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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 constituents are still not clearly analyzed, leading to a challenge when combining all extracts for study³. The interactions of numerous constituents/extracts mediate the therapeutic effects of herbal medicines. To retain this advantage, the traditional extract form—decoction—is still widely used in the clinic. Since the extract procedure has a great impact on the therapeutic efficacy, a standard protocol of traditional water decoction is necessary, especially for in vivo studies^4,5.

On the other hand, when exploring pharmacological mechanisms, the administration of herbal decoctions during in vitro or ex vivo studies is also a challenge. The concept of a drug-containing serum was first proposed by Tashino in 1988⁶. Since then, increasing numbers of researchers have applied it to herbal medicine^7,8,9. Although the method of drug-containing serum has some limitations, such as the influence of certain components of the serum, it is still considered to be a method that closely mimics physiological conditions.

The er-xian decoction was developed in the early 1950s to relieve menopausal symptoms^10,11,12,13. It has also been applied to the treatment of aplastic anemia¹⁴, menopausal osteoporosis^15,16,17, premature ovarian failure¹⁸, breast cancer¹⁹, ovarian cancer²⁰, and delayed puberty²¹. Here, we present detailed protocols for the preparation of both the er-xian decoction (EXD) and its drug-containing serum. In addition, we describe the application of EXD and EXD-containing serum to murine menopausal osteoporotic models.

An EXD is composed of 9 g each of Curculigo orchioides Gaertn, Herbaa Epimedii, Radix Morindae Officinalis, and Radix Angelicae Sinensis and 6 g each of Cortex Phellodendri and Rhizoma Anemarrhenae per adult patient per day. The equivalent dose for a mouse is 0.1418 EXD/kg/day based on the following equation²²: dB = dA * RB / RA * (WA / WB) ^1/3. dA and dB refer to the dose per bodyweight (mg/kg) of the human and mouse, respectively. The dose, in mg/kg, is replaced by the number of EXD/kg. RA and RB represent the human body factor and mouse body factor, respectively, which are proportional to the (body surface area (m²) / body weight (kg))^2/3 (See Table 1). WA and WB indicate the bodyweight (kg) of the human and mouse, respectively.

Protokół

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•day) * (0.02 kg/mouse) * 50 mL/EXD = 0.14 mL/mouse•day).
2. Soaking
   1. Put all the raw materials of two EXDs (ie., Curculigo orchioides Gaertn (18 g), Herbaa Epimedii (18 g), Radix Morindae Officinalis (18 g), Radix Angelicae Sinensis (18 g), Cortex Phellodendri (12 g), and Rhizoma Anemarrhenae (12 g), 96 g in total) into a container with a lid.
      NOTE: A ceramic container is recommended.
   2. Macerate the raw herbs with 500 mL of distilled water for 1 h; the water should cover the herbs by about an inch. Let all the herbs thoroughly soak.
3. First water decoction
   1. Heat the herbs using a gas cooker or an induction cooker on high power until the water boils (about 5 - 10 min; the time span depends on the heating power, the container, and the quantity of herbs and water). Turn down the power to a low simmer for 2 h.
4. First filtration
   1. Cover a 500-mL glass beaker with regular filter paper or with gauze and cotton. Carefully pour off the decoction into the beaker through the filter. Leave the herbs in the container.
   2. Return the remaining herbal residue on the filter paper to the container.
5. Second water decoction
   1. Add distilled water to the container with the herbs; let it cover the herbs by about an inch.
   2. Repeat step 1.3.
6. Second filtration
   1. Repeat steps 1.4.1 and 1.4.2.
   2. Pour off the second decoction into the same beaker. Mix the first and second decoctions together.
7. Concentration
   1. Put the beaker onto a gas cooker with asbestos-free wire gauze between them, heat the decoction using a low simmer, and slowly and continuously stir with a glass rod.
   2. When the decoction reduces to 200 mL, transfer it to a 500-mL beaker.
   3. Repeat step 1.7.1 until the decoction reduces to 100 mL.
8. Storage and administration
   1. Transfer the concentrated decoction to a sterile glass bottle. Let it cool to room temperature (RT). Store at 4 °C if using within one week or at -70 °C for long-term storage.
   2. Using a gavage needle, administer 0.14 mL/mouse of the decoction to ovariectomized (OVX) mice once per day, 5 days a week for 12 weeks.

2. Protocol II: Preparation of EXD-containing Serum

1. Calculation
   1. Calculate the rat-equivalent dose (RED) using 0.15 kg as the body weight of each rat (1 month old): dB = (1/60) * (90/100) * (60/0.15)^1/3 = 0.111 EXD/kg/day.
      NOTE: A total of 10 mL of EXD-containing serum is needed to prepare 100 mL of culture medium (10% EXD-containing serum). Each rat is expected to provide 2 mL of serum. Thus, 6 rats (a 20% loss is taken into account) are needed for EXD administration once a day for 3 days. The number of EXDs is: (0.111 EXD/kg•day) * (0.15 kg/rat) * (6 rats) * (3 days) = 0.300 EXD. Again, because EXDs are measured in discrete increments, use 1 EXD.
   2. Calculate the volume of EXD applied per rat (i.e., V = (0.111 EXD/kg•day) * (0.15 kg/rat) * (50 mL/EXD) = 0.83 mL/rat•day).
2. Soaking
   1. Put the raw materials for 1 EXD into a container with a lid. Macerate the raw herbs in distilled water for 1 h; the water should cover the herbs by about an inch. Let all the herbs thoroughly soak.
3. First water decoction
   1. Heat the herbs using an induction cooker on high power until the water boils (about 5 - 10 min; the time span depends on the heat power, the container, and the quantity of herbs and water). Turn the power down to a low simmer for 2 h.
4. First filtration
   1. Cover a 500-mL glass beaker with regular filter paper or with gauze and cotton. Carefully pour off the decoction into the beaker through the filter. Leave the herbs in the container.
   2. Return the herbal residue on the filter paper to the container.
5. Second water decoction
   1. Add distilled water to the container with the herbs; let it cover the herbs by about an inch.
   2. Repeat step 2.4.
6. Second filtration
   1. Repeat steps 2.5.1 and 2.5.2.
   2. Pour off the second decoction into the same beaker as in step 2.4.1 and mix the first and second decoctions together.
7. Concentration
   1. Put the beaker on a gas cooker with asbestos-free wire gauze between them, heat the decoction using a low simmer, and slowly and continuously stir with a glass rod.
   2. When the decoction reduces to 100 mL, transfer it to a 100-mL beaker.
   3. Repeat step 2.7.1 until the decoction reduces to 50 mL.
8. Storage and administration
   1. Transfer the concentrated decoction into a sterile glass bottle. Let it cool to RT. Store at 4 °C if using within one week or at -70 °C for long-term storage.
   2. Intragastrically administer 0.83 mL/rat once per day for 3 days.
9. EXD-containing serum preparation
   1. Anesthetize the rats by intraperitoneally injecting of 300 mL/100 g of 80 mg/kg ketamine and 10 mg/kg xylazine  at 1 h after the last administration of EXD. Confirm proper anesthetization by toe pinch.
   2. Incise the skin and peritoneum of the rat from the abdomen to the bottom of the thorax using a scalpel (or straight operating scissors). Create an incision of length and depth of about 5 cm and 0.5 cm, respectively. Move the abdominal viscera to the left using tissue paper.
   3. Using tissue paper, remove the connective tissue of the abdominal aorta to clearly expose the vessel.
   4. Draw the blood slowly from the abdominal aorta using a 10-mL, 22-gauge syringe. Transfer it to a 15-mL sterile tube after removing the needle; one rat can produce 8-10 mL of blood.
      NOTE: The rat should be alive (i.e., with the abdominal aorta pulsating) when beginning the blood drawing and dead after the drawing completes.
   5. Clot the blood in an upright position for 30 - 60 min at room RT. Centrifuge at 500-600 x g for 20 min. Carefully place all the supernatant (serum) in one 50-mL sterile tube and mix all the serum (from different animals) together.
   6. Perform heat inactivation by incubating the serum in a 56 °C water bath for 30 min. Filter the serum using a syringe filter with a 0.22-µm pore size hydrophilic polyethersulfone membrane. Use the serum fresh or store it at -20 °C.
10. Control drug-containing serum preparation (used in the control group)
    1. Administer each rat with the same volume of saline (0.83 mL) once per day for 3 days. The other steps are the same as in step 2.9.
11. Application
    1. Add 10 mL of EXD-containing serum or drug-containing serum to each 100 mL of medium, plus 1% penicillin-streptomycin (PS)²³.

Wyniki

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 (Figur...

Dyskusje

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...

Materiały

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