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

Summary

Here, we present the preparation of an er-xian decoction (EXD) in four steps—soaking, decoction, filtration, and concentration—and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

Abstract

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Introduction

Interest in the study and application of traditional herbal medicine is currently growing. As opposed to modern drugs, in which chemical ingredients are definite, herbal formulas have some unknown ingredients and require extraction processes to enable the delivery of their active compounds. Although many studies try to select one small compound with a well-known structure as a representative of the whole herb or herbal formula, neither the pharmacological efficacies nor mechanisms can be considered equivalent^1,2. While fingerprinting enables the analysis of the constituents of complex herbal formulas, some con....

Protocol

The protocol follows the animal care guidelines of Shanghai University of Traditional Chinese Medicine and is approved by the animal experiment Shanghai Animal Ethics Committee.

1. Protocol I: Preparation of EXD

1. Calculation
   1. Administer the EXD to a treatment group of 10 Six month-old female Imprinting Control Region (ICR) mice (0.02 kg/mouse), 5 days per week for 12 weeks.
      NOTE: A total of 1.702 EXDs are needed. Because EXDs are measured in discrete increments, in real practice, administer 2 EXDs.
   2. Calculate the volume of EXD applied per mouse (i.e. V = (0.1418 EXD/kg&#....

Results

The effect of EXD on the bone density of OVX mice

Hematoxylin and eosin (H&E) staining of the lumbar vertebra section shows increased bone trabeculae after EXD treatment in vivo (Figure 1A, right panel) compared with those in the OVX group (Figure 1A, left panel). Figure 1B shows the representative µCT images of the 4^th lumbar in OVX mice (Figure 1B<.......

Discussion

In recent years, more attention has been paid to herbal medicines, one type of alternative medicine that has been applied in the clinical setting in the Eastern world for thousands of years. Different from the "bench to bed" pattern of modern medicine, traditional herbal medicine first require the "bed to bench" pattern to explain their mechanisms. This can be followed by validation performed at the bench stage and processing for the development of newly optimized drugs. So far, there are several extracti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Curculigo orchioides Gaertn (9 g), Herbaa Epimedii (9 g), Radix Morindae Officinalis (9 g), Radix Angelicae Sinensis (9 g), Cortex Phellodendri (6 g), and Rhizoma Anemarrhenae (6 g)	Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)	160922	EXD components
Filter paper	GElifesciences	99-103-952	Filter EXD decoction before concentration
Imprinting Control Region (ICR) mice	Shanghai Laboratory Animal Center	SCXK 2007-0005	In vivo study
Sprague Dawley rats	Shanghai Laboratory Animal Center	SCXK 2007-0005	EXD-containing serum preparation
Syringe filter	Millipore	SLGP033RB	0.22 µm
gavage needles (10 ml)	Shanghai BO Ge trade sales department	59104274	Adminstration of EXD
Ketamine (80 mg/kg)	Fujian Gutian Pharma Co. Ltd	H35020148	Anesthesia
Xylazine (10 mg/kg)	Sunway Pharma Co. Ltd	CB07591	Anesthesia
Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM) culture medium	Gibco	12800-116	DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides
Streptomycin	Sigma	1277	100 µg / ml
Penicillin	Sigma	4687	100 µg / ml