This work was supported by the National Natural Science Foundation of China (81573992). We thank Emily K. Lo and Kathleen DiNapoli for their language editing assistance.

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
<ol>
	<li>Calculation
	<ol>
		<li>Administer the EXD to a treatment group of 10 Six month-old female Imprinting Control Region (ICR)&#160;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.</li>
		<li>Calculate the volume of EXD applied per mouse (i.e. V = (0.1418 EXD/kg&#8226;day) * (0.02 kg/mouse) * 50 mL/EXD = 0.14 mL/mouse&#8226;day).</li>
	</ol>
	</li>
	<li>Soaking
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>First water decoction
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>First filtration
	<ol>
		<li>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.</li>
		<li>Return the remaining herbal residue on the filter paper to the container.</li>
	</ol>
	</li>
	<li>Second water decoction
	<ol>
		<li>Add distilled water to the container with the herbs; let it cover the herbs by about an inch.</li>
		<li>Repeat step 1.3.</li>
	</ol>
	</li>
	<li>Second filtration
	<ol>
		<li>Repeat steps 1.4.1 and 1.4.2.</li>
		<li>Pour off the second decoction into the same beaker. Mix the first and second decoctions together.</li>
	</ol>
	</li>
	<li>Concentration
	<ol>
		<li>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.</li>
		<li>When the decoction reduces to 200 mL, transfer it to a 500-mL beaker.</li>
		<li>Repeat step 1.7.1 until the decoction reduces to 100 mL.</li>
	</ol>
	</li>
	<li>Storage and administration
	<ol>
		<li>Transfer the concentrated decoction to a sterile glass bottle. Let it cool to room temperature (RT). Store at 4 &#176;C if using within one week or at -70 &#176;C for long-term storage.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Protocol II: Preparation of EXD-containing Serum
<ol>
	<li>Calculation
	<ol>
		<li>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&#8226;day) * (0.15 kg/rat) * (6 rats) * (3 days) = 0.300 EXD. Again, because EXDs are measured in discrete increments, use 1 EXD.</li>
		<li>Calculate the volume of EXD applied per rat (i.e., V = (0.111 EXD/kg&#8226;day) * (0.15 kg/rat) * (50 mL/EXD) = 0.83 mL/rat&#8226;day).</li>
	</ol>
	</li>
	<li>Soaking
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>First water decoction
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>First filtration
	<ol>
		<li>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.</li>
		<li>Return the herbal residue on the filter paper to the container.</li>
	</ol>
	</li>
	<li>Second water decoction
	<ol>
		<li>Add distilled water to the container with the herbs; let it cover the herbs by about an inch.</li>
		<li>Repeat step 2.4.</li>
	</ol>
	</li>
	<li>Second filtration
	<ol>
		<li>Repeat steps 2.5.1 and 2.5.2.</li>
		<li>Pour off the second decoction into the same beaker as in step 2.4.1 and mix the first and second decoctions together.</li>
	</ol>
	</li>
	<li>Concentration
	<ol>
		<li>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.</li>
		<li>When the decoction reduces to 100 mL, transfer it to a 100-mL beaker.</li>
		<li>Repeat step 2.7.1 until the decoction reduces to 50 mL.</li>
	</ol>
	</li>
	<li>Storage and administration
	<ol>
		<li>Transfer the concentrated decoction into a sterile glass bottle. Let it cool to RT. Store at 4 &#176;C if using within one week or at -70 &#176;C for long-term storage.</li>
		<li>Intragastrically administer 0.83 mL/rat once per day for 3 days.</li>
	</ol>
	</li>
	<li>EXD-containing serum preparation
	<ol>
		<li>Anesthetize the rats by intraperitoneally injecting of 300 mL/100 g of 80 mg/kg ketamine and 10 mg/kg xylazine&#160;&#160;at 1 h after the last administration of EXD. Confirm proper anesthetization by toe pinch.</li>
		<li>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.</li>
		<li>Using tissue paper, remove the connective tissue of the abdominal aorta to clearly expose the vessel.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Perform heat inactivation by incubating the serum in a 56 &#176;C water bath for 30 min. Filter the serum using a syringe filter with a 0.22-&#181;m pore size hydrophilic polyethersulfone membrane. Use the serum fresh or store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Control drug-containing serum preparation (used in the control group)
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Application
	<ol>
		<li>Add 10 mL of EXD-containing serum or drug-containing serum to each 100 mL of medium, plus 1% penicillin-streptomycin (PS)23.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zhang, L. G., Ouyang, X. W., Wu, T. T., Ni, L. J., Shi, W. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+in+vitro+effects+and+interactions+of+active+fractions+in+a+Chinese+medicinal+formula+(Yaotongning+Capsule)+on+rat+chondrocytes.">Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes.</a> J Ethnopharmacol. 155 (3), 1424-1432 (2014).</li><li>Ogawa, Y., Fujii, Y., Sugiyama, R., Konishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+seven+crude+drug+components+in+the+sleep-promoting+effect+of+Yokukansan.">The role of the seven crude drug components in the sleep-promoting effect of Yokukansan.</a> J Ethnopharmacol. 177, 19-27 (2016).</li><li>Kim, J. H., Doh, E. J., Lee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Medicinal+Categorization+of+Atractylodes+japonica+Koidz.+by+Using+Internal+Transcribed+Spacer+Sequencing+Analysis+and+HPLC+Fingerprinting+Combined+with+Statistical+Tools.">Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools.</a> Evid Based Complement Alternat Med. 2016, 2926819 (2016).</li><li>Sheridan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+metabolic+fingerprinting+as+a+tool+for+the+modernisation+of+TCM+preparations.">The potential of metabolic fingerprinting as a tool for the modernisation of TCM preparations.</a> J Ethnopharmacol. 140 (3), 482-491 (2012).</li><li>Planas, G. M., Kucacute, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraceptive+Properties+of+Stevia+rebaudiana.">Contraceptive Properties of Stevia rebaudiana.</a> Science. 162 (3857), 1007 (1968).</li><li>Tashino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Serum+pharmacology"+and+"serum+pharmaceutical+chemistry":+from+pharmacology+of+Chinese+traditional+medicines+to+start+a+new+measurement+of+drug+concentration+in+blood.">"Serum pharmacology" and "serum pharmaceutical chemistry": from pharmacology of Chinese traditional medicines to start a new measurement of drug concentration in blood.</a> Ther Drug Monit Res. 5, 54-64 (1988).</li><li>Cao, Y., Liu, F., Huang, Z., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Guanxin+Shutong+capsule+drug-containing+serum+on+tumor+necrosis+factor-alpha-induced+endothelial+dysfunction+through+nicotinamide+adenine+dinucleotide+phosphate+oxidase+and+the+nitric+oxide+pathway.">Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-alpha-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway.</a> Exp Ther Med. 8 (3), 998-1004 (2014).</li><li>Fu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+Vivo+Stromal+Cell-Derived+Factor+1-Mediated+Differentiation+of+Mouse+Bone+Marrow+Mesenchymal+Stem+Cells+into+Hepatocytes+Is+Enhanced+by+Chinese+Medicine+Yiguanjian+Drug-Containing+Serum.">Ex Vivo Stromal Cell-Derived Factor 1-Mediated Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Hepatocytes Is Enhanced by Chinese Medicine Yiguanjian Drug-Containing Serum.</a> Evid Based Complement Alternat Med. 2016, 7380439 (2016).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+serum+pharmacology+in+evaluating+the+antitumor+effect+of+Fuzheng+Yiliu+Decoction+from+Chinese+Medicine.">Application of serum pharmacology in evaluating the antitumor effect of Fuzheng Yiliu Decoction from Chinese Medicine.</a> Chin J Integr Med. 20 (6), 450-455 (2014).</li><li>Sze, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mechanism:+Erxian+Decoction,+a+Chinese+medicine+formula,+for+relieving+menopausal+syndrome.">A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome.</a> J Ethnopharmacol. 123 (1), 27-33 (2009).</li><li>Sze, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Erxian+decoction,+a+Chinese+medicinal+formulation,+on+serum+lipid+profile+in+a+rat+model+of+menopause.">Effects of Erxian decoction, a Chinese medicinal formulation, on serum lipid profile in a rat model of menopause.</a> Chin Med. 6, 40 (2011).</li><li>Zhong, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind,+controlled+trial+of+a+Chinese+herbal+formula+(Er-Xian+decoction)+for+menopausal+symptoms+in+Hong+Kong+perimenopausal+women.">A randomized, double-blind, controlled trial of a Chinese herbal formula (Er-Xian decoction) for menopausal symptoms in Hong Kong perimenopausal women.</a> Menopause. 20 (7), 767-776 (2013).</li><li>Wang, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroidogenic+effect+of+Erxian+decoction+for+relieving+menopause+via+the+p-Akt/PKB+pathway+in+vitro+and+in+vivo.">Steroidogenic effect of Erxian decoction for relieving menopause via the p-Akt/PKB pathway in vitro and in vivo.</a> J Ethnopharmacol. , (2016).</li><li>Lin, L., Wu, S., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+and+experimental+study+of+the+treatment+of+aplastic+anemia+by+warming+and+tonifying+the+spleen+and+kidney].">[Clinical observation and experimental study of the treatment of aplastic anemia by warming and tonifying the spleen and kidney].</a> Zhong Xi Yi Jie He Za Zhi. 10 (5), 272-274 (1990).</li><li>Nian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiosteoporotic+activity+of+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+in+ovariectomized+rats.">Antiosteoporotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in ovariectomized rats.</a> J Ethnopharmacol. 108 (1), 96-102 (2006).</li><li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiosteoporotic+chemical+constituents+from+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula.">Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula.</a> J Ethnopharmacol. 118 (2), 271-279 (2008).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+HNMR-based+metabonomics+study+of+postmenopausal+osteoporosis+and+intervention+effects+of+Er-Xian+Decoction+in+ovariectomized+rats.">A HNMR-based metabonomics study of postmenopausal osteoporosis and intervention effects of Er-Xian Decoction in ovariectomized rats.</a> Int J Mol Sci. 12 (11), 7635-7651 (2011).</li><li>Lu, X. N., Xu, X. R., Lin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+of+bushen+er'xian+decoction+in+treating+premature+ovarian+failure].">[Clinical observation of bushen er'xian decoction in treating premature ovarian failure].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 28 (7), 594-596 (2008).</li><li>Yu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-angiogenic+activity+of+Erxian+Decoction,+a+traditional+Chinese+herbal+formula,+in+zebrafish.">Anti-angiogenic activity of Erxian Decoction, a traditional Chinese herbal formula, in zebrafish.</a> Biol Pharm Bull. 35 (12), 2119-2127 (2012).</li><li>Chu, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+and+in+vivo+investigation+of+the+antimetastatic+effects+of+a+Chinese+medicinal+decoction,+erxian+decoction,+on+human+ovarian+cancer+models.">An in vitro and in vivo investigation of the antimetastatic effects of a Chinese medicinal decoction, erxian decoction, on human ovarian cancer models.</a> Integr Cancer Ther. 12 (4), 336-346 (2013).</li><li>Zhu, Z., Li, L., Jin, X., Fang, J., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+intervening+early+in+hypothalamic-pituitary+axis+of+male+rats+with+delayed+puberty.">Er-Xian Decoction, a traditional Chinese herbal formula, intervening early in hypothalamic-pituitary axis of male rats with delayed puberty.</a> Pharmacogn Mag. 10 (40), 517-521 (2014).</li><li>Miao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental animals and technology. 1997, 145 (1997).</li><li>Soleimani, M., Nadri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Nat Protoc. 4 (1), 102-106 (2009).</li><li>Lu, S., Huang, J., Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Er-Xian+Decoction+Stimulates+Osteoblastic+Differentiation+of+Bone+Mesenchymal+Stem+Cells+in+Ovariectomized+Mice+and+Its+Gene+Profile+Analysis.">Er-Xian Decoction Stimulates Osteoblastic Differentiation of Bone Mesenchymal Stem Cells in Ovariectomized Mice and Its Gene Profile Analysis.</a> Stem Cells International. 2016, 4079210 (2016).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+seven+flavonoids+in+dog+plasma+by+ultra-performance+liquid+chromatography-tandem+mass+spectrometry+and+its+application+to+a+bioequivalence+study+of+bioactive+components+in+Herba+Epimedii+and+Er-Xian+Decoction.">Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction.</a> J Pharm Biomed Anal. 54 (1), 186-191 (2011).</li><li>Hu, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+major+chemical+constituents+and+their+metabolites+in+rat+plasma+and+various+organs+after+oral+administration+of+effective+Erxian+Decoction+(EXD)+fraction+by+liquid+chromatography-mass+spectrometry.">Identification of the major chemical constituents and their metabolites in rat plasma and various organs after oral administration of effective Erxian Decoction (EXD) fraction by liquid chromatography-mass spectrometry.</a> Biomed Chromatogr. 24 (5), 479-489 (2010).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+interaction+of+icariin,+curculigoside,+and+berberine+in+er-xian+decoction,+a+traditional+chinese+medicinal+formula,+on+osteoclastic+bone+resorption.">Effects and interaction of icariin, curculigoside, and berberine in er-xian decoction, a traditional chinese medicinal formula, on osteoclastic bone resorption.</a> Evid Based Complement Alternat Med. , (2012).</li><li>Nair, A. B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> J Basic Clin Pharm. 7 (2), 27-31 (2016).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+the+traditional+Chinese+medicine+xuesaitong+on+intestinal+ischemia-reperfusion+injury+in+rats.">Protective effect of the traditional Chinese medicine xuesaitong on intestinal ischemia-reperfusion injury in rats.</a> Int J Clin Exp Med. 8 (2), 1768-1779 (2015).</li><li>Li, Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methods+for+Chinese+herbs+serum+pharmcology.">On the methods for Chinese herbs serum pharmcology.</a> Zhong Guo Zhong Yi Yao Xing Xi Za Zhi. 9 (2), 5-6 (2002).</li><li>Jiang, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chinese+herbal+drug-containing+serum+for+activating-blood+and+dispelling-toxin+on+ox-LDL-induced+inflammatory+factors'+expression+in+endothelial+cells.">Effect of chinese herbal drug-containing serum for activating-blood and dispelling-toxin on ox-LDL-induced inflammatory factors' expression in endothelial cells.</a> Chin J Integr Med. 18 (1), 30-33 (2012).</li><li>Guo, C. Y., Ma, X. J., Liu, Q., Yin, H. J., Shi, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

The authors declare that they have no competing financial interests.

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 &#34;bench to bed&#34; pattern of modern medicine, traditional herbal medicine first require the &#34;bed to bench&#34; 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 extraction methods that have been reported to the release active components from whole formulas or herbs. Among them, water extraction is the most broadly used.
The water extraction of herbal medicines has four basic steps. The first step is soaking. A complex herbal medicine is macerated in water for a period of time, usually from 0.5 to 1 h. The time span for soaking depends on the quantity and property of the raw herbs. To ensure that the raw herbs are thoroughly soaked, the center of every piece or block should be steeped. The volume of water depends on the total volume of herbs. Sometimes, the weight of the herbs is used to determine how much water is needed for soaking. However, herb densities vary widely. Since the purpose of soaking is to macerate all the herbs, facilitating the release of the active components during the following steps, the volume of herbs is preferred for use as a reference standard for the volume of water. The second step is cooking. The boiling and simmering processes have been developed over thousands of years. The variation in the cooking time depends mostly on the kind of herbs, due to their different physical, chemical, and pharmacological characteristics12. Diaphoretics are suggested to cook for a short period of time, usually less than 20 min, while aromatics should be added only a few minutes before the first boiling to avoid volatile reactions. For tonic herbs, a long period of time is required to leach out their therapeutic constituents. In this case, EXD is used for the treatment of postmenopausal osteoporosis over decades12. In traditional Chinese medicine theory, this syndrome is considered to be caused by a kidney-related deficiency. EXD is mainly designed to tonify the kidney. Therefore, the EXD was cooked for 2 - 3 h to allow the EXD to exert its anti-osteoporotic effects15. The third step is filtration. Filtration helps to prevent the gavage needle from being clogged with the herbal granules. The fourth step is concentration. The volume of gavage administration should be carefully considered. It has been reported that large volumes, such as 10 mL/kg or more, administered by oral gavage can result in several problems related to absorption, including the rapid shunting of the compounds to the duodenum or aspiration pneumonia associated with the passive reflux of the material into the esophagus25. Thus, the volumes chosen for the mice and rats were around 7.5 mL/kg and 5.5 mL/kg, respectively.
It is important to confirm whether the effective components of digested and metabolized EXD in the blood are similar to those of the raw extract. In terms of EXD bioequivalence, Wu et al. reported the determination of 7 components in dog plasma: epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2&#8243;-O-rhamnosyl icariside II, and baohuoside I26. Hu et al. found 21 compounds in rat plasma after oral administration27. So far, the raw extract from EXD has not been reported. However, some of the effective components tested in blood, such as icariin and berberine, have been directly applied to animals for comparative observation28.
There are multiple approaches to calculate the animal bioequivalent dose. The traditional method based on bodyweight (mg/kg) is not appropriate because the pharmacokinetics vary in different species. The calculation based on body surface area (mg/m2), in which the metabolic rate is related to the individual animal size, is frequently used29. The equation applied here takes both body surface area and body weight into account and is commonly utilized in traditional Chinese medicine30.
Many kinds of animals, such as rabbits, guinea pigs, rats, and mice, can be chosen for drug-containing serum preparation. The same species as that of the in vitro-treated cells is preferred. In this study, rats were chosen because they provide more serum than mice do and are closer to mice in terms of species compared to other animals. In addition, the dose equivalent in in vivo or clinic usage is recommended, as the in vitro protocol showed. The dilution of serum (1:10 is recommended) is not taken into account; that is, 10-fold of the equivalent dose is not applied to the serum-provided animals due to the potential toxic reaction caused by treated cells or organs31. The drug administration frequency varies from once a day for 3 to 14 days to two times per day (2 h between each administration)7,8,32. The collection time usually occurs between hours 1 and 2 (before hour 6) after the last administration33,34. The aim is to keep the drug concentration in the blood relatively stable and at its peak level when the samples are collected35. The administration routines can include injection, skin administration, or inhalation, in accordance with the in vivo administration routines.
The inactivation of drug-containing serum is still controversial. Supporters think that the presence of many active components, such as hormones, enzymes, antibodies, and complements in the serum itself, may result in unexpected reactions that affect the results36. The opposition believes that the active components produced by the drugs might also be removed by the inactivation process37. To balance this, people design the control group such that the serum from saline-treated animals is used.
There are some limitations to the protocol. The quality of the extract is not evaluated before starting in vivo and in vitro experimentation. Second, antibiotics are used in the culture medium, which may cause herb-drug interactions and requires further testing. Third, the soaking and simmer time are determined based on the experience garnered from clinical practice and animal experimentation. More periods of time can be chosen for comparison. The soaking and simmer times can be modified for different herbal formulas. The decoction volume for animal administration can be modified according to the species and the age and weight of the animal. The animal chosen for the preparation of drug-containing serum and for the administration routines can be changed, as discussed above.
Taken together, the protocol and results provide an example for the preparation and application of herbal decoction in in vivo and in vitro studies. In different cases, some of the details must be optimized, including the treatment periods, species, and administration routines, based on the herbal characteristics.

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 equivalent1,2. 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&#8212;decoction&#8212;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 studies4,5.
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 medicine7,8,9. 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 symptoms10,11,12,13. It has also been applied to the treatment of aplastic anemia14, menopausal osteoporosis15,16,17, 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.

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

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

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.

The effect of EXD on the bone density of OVX mice
Hematoxylin and eosin (H&#38;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 &#181;CT images of the 4th lumbar in OVX mice (Figure 1B, left panel) and OVX mice with EXD treatment for 12 weeks (Figure 1B, right panel). More trabecular bones are seen inside the lumbar vertebra of EXD-treated mice than those of control OVX mice. The data from the &#181;CT imaging indicates an increased bone volume/tissue volume (BV/TV), trabecular number (Tb. N), and trabecular thickness (Tb. Th) and decreased trabecular spacing (Tb. Sp) of the 4th lumbar in EXD mice (Figure 1C).
The effect of EXD on the osteogenesis of bone mesenchymal stem cells (bMSCs) from OVX mice
Figure 2 represents the osteogenic potential of bMSCs. Fat droplets (asterisk, irregular shape of a lipid droplet) are automatically formed when bMSCs of OVX mice are cultured for 7 days (Figure 2A, left panel). In EXD-treated mice, bone nodules (arrow) are formed instead of a fat droplet (Figure 2B, right panel). An alkaline phosphatase (ALP) assay demonstrates that more ALP-positive cells (purple) can be detected in EXD-treated bMSCs (Figure 2B, right panel) compared with those in bMSCs from control OVX mice (Figure 2B, left panel).
The changes in gene expression between OVX and EXD mice24
Hierarchical clustering shows that the expression of 389 genes revealed by microarray in bMSCs was fold changed (&#62; 1.5, normalized by non-OVX mice) between OVX and EXD-treated mice in vivo (total: 26,991 genes) (Figure 3). Green indicates that the expression was upregulated, while red indicates that the expression was downregulated. Three samples in the same group are clustered together first, indicating the reliable qualities of the profile. Figure 4 shows the overlapped signaling pathway targeted by EXD, both in vivo and in vitro.
<img alt="Figure 1" src="/files/ftp_upload/55654/55654fig1.jpg" /> 
Figure 1: The Effect of EXD on Bone Morphology. 
(A) H &#38; E staining of the lumbar 4th section of OVX and EXD-treated mice. (B) Three-dimensional &#181;CT reconstructive images of the lumbar 4th trabecular bone in control OVX and EXD-treated mice. (C) Quantification of &#181;CT. BV: bone volume, TV: tissue volume, Tb.N: trabecular number, Tb.Th: trabecular thickness, and Tb.Sp: trabecular spacing. The columns represent the means &#177; SE. n = 6 per group. * p &#60; 0.05, ** p &#60; 0.01 EXD versus OVX (Student&#39;s t-test). (A-C) have been modified from Shufen Liu et al.24. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/55654fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55654/55654fig2.jpg" /> 
Figure 2: The Effect of EXD on bMSC Differentiation. 
(A) Images of bMSCs cultured for 7 days after being isolated from the femur of OVX or EXD-treated mice. * indicates a fat droplet. An arrow indicates a bone nodule. (B) ALP staining of OVX and EXD-treated bMSCs cultured for 7 days (purple represents ALP positive). (C) Quantification of (B). The columns represent the means &#177; SE from three dishes (six mice) per group. ** p &#60; 0.01 EXD versus OVX (Student&#39;s t-test). (A-C) have been modified from Shufen Liu et al.24. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/55654fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55654/55654fig3.jpg" /> 
Figure 3: The Effect of EXD on the Gene Expression Profile. 
Heatmap of the hierarchical clustering of expression of 389 genes in OVX and EXD bMSCs harvested on the 7th day post-disassociation. The scale (small image) indicates fold changes normalized by non-OVX bMSCs. The list of genes is provided in Supplemental File 1. The figure has been modified from Shufen Liu et al.24. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/55654fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55654/55654fig4.jpg" /> 
Figure 4: The Effect of EXD on Overlapped Signaling Pathway in Both In Vivo and In Vitro Experiments. 
The first 10 overlapped signaling pathways that are reversed by EXD in vivo and EXD-containing serum in vitro based on the KEGG pathway. The genes related to the pathways are shown in Supplemental File 2. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/55654fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Mouse</td>
			<td>Rat</td>
			<td>Guinea pig</td>
			<td>Rabbit</td>
			<td>Cat</td>
			<td>Monkey</td>
			<td>Dog</td>
			<td>Human</td>
		</tr>
		<tr>
			<td>R</td>
			<td>59</td>
			<td>90</td>
			<td>99</td>
			<td>93</td>
			<td>82</td>
			<td>111</td>
			<td>104</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 1: R-factor in Different Species. 
The R-factor in 8 species that are commonly used in pharmacological research. The R-factor is proportional to: (body surface area (m2) / body weight (kg)) 2/3.
Supplemental File 1. 
Information on 389 genes that are upregulated or downregulated &#8805;1.5-fold but are rescued by EXD in vivo. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/Supplemental_file_1.xls" target="_blank">Please click here to download this file.</a>
Supplemental File 2. 
Information on the signaling pathway (based on the KEGG pathway) and related genes in both in vivo and in vitro experiments. <a href="http://cloudflare.jove.com/files/ftp_upload/55654/Supplemental_file_2.xls" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

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

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;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.

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

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Nanyang Technological University

Research

JoVE Journal

Medicine

10.9K Views.  Shanghai University of Traditional Chinese Medicine. 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.

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

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Chemistry

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

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.&#160;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.

Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

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 ...

Construction of Lung Adenocarcinoma Xenograft Model in Mice and Evaluation of the Efficiency of XY Decoction through MAPK Pathway

Lung adenocarcinoma is increasing worldwide, prompting the exploration and development of effective treatment methods. In recent years, the therapeutic potential of traditional Chinese medicine for lung adenocarcinoma, such as the XiaoYi (XY) decoction, has been increasingly recognized. The mechanism of action of traditional Chinese medicine in treating lung adenocarcinoma continues to be elucidated. In this study, animal models were used to investigate the therapeutic effects of XY decoction in the treatment of lung adenocarcinoma. The blood-entry chemical components of XY decoction in mice were separated and analyzed in both positive and negative ion modes using UHPLC-QE-MS. The findings were then validated through in vivo experiments, which demonstrated reduced tumor size, improved pathological changes, and increased apoptosis. As a result, fifteen components absorbed into the blood following XY decoction administration were identified: Stachyose, Quercetin-3-O-beta-glucopyranosyl-7-O-alpha-rhamnopyranoside; Imperialine; Spinosin B; Peimine; Flavone base + 3O, 2MeO, O-Hex; Picropodophyllotoxin; (2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-4,5-dihydroxy-2 [[(3S,5R,8R,10R,12R,13R,14R,17S)-12-hydroxy-17-(2-hydroxy-6-methylhept-5-en-2-yl) 4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol; Tricin; Ginsenoside Rg1; (20R)-Ginsenoside Rh1; Jujuboside B; Aucubin; Ginsenoside Rg3; and beta-Elemonic acid. Animal experiments demonstrated that XY decoction inhibited tumor growth, improved pathological changes, and promoted tumor cell apoptosis in a dose-dependent manner. The mechanisms involved may be related to the upregulation of p-JNK and p-P38 protein expression in the MAPK pathway and the downregulation of p-ERK protein expression in the MAPK pathway. In conclusion, XY decoction has the potential to regulate the MAPK pathway and exert therapeutic effects against lung adenocarcinoma.

This study demonstrates that XY decoction exerts therapeutic effects against lung adenocarcinoma by regulating the MAPK pathway.

Lung adenocarcinoma is increasing worldwide, prompting the exploration and development of effective treatment methods. In recent years, the therapeutic ...

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

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Chapters in this video

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Preparation of Naringenin Solution for In Vivo Application

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

Construction of Lung Adenocarcinoma Xenograft Model in Mice and Evaluation of the Efficiency of XY Decoction through MAPK Pathway