JP XVII Crude Drugs and Related Drugs / Bofutsushosan Extract 1813

Bofutsushosan Extract
防風通聖散エキス

Bofutsushosan Extract contains not less than 9 mg

and not more than 36 mg of paeoniflorin (C23H28O11:
480.46), not less than 4 mg and not more than 12 mg
of total alkaloids [ephedrine (C10H15NO: 165.23) and
pseudoephedrine (C10H15NO: 165.23)], not less than 54
mg and not more than 162 mg of baicalin (C21H18O11:
446.36), and not less than 16 mg and not more than 48
mg of glycyrrhizic acid (C42H62O16: 822.93), per extract
prepared with the amount specified in the Method of
preparation.
Method of preparation
1) 2) 3) 4) 5) 6)
Japanese Angelica
Root 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Peony Root 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Cnidium Rhizome 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Gardenia Fruit 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Forsythia Fruit 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Mentha Herb 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Ginger 0.3 g 0.3 g 0.4 g 0.4 g 1.2 g 0.3 g
Schizonepeta Spike 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Saposhnikovia Root
and Rhizome 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g —
Glehnia Root and
Rhizome — — — — — 1.2 g
Ephedra Herb 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Rhubarb 1.5 g 1.5 g 1.5 g 1.5 g 1.5 g 1.5 g
Sodium Sulfate — 1.5 g — 1.5 g — —
Anhydrous Sodium
Sufate 0.7 g — 0.75 g — 1.5 g 0.75 g
Atractylodes
Rhizome 2g 2g 2g 2g 2g 2g
Platycodon Root 2g 2g 2g 2g 2g 2g
Scutellaria Root 2g 2g 2g 2g 2g 2g
Glycyrrhiza 2g 2g 2g 2g 2g 2g
Gypsum 2g 2g 2g 2g 2g 2g
Aluminum Silicate
Hydrate with
Silicon Dioxide 3g 3g 3g 3g 3g 3g

Prepare a dry extract or viscous extract as directed under

Extracts, according to the prescription 1) to 6), using the
crude drugs shown above.
Description Bofutsushosan Extract is a yellow-brown to
brown powder or blackish brown viscous extract. It has a
slightly odor and a sweet and slightly bitter taste.
Identification (1) To 2.0 g of the dry extract (or 6.0 g of
the viscous extract) add 10 mL of water, shake, then add 10
mL of diethyl ether, shake, and centrifuge. Separate the
diethyl ether layer, add 10 mL of sodium hydroxide TS,
shake, centrifuge, separate the diethyl ether layer, and use
this solution as the sample solution. Separately, dissolve 1
mg of (Z )-ligustilide for thin-layer chromatography in 10 mL
of methanol, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 20 mL of the sample so-
lution and 10 mL of the standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
1814 Bofutsushosan Extract / Crude Drugs and Related Drugs JP XVII
mixture of butyl acetate and hexane (2:1) to a distance of lution. Perform the test with these solutions as directed
about 7 cm, and air-dry the plate. Examine under ultraviolet under Thin-layer Chromatography <2.03>. Spot 20 mL each
light (main wavelength: 365 nm): one of the spot among the of the sample solution and standard solution on a plate of
several spots obtained from the sample solution has the same silica gel for thin-layer chromatography. Develop the plate
color tone and R f value with the bluish white fluorescent with a mixture of acetone, ethyl acetate, water, and acetic
spot obtained from the standard solution (Japanese Angelica acid (100) (10:10:3:1) to a distance of about 7 cm, and air-
Root; Cnidium Rhizome). dry the plate. Spray evenly 2,6-dibromo-N-chloro-1,4-benzo-
(2) To 1.0 g of the dry extract (or 3.0 g of the viscous quinone monoimine TS on the plate, heat at 1059C for 5
extract) add 10 mL of water, shake, then add 10 mL of 1- minutes: one of the spot among the several spots obtained
butanol, shake, centrifuge, and use the supernatant liquid as from the sample solution has the same color tone and R f
the sample solution. Separately, dissolve 1 mg of paeoniflo- value with the red-brown spot (R f value: around 0.4) ob-
rin for thin-layer chromatography in 1 mL of methanol, and tained from the standard solution (Mentha Herb).
use this solution as the standard solution. Perform the test (6) Perform the test according to the following (i) or (ii)
with these solutions as directed under Thin-layer Chroma- (Ginger).
tography <2.03>. Spot 10 mL of the sample solution and 5 mL (i) To 1.0 g of the dry extract (or 3.0 g of the viscous ex-
of the standard solution on a plate of silica gel for thin-layer tract) add 10 mL of water, shake, then add 25 mL of diethyl
chromatography. Develop the plate with a mixture of ethyl ether, and shake. Separate the diethyl ether layer, evaporate
acetate, methanol and ammonia solution (28) (6:3:2) to a the solvent under reduced pressure, dissolve the residue in 2
distance of about 7 cm, and air-dry the plate. Spray evenly 4- mL of diethyl ether, and use the solution as the sample solu-
methoxybenzaldehyde-sulfuric acid TS on the plate, heat at tion. Separately, dissolve 1 mg of [6]-gingerol for thin-layer
1059C for 1 minute: one of the spot among the several spots chromatography in 1 mL of methanol, and use this solution
obtained from the sample solution has the same color tone as the standard solution. Perform the test with these solu-
and R f value with the red-purple to purple spot obtained tions as directed under Thin-layer Chromatography <2.03>.
from the standard solution (Peony Root). Spot 20 mL of the sample solution and 5 mL of the standard
(3) To 1.0 g of the dry extract (or 3.0 g of the viscous solution on a plate of silica gel for thin-layer chromatogra-
extract) add 10 mL of water, shake, then add 10 mL of 1- phy. Develop the plate with a mixture of ethyl acetate and
butanol, shake, centrifuge, and use the supernatant liquid as hexane (1:1) to a distance of about 7 cm, and air-dry the
the sample solution. Separately, dissolve 1 mg of geniposide plate. Spray evenly 4-dimethylaminobenzaldehyde TS for
for thin-layer chromatography in 1 mL of methanol, and use spraying on the plate, heat at 1059C for 5 minutes, allow to
this solution as the standard solution. Perform the test with cool, and spray water: one of the spot among the several
these solutions as directed under Thin-layer Chromatogra- spots obtained from the sample solution has the same color
phy <2.03>. Spot 10 mL of the sample solution and 5 mL of tone and R f value with the blue-green to grayish green spot
the standard solution on a plate of silica gel for thin-layer obtained from the standard solution.
chromatography. Develop the plate with a mixture of ethyl (ii) To 1.0 g of the dry extract (or 3.0 g of the viscous ex-
acetate, methanol and ammonia solution (28) (6:3:2) to a tract) add 10 mL of water, shake, then add 25 mL of diethyl
distance of about 7 cm, and air-dry the plate. Spray evenly 4- ether, and shake. Separate the diethyl ether layer, evaporate
methoxybenzaldehyde-sulfric acid TS on the plate, and heat the solvent under reduced pressure, dissolve the residue in 2
at 1059 C for 1 minute: one of the spot among the several mL of diethyl ether, and use the solution as the sample solu-
spots obtained from the sample solution has the same color tion. Separately, dissolve 1 mg of [6]-shogaol for thin-layer
tone and R f value with the red-purple to purple spot ob- chromatography in 1 mL of methanol, and use this solution
tained from the standard solution (Gardenia Fruit). as the standard solution. Perform the test with these solu-
(4) To 1.0 g of the dry extract (or 3.0 g of the viscous tions as directed under Thin-layer Chromatography <2.03>.
extract) add 10 mL of water, shake, then add 5 mL of 1- Spot 20 mL of the sample solution and 5 mL of the standard
butanol, shake, centrifuge, and use the supernatant liquid as solution on a plate of silica gel for thin-layer chromatogra-
the sample solution. Separately, to 1.0 g of pulverized for- phy. Develop the plate with a mixture of ethyl acetate and
sythia fruit add 10 mL of methanol, shake, centrifuge, and hexane (1:1) to a distance of about 7 cm, and air-dry the
use the supernatant liquid as the standard solution. Perform plate. Spray evenly 4-dimethylaminobenzaldehyde TS for
the test with these solutions as directed under Thin-layer spraying on the plate, heat at 1059C for 5 minutes, allow to
Chromatography <2.03>. Spot 20 mL of the sample solution cool, and spray water: one of the spot among the several
and 10 mL of the standard solution as bands on the original spots obtained from the sample solution has the same color
line on a plate of silica gel for thin-layer chromatography. tone and R f value with the blue-green to grayish green spot
Develop the plate with a mixture of ethyl acetate, methanol obtained from the standard solution.
and ammonia solution (28) (10:2:1) to a distance of about (7) To 1.0 g of the dry extract (or 3.0 g of the viscous ex-
7 cm, and air-dry the plate. Spray evenly 4-methoxybenzal- tract) add 10 mL of 0.1 mol/L hydrochloric acid TS, shake,
dehyde-sulfric acid TS on the plate, heat at 1059C for 5 then add 25 mL of diethyl ether, and shake. Separate the
minutes, and allow to cool: one of the spot among the diethyl ether layer, evaporate the solvent under reduced pres-
several spots obtained from the sample solution has the same sure, dissolve the residue in 1 mL of methanol, and use the
color tone and R f value with the red-purple spot (R f value: solution as the sample solution. Separately, dissolve 1 mg of
about 0.4) obtained from the standard solution (Forsythia rosmarinic acid for thin-layer chromatography in 1 mL of
Fruit). methanol, and use this solution as the standard solution.
(5) To 2.0 g of the dry extract (or 6.0 g of the viscous ex- Perform the test with these solutions as directed under Thin-
tract) add 10 mL of diluted phosphoric acid (1 in 30), shake, layer Chromatography <2.03>. Spot 5 mL each of the sample
then add 15 mL of ethyl acetate, shake, centrifuge, and use solution and standard solution on a plate of silica gel for
the supernatant liquid as the sample solution. Separately, thin-layer chromatography. Develop the plate with a mixture
shake 0.2 g of pulverized mentha herb with 10 mL of diluted of ethyl acetate, water and acetic acid (100) (60:1:1) to a dis-
phosphoric acid (1 in 30), add 15 mL of ethyl acetate, shake, tance of about 10 cm, and air-dry the plate. Spray evenly
centrifuge, and use the supernatant liquid as the standard so- iron (III) chloride TS: one of the spot among the several
JP XVII Crude Drugs and Related Drugs / Bofutsushosan Extract 1815

spots obtained from the sample solution has the same color tate, methanol and water (20:3:2) to a distance of about 7
tone and R f value with the greenish brown spot obtained cm, and air-dry the plate. Examine under ultraviolet light
from the standard solution (Schizonepeta Spike; Mentha (main wavelength: 365 nm): one of the spot among the sev-
Herb). eral spots obtained from the sample solution has the same
(8) For preparation prescribed Saposhnikovia Root and color tone and R f value with the orange fluorescent spot ob-
Rhizome—To 2.0 g of the dry extract (or 6.0 g of the viscous tained from the standard solution (Rhubarb).
extract) add 10 mL of sodium hydroxide TS, shake, then add (12) To 1.0 g of the dry extract (or 3.0 g of the viscous
5 mL of 1-butanol, shake, centrifuge, and use the superna- extract) add 10 mL of water, shake, then add 25 mL of
tant liquid as the sample solution. Separately, dissolve 1 mg diethyl ether, and shake. Separate the diethyl ether layer,
of 4?-O-glycosyl-5-O-methylvisamminol in 1 mL of metha- evaporate the solvent under reduced pressure, then dissolve
nol, and use this solution as the standard solution. Perform the residue in 2 mL of diethyl ether, and use this solution as
the test with these solutions as directed under Thin-layer the sample solution. Separately, dissolve 1 mg of atrac-
Chromatography <2.03>. Spot 10 mL of the sample solution tylenolide III for thin-layer chromatography in 2 mL of
and 5 mL of the standard solution on a plate of silica gel for methanol, and use this solution as the standard solution.
thin-layer chromatography. Develop the plate with a mixture Perform the test with these solutions as directed under Thin-
of ethyl acetate, 1-propanol, water and acetic acid (100) layer Chromatography <2.03>. Spot 20 mL of the sample so-
(7:5:4:1) to a distance of about 7 cm, and air-dry the plate. lution and 5 mL of the standard solution on a plate of silica
Spray evenly diluted sulfuric acid on the plate, heat at 1059 C gel for thin-layer chromatography. Develop the plate with a
for 2 minutes, then examine under ultraviolet light (main mixture of hexane and ethyl acetate (2:1) to a distance of
wavelength: 365 nm): one of the spot among the several about 7 cm, and air- dry the plate. Spray evenly 1-naphthol-
spots obtained from the sample solution has the same color sulfuric acid TS on the plate, heat at 1059 C for 5 minutes,
tone and R f value with the bluish white fluorescent spot ob- and allow to cool: one of the spot among the several spots
tained from the standard solution (Saposhnikovia Root and obtained from the sample solution has the same color tone
Rhizome). and R f value with the red to red-purple spot obtained from
(9) For preparation prescribed Glehnia Root and the standard solution (Atractylodes Rhizome).
Rhizome—To 0.5 g of the dry extract (or 1.5 g of the viscous (13) To 2.0 g of the dry extract (or 6.0 g of the viscous
extract) add 5 mL of ethyl acetate, and heat on a water bath extract) add 10 mL of sodium carbonate TS, shake, then add
under a reflux condenser for 30 minutes. After cooling, 5 mL of 1-butanol, shake, centrifuge, and use the superna-
filter, and use the filtrate as the sample solution. Separately, tant liquid as the sample solution. Separately, to 2.0 g of
dissolve 1 mg of scopoletin for thin-layer chromatography in pulverized platycodon root add 10 mL of sodium carbonate
10 mL of methanol, and use this solution as the standard so- TS, shake, then add 5 mL of 1-butanol, shake, centrifuge,
lution. Perform the test with these solutions as directed and use the supernatant liquid as the standard solution. Per-
under Thin-layer Chromatography <2.03>. Spot 20 mL of the form the test with these solutions as directed under Thin-
sample solution and 2 mL of the standard solution on a plate layer Chromatography <2.03>. Spot 10 mL each of the sample
of silica gel for thin-layer chromatography. Develop the solution and standard solution on a plate of silica gel for
plate with a mixture of ethyl acetate and hexane (3:1) to a thin-layer chromatography. Develop the plate with a mixture
distance of about 7 cm, and air-dry the plate. Spray evenly of 1-propanol, ethyl acetate and water (4:4:3) to a distance
diluted sulfuric acid, heat at 1059C for 5 minutes, and exa- of about 10 cm, and air-dry the plate. Spray evenly 1,3-
mine under ultraviolet light (main wavelength: 365 nm): one naphthalenediol TS on the plate, heat at 1059 C for 5
of the spot among the several spots obtained from the sam- minutes: one of the spot among the several spots obtained
ple solution has the same color tone and R f value with the from the sample solution has the same color tone and R f
bluish white fluorescent spot obtained from the standard so- value with the blue-purple spot (R f value: about 0.4) ob-
lution (Glehnia Root and Rhizome). tained from the standard solution (Platycodon Root).
(10) To 1.0 g of the dry extract (or 3.0 g of the viscous (14) To 1.0 g of the dry extract (or 3.0 g of the viscous
extract) add 10 mL of sodium hydroxide TS, shake, then add extract) add 10 mL of water, centrifuge, then add 25 mL of
10 mL of diethyl ether, shake, centrifuge, and use the super- diethyl ether, shake, and centrifuge. Separate the diethyl
natant liquid as the sample solution. Perform the test with ether layer, evaporate the solvent under reduced pressure,
the sample solution as directed under Thin-layer Chromatog- dissolve the residue in 2 mL of diethyl ether, and use the so-
raphy <2.03>. Spot 15 mL of the sample solution on a plate of lution as the sample solution. Separately, dissolve 1 mg of
silica gel for thin-layer chromatography. Develop the plate wogonin for thin-layer chromatography in 1 mL of metha-
with a mixture of 1-propanol, ethyl acetate, water and acetic nol, and use this solution as the standard solution. Perform
acid (100) (4:4:2:1) to a distance of about 7 cm, and air-dry the test with these solutions as directed under Thin-layer
the plate. Spray evenly ninhydrin-ethanol TS for spraying on Chromatography <2.03>. Spot 20 mL of the sample solution
the plate, and heat at 1059C for 5 minutes: a red-purple spot and 2 mL of the standard solution on a plate of silica gel for
is observed at about 0.5 of R f value (Ephedra Herb). thin-layer chromatography. Develop the plate with a mixture
(11) To 1.0 g of the dry extract (or 3.0 g of the viscous of ethyl acetate, hexane and acetic acid (100) (10:10:1) to a
extract) add 10 mL of water, shake, then add 25 mL of distance of about 7 cm, and air-dry the plate. Spray evenly
diethyl ether, and shake. Separate the diethyl ether layer, iron (III) chloride-methanol TS on the plate: one of the spot
evaporate the solvent under reduced pressure, dissolve the among the several spots obtained from the sample solution
residue in 2 mL of diethyl ether, and use this solution as the has the same color tone and R f value with the yellow-brown
sample solution. Separately, dissolve 1 mg of rhein for thin- to grayish brown spot obtained from the standard solution
layer chromatography in 10 mL of acetone, and use this so- (Scutellaria Root).
lution as the standard solution. Perform the test with these (15) To 1.0 g of the dry extract (or 3.0 g of the viscous
solutions as directed under Thin-layer Chromatography extract) add 10 mL of water, shake, then add 10 mL of 1-
<2.03>. Spot 10 mL of the sample solution and 5 mL of the butanol, shake, centrifuge, and use the supernatant liquid as
standard solution on a plate of silica gel for thin-layer chro- the sample solution. Separately, dissolve 1 mg of liquiritin
matography. Develop the plate with a mixture of ethyl ace- for thin-layer chromatography in 1 mL of methanol, and use
1816 Bofutsushosan Extract / Crude Drugs and Related Drugs JP XVII
this solution as the standard solution. Perform the test with MS: Amount (mg) of Paeoniflorin RS taken, calculated on
these solutions as directed under Thin-layer Chromatogra- the anhydrous basis
phy <2.03>. Spot 5 mL each of the sample solution and stand-
Operating conditions—
ard solution on a plate of silica gel for thin-layer chromatog-
Detector: An ultraviolet absorption photometer (wave-
raphy. Develop the plate with a mixture of ethyl acetate,
length: 232 nm).
methanol and water (20:3:2) to a distance of about 7 cm, and
Column: A stainless steel column 4.6 mm in inside diame-
air-dry the plate. Spray evenly dilute sulfuric acid on the
ter and 15 cm in length, packed with octadecylsilanized silica
plate, and heat at 1059C for 5 minutes, then examine under
gel for liquid chromatography (5 mm in particle diameter).
ultraviolet light (main wavelength: 365 nm): one of the spot
Column temperature: A constant temperature of about
among the several spots obtained from the sample solution
209C.
has the same color tone and R f value with the yellow-green
Mobile phase: A mixture of water, acetonitrile and phos-
fluorescent spot obtained from the standard solution
phoric acid (850:150:1).
(Glycyrrhiza).
Flow rate: 1.0 mL per minute (the retention time of
(16) Place 2.0 g of the dry extract (or 6.0 g of the viscous
paeoniflorin is about 9 minutes).
extract) in a porcelain crucible, ignite to incinerate at 5509C,
System suitability—
then to the residue add 60 mL of water, shake, centrifuge,
System performance: Dissolve 1 mg each of Paeoniflorin
and use the supernatant as the sample solution. Add ammo-
RS and albiflorin in diluted methanol (1 in 2) to make
nium oxalate TS to the sample solution: a white precipitate is
10 mL. When the procedure is run with 10 mL of this solu-
formed. The precipitate does not dissolve in diluted acetic
tion under the above operating conditions, albiflorin and
acid, but dissolve on the addition of diluted hydrochloric
paeoniflorin are eluted in this order with the resolution be-
acid (Gypsum).
tween these peaks being not less than 2.5.
(17) Place 2.0 g of the dry extract (or 6.0 g of the viscous
System repeatability: When the test is repeated 6 times
extract) in a porcelain crucible, ignite to incinerate at 5509C.
with 10 mL of the standard solution under the above operat-
To the residue add 60 mL of water, shake well, centrifuge,
ing conditions, the relative standard deviation of the peak
and use the supernatant as the sample solution. The sample
area of paeoniflorin is not more than 1.5z.
solution responds to the Qualitative Tests <1.09> (1) for
(2) Total alkaloids (ephedrine and pseudoephedrine)—
sulfate (Gypsum; Sodium Sulfate or Anhydrous Sodium Sul-
Weigh accurately about 0.5 g of the dry extract (or an
fate).
amount of the viscous extract, equivalent to about 0.5 g of
Purity (1) Heavy metals <1.07>—Prepare the test solution the dried substance), add 20 mL of diethyl ether, shake, then
with 1.0 g of the dry extract (or an amount of the viscous ex- add 3.0 mL of 0.1 mol/L hydrochloric acid TS, and shake
tract, equivalent to 1.0 g of the dried substance) as directed for 10 minutes. After centrifugation, remove the upper
under Extracts (4), and perform the test (not more than 30 layer, add 20 mL of diethyl ether, proceed in the same man-
ppm). ner as above, and remove the upper layer. To the aqueous
(2) Arsenic <1.11>—Prepare the test solution with 0.67 g layer add 1.0 mL of ammonia TS and 20 mL of diethyl
of the dry extract (or an amount of the viscous extract, ether, shake for 30 minutes, centrifuge, and separate the su-
equivalent to 0.67 g of the dried substance) according to pernatant liquid. In addition, repeat twice in the same man-
Method 3, and perform the test (not more than 3 ppm). ner for the aqueous layer using 1.0 mL of ammonia TS and
20 mL of diethyl ether. Combine all the supernatant liquids,
Loss on drying <2.41> The dry extract: Not more than
evaporate the solvent under reduced pressure, dissolve the
9.0z (1 g, 1059C, 5 hours).
residue in diluted methanol (1 in 2) to make exactly 50 mL,
The viscous extract: Not more than 66.7z (1 g, 1059C,
centrifuge, and use the supernatant liquid as the sample solu-
5 hours).
tion. Separately, weigh accurately about 10 mg of ephedrine
Total ash <5.01> Not less than 10.0z and more than hydrochloride for assay of crude drug, previously dried at
22.0z, calculated on the dried basis. 1059C for 3 hours, dissolve in diluted methanol (1 in 2) to
make exactly 100 mL. Pipet 10 mL of this solution, add
Assay (1) Paeoniflorin—Weigh accurately about 0.5 g of
diluted methanol (1 in 2) to make exactly 50 mL, and use this
the dry extract (or an amount of the viscous extract, equiva-
solution as the standard solution. Perform the test with ex-
lent to about 0.5 g of the dried substance), add exactly 50
actly 10 mL each of the sample solution and standard solu-
mL of diluted methanol (1 in 2), shake for 15 minutes, and
tion as directed under Liquid Chromatography <2.01> ac-
filter. Pipet 5 mL of the filtrate, elute through a column
cording to the following conditions, and determine the peak
packed with 2 g of polyamide for column chromatography
areas, ATE and ATP, of ephedrine and pseudoephedrine ob-
using 20 mL of water, then add 1 mL of acetic acid (100),
tained with the sample solution, and the peak area, AS, of
add water to make exactly 25 mL, and use this solution as
ephedrine obtained with the standard solution.
the sample solution. Separately, weigh accurately about 10
mg of Paeoniflorin RS (separately determine the water Amount (mg) of total alkaloids [ephedrine(C10H15NO)
<2.48> by coulometric titration, using 10 mg), and dissolve in and pseudoephedrine(C10H15NO)]
diluted methanol (1 in 2) to make exactly 100 mL. Pipet 5 ＝ MS × (ATE ＋ ATP)/AS × 1/10 × 0.819
mL of this solution, add diluted methanol (1 in 2) to make
MS: Amount (mg) of ephedrine hydrochloride for assay of
exactly 20 mL, and use this solution as the standard solution.
crude drug taken
Perform the test with exactly 10 mL each of the sample solu-
tion and standard solution as directed under Liquid Chroma- Operating conditions—
tography <2.01> according to the following conditions, and Detector: An ultraviolet absorption photometer (wave-
determine the peak areas, AT and AS, of paeoniflorin in each length: 210 nm).
solution. Column: A stainless steel column 4.6 mm in inside diame-
ter and 15 cm in length, packed with octadecylsilanized silica
Amount (mg) of paeoniflorin (C23H28O11)
gel for liquid chromatography (5 mm in particle diameter).
＝ MS × AT/AS × 5/8
Column temperature: A constant temperature of about
JP XVII Crude Drugs and Related Drugs / Boiogito Extract 1817

409 C. remove the upper layer. To the resultant aqueous layer add
Mobile phase: To 5 g of sodium lauryl sulfate add 350 mL 10 mL of methanol, shake for 30 minutes, centrifuge, and
of acetonitrile, shake, then add 650 mL of water and 1 mL separate the supernatant liquid. To the residue add 20 mL of
of phosphoric acid. diluted methanol (1 in 2), shake for 5 minutes, and centri-
Flow rate: 1.0 mL per minute (the retention time of ephe- fuge. Separate the supernatant liquid, combine all the super-
drine is about 27 minutes). natant liquids, add diluted methanol (1 in 2) to make exactly
System suitability— 50 mL, and use this solution as the sample solution. Sepa-
System performance: Dissolve 1 mg each of ephedrine hy- rately, weigh accurately about 10 mg of Glycyrrhizic Acid
drochloride for assay of crude drug and pseudoephedrine hy- RS (separately determine the water <2.48> by coulometric
drochloride in diluted methanol (1 in 2) to make 10 mL. titration, using 10 mg), dissolve in diluted methanol (1 in 2)
When the procedure is run with 10 mL of this solution under to make exactly 100 mL, and use this solution as the stand-
the above operating conditions, pseudoephedrine and ephe- ard solution. Perform the test with exactly 10 mL each of the
drine are eluted in this order with the resolution between sample solution and standard solution as directed under
these peaks being not less than 1.5. Liquid Chromatography <2.01> according to the following
System repeatability: When the test is repeated 6 times conditions, and determine the peak areas, AT and AS, of
with 10 mL of the standard solution under the above operat- glycyrrhizic acid in each solution.
ing conditions, the relative standard deviation of the peak
Amount (mg) of glycyrrhizic acid (C42H62O16)
area of ephedrine is not more than 1.5z.
＝ MS × AT/AS × 1/2
(3) Baicalin—Weigh accurately about 0.1 g of the dry
extract (or an amount of the viscous extract, equivalent to MS: Amount (mg) of Glycyrrhizic Acid RS taken, calcu-
about 0.1 g of the dried substance), add exactly 50 mL of lated on the anhydrous basis
diluted methanol (7 in 10), shake for 15 minutes, filter, and
Operating conditions—
use the filtrate as the sample solution. Separately, weigh ac-
Detector: An ultraviolet absorption photometer (wave-
curately about 10 mg of Baicalin RS (separately determine
length: 254 nm).
the water <2.48> by coulometric titration, using 10 mg), dis-
Column: A stainless steel column 4.6 mm in inside diame-
solve in methanol to make exactly 100 mL. Pipet 5 mL of
ter and 15 cm in length, packed with octadecylsilanized silica
this solution, add diluted methanol (7 in 10) to make exactly
gel for liquid chromatography (5 mm in particle diameter).
10 mL, and use this solution as the standard solution. Per-
Column temperature: A constant temperature of about
form the test with exactly 10 mL each of the sample solution
409C.
and standard solution as directed under Liquid Chromatog-
Mobile phase: A mixture of diluted acetic acid (31) (1 in
raphy <2.01> according to the following conditions, and
15) and acetonitrile (13:7).
determine the peak areas, AT and AS, of baicalin in each
Flow rate: 1.0 mL per minute (the retention time of glycyr-
solution.
rhizic acid is about 12 minutes).
Amount (mg) of baicalin (C21H18O11) System suitability—
＝ MS × AT/AS × 1/4 System performance: When the procedure is run with 10
mL of the standard solution under the above operating con-
MS: Amount (mg) of Baicalin RS taken, calculated on the
ditions, the number of theoretical plates and the symmetry
anhydrous basis
factor of the peak of glycyrrhizic acid are not less than 5000
Operating conditions— and not more than 1.5, respectively.
Detector: An ultraviolet absorption photometer (wave- System repeatability: When the test is repeated 6 times
length: 277 nm). with 10 mL of the standard solution under the above operat-
Column: A stainless steel column 4.6 mm in inside diame- ing conditions, the relative standard deviation of the peak
ter and 15 cm in length, packed with octadecylsilanized silica area of glycyrrhizic acid is not more than 1.5z.
gel for liquid chromatography (5 mm in particle diameter).
Containers and storage Containers—Tight containers.
Column temperature: A constant temperature of about
409 C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
200) and acetonitrile (19:6).
Flow rate: 1.0 mL per minute (the retention time of baica-
lin is about 10 minutes).
System suitability—
System performance: When the procedure is run with 10
mL of the standard solution under the above operating con-
ditions, the number of theoretical plates and the symmetry
factor of the peak of baicalin are not less than 5000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times
with 10 mL of the standard solution under the above operat-
ing conditions, the relative standard deviation of the peak
area of baicalin is not more than 1.5z.
(4) Glycyrrhizic acid—Weigh accurately about 0.5 g of
the dry extract (or an amount of the viscous extract, equiva-
lent to about 0.5 g of the dried substance), add 20 mL of
ethyl acetate and 10 mL of water, and shake for 10 minutes.
After centrifugation, remove the upper layer, add 20 mL of
ethyl acetate, proceed in the same manner as above, and