebral section of an ovariectomized or OVX mouse reveals an increase in bone trabeculae after er-xian decoction treatment in vivo. Compare to control OVX animals. Representative micro-CT images of the fourth lumbar of a control OVX mouse and an OVX mouse treated with the er-xian decoction for two weeks reveals that more trabecular bones are observed on the inside of the lumbar vertebra of the er-xian decoction-treated animals than those of the control OVX mice.

Indeed, quantitative analysis of these micro-CT data indicates an increase in bone to tissue volume, trabecular number and thickness, and a decrease in the trabecular spacing of the fourth lumbar of the er-xian decoction-treated animals. Fat droplets automatically form when bone mesenchymal stem cells from OVX mice are cultured for seven days. In cultures from er-xian decoction-treated animals, bone nodules form instead.

Further, more alkaline phosphatase-positive cells can be detected in er-xian decoction-treated bone mesenchymal stem cells than in stem cells from control OVX animals. In addition, hierarchical clustering demonstrates that the expression of 389 genes in bone mesenchymal cells as reveal from microarray is full change greater than 1.5 times between OVX and er-xian decoction-treated mice in vivo. Once mastered, the er-xian decoction can be prepared in eight hours, and the contained herb serum can be prepared in two hours.

For internal gastric demonstration, first confirm that the IG tube is properly positioned within the esophagus, then gently delivers the decoction to the animal. According to tradition, preparation method of a Chinese herbal medicine, the medicine or materials should be extracted with water rather than external. Rotary evaporators are commonly used for concentrating the decoction but gas can be used as necessary.

Traditional Chinese medicine requires the standardized preparation procedure and general medicinal materials for in this procedure, the consistent quality will be controlled. After watching this video, you should have a good understanding of how to standardize medicinal materials extraction, and to prepare contained herbal serum. Please keep in mind that the boiling decoction is very hot.

Wear waterproof heat-resistant gloves as necessary.