Eleuthero
DEFINITION
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Eleuthero is the dried rhizome with roots of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (Fam. Araliaceae) [Acanthopanax senticosus (Rupr. & Maxim.) Harms]. It contains NLT 0.08% of the sum of eleutheroside B and eleutheroside E, calculated on the dried basis.USP35
IDENTIFICATION
Change to read:
•  A.USP35 Thin-Layer Chromatographic Identification Test 201
Standard solution A:  1 mg/mL of USP Eleutheroside E RS in methanol
Standard solution B:  1 mg/mL of USP Eleutheroside B RS in methanol
Standard solution C:  0.1 g of USP Powdered Eleuthero Extract RS in 5 mL of aqueous ethanol 50%. Sonicate for 10 min, centrifuge, and use the supernatant.
Sample solution:  Transfer about 1 g of finely powdered Eleuthero to a centrifuge tube, add 5 mL of aqueous ethanol 50%, and mix well. Sonicate for 10 min. Centrifuge or filter the solution, and use the supernatant or the filtrate.
Adsorbent:  Chromatographic silica gel with an average particle size of 5 µm (HPTLC plates)
Application volume:  10 µL, as bands
Developing solvent system:  Chloroform, methanol, and water (35:15:2)
Spray reagent:  Place 18 mL of methanol in a glass flask, and cool in a water–ice–salt bath or in a freezer. To the ice-cold methanol, slowly and carefully add 2 mL of sulfuric acid, and mix well. Allow the mixture to adjust to room temperature.
Analysis 
Samples:  Standard solution A, Standard solution B, Standard solution C, and Sample solution
Before the development of the chromatogram, saturate the chamber for 20 min with Developing solvent system. Record the temperature and humidity in the laboratory. If the relative humidity exceeds 50%, condition the plate to about 30% relative humidity, using a suitable device. Develop the plate over a path of 6 cm, dry, and spray with Spray reagent. Heat the plate at 100 for 5 min, and examine under visible light and UV light at 365 nm.
Acceptance criteria:  Under visible light, the Sample solution exhibits two brown bands due to eleutheroside E and eleutheroside B at RF values of about 0.34 and 0.45, corresponding in color and RF to the bands exhibited by Standard solution A and Standard solution B, respectively. The Sample solution also exhibits two additional brown bands near the application zone, corresponding in color and RF values to the bands exhibited by Standard solution C. Other bands may be observed in the Sample solution and Standard solution C chromatograms. Under UV light, the Sample solution shows a brown band due to eleutheroside E corresponding in color and RF to the band exhibited by Standard solution A.USP35
Add the following:
•  B. HPLC: The chromatogram of the Sample solution obtained in the test for Content of Eleutherosides B and E shows a peak at a retention time corresponding to that of eleutheroside B in the chromatogram of Standard solution B and a peak at a retention time corresponding to that of eleutheroside E in the chromatogram of Standard solution A.USP35
COMPOSITION
Change to read:
•  Content of Eleutherosides B and E
Solvent:  Methanol and water (1:1)
Solution A:  Acetonitrile and water (5:95)
Solution B:  Acetonitrile and water (60:40)
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 97 3
5 97 3
30 60 40
31 5 95
45 5 95
45.1 97 3
60 97 3
Standard solution A:  0.1 mg/mL of USP Eleutheroside E RS in methanol. Transfer 2.0 mL to a 5-mL volumetric flask, and dilute with Solvent to volume.
Standard solution B:  0.1 mg/mL of USP Eleutheroside B RS in methanol. Transfer 2.0 mL to a 5-mL volumetric flask, and dilute with Solvent to volume.
Standard solution C:  5.0 mg/mL of USP Powdered Eleuthero Extract RS in Solvent. Sonicate for 30 min, cool to room temperature, decant, and pass through a nylon filter of 0.45-µm or finer pore size.USP35
Sample solution:  Transfer about 5.0 g of finely ground Eleuthero, accurately weighed, to a round-bottom flask equipped with a condenser. Add 50 mL of Solvent, and heat under reflux for 30 min. Filter the supernatant through cotton wool into a 100-mL volumetric flask. Transfer the cotton wool to the round-bottom flask, and repeat the extraction twice, using 22 mL of Solvent for each extraction. Filter through cotton wool into the volumetric flask, wash the residue and the cotton wool with Solvent, cool to room temperature, dilute with Solvent to volume, and mix. Before injection, pass through a nylon filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.USP35
Chromatographic system 
Mode:  LC
Detector:  UV 220 nm
Column:  4.0-mm × 25-cm; 5-µm packing L1
Flow rate:  1 mL/min
Injection size:  10 µL
System suitability 
Samples:  Standard solution B and Standard solution C
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution C is similar to the reference chromatogram provided with the lot of USP Powdered Eleuthero Extract RS being used.
Relative standard deviation:  NMT 2.0% determined from the eleutheroside B peak in repeated injections, Standard solution BUSP35
Analysis 
Samples:  Standard solution A, Standard solution B, Standard solution C, and Sample solution
Identify the eleutheroside B and eleutheroside E peaks in the Sample solution by comparison with the chromatograms of Standard solution B and Standard solution A, respectively, and measure the peak responses.
Separately calculate the percentages of eleutheroside B and eleutheroside E in the portion of Eleuthero taken:
Result = (rU/rS) × CS × (V/W) × 100
rU== peak response of the relevant analyte from the Sample solution
rS== peak response of eleutheroside E or eleutheroside B from Standard solution A or Standard solution B, respectively
CS== concentration of eleutheroside E or eleutheroside B in Standard solution A or Standard solution B, respectively (mg/mL)
V== volume of the Sample solution (mL)
W== weight of Eleuthero taken to prepare the Sample solution (mg)
USP35
Acceptance criteria:  Add the percentages of eleutheroside B and eleutheroside E: NLT 0.08% on the dried basis.
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 20 ppm
Change to read:
•  Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.USP35
Add the following:
•  Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coliUSP35
SPECIFIC TESTS
•  Botanic Characteristics
Macroscopic:  The rhizome is knotty and of irregular cylindrical form with a diameter of 15–40 mm. The heartwood area is light brown, and the connecting splint wood is pale yellow. The bark is approximately 2 mm thick and is firmly affixed to the xylem. The surface is gray-brown or black-brown, coarse, and longitudinally valleculate and plicate. A broken rhizome is coarse and fibrous, particularly inside the xylem. The fractured surface of the bark shows short, thin fibers. Numerous roots spring from the underside of the rhizome. These roots are 35–150 mm long, cylindrical, and knotty, with a diameter of 3–15 mm. The surface of the roots is gray-brown to black-brown, is smoother than the rhizome, and has longitudinal stripes. A 0.5-mm thin bark is tightly affixed to the pale yellow xylem. A broken root is sparsely fibrous and appears yellowish-gray where the thin epidermis is flaked off.
Histology:  The roots have five to seven rows of brown cork cells. Secretory canals with brown contents appear in groups of four or five and are not more than 20 µm in diameter. Phloem fibers with thick lignified walls occur singly or in small groups; there are cluster crystals of calcium oxalate in the phloem parenchyma. Parenchymatous cells surround the secretory cells, and medullary ray cells contain small starch granules. The xylem shows reticulately thickened and pitted vessels. The rhizome is similar to the roots except for its larger secretory canals, up to 25 µm in diameter, and the presence of a pith with parenchymatous cells containing starch granules.
•  Loss on Drying 731: Dry a sample at 105 to constant weight: it loses NMT 14.0% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed, light-resistant containers.
•  Labeling: The label states the Latin binomial and, following the official name, the parts of the plant contained in the article.
Change to read:
•  USP Reference Standards 11
USP Eleutheroside B RS
-d-Glucopyranoside, 4-(3-hydroxy-1-propenyl)-2,6-dimethoxyphenyl.
    C17H24O9        372.37
USP Eleutheroside E RS
-d-Glucopyranoside, (tetrahydro-1H,3H-furo(3,4-c)furan-1,4-diyl)bis(2,6-dimethoxy-4,1-phenylene)bis-.
    C34H46O18        742.70
USP35
USP Powdered Eleuthero Extract RS
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Pharmacopeial Forum: Volume No. 36(6) Page 1588