VI. E L LEGADO DE D ESCARTES
2. El triunfo de Descartes
(1) (2)
examined, and reflux with methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Dissolve the residue in hot water and filter. Extract the filtrate repeatedly with 50, 50, 50, 25, 25 ml of ethyl acetate. Combine all the ethyl
acetate extracts and filter and evaporate to
dryness under reduced pressure. Dry the residue at 1000 for one hour and weigh the residue.
Calculate the content of bitters from the weight of the residue and from the weight of substance taken for the test.
Assay:
Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.2 g, accurately weighed, of the substance being examined and reflux with water (25 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml volumetric flask and make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of picroside-I RS and
picroside-II RS in 25-ml volumetric flask and
dissolve in about 20 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and
stationary phase: C18 (250 mm x 4.6 mm, 5 m).
Mobile phase: Filtered and degassed mixture of
17 volumes of acetonitrile and 83 volumes of
water containing 0.1 per cent phosphoric acid. Injection volume: 20 l. Flow rate: 1 ml per min.
Detector: UV 262 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the responses for the analyte peaks.
Analyte Relative retention time
Picroside-II 1.0
Picroside-I 2.5
Fig. 2: HPLC chromatograms of Ka¶uk¡
water extract with Picroside- I and
Picroside-II as RS
Calculate the content of picroside-I and picroside-
II in the substance being examined from peak
response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.
Additional requirements:
Storage: Store in well closed container protected
from heat, light, moisture and against attack by insects and rodents.
Labelling: The label states the official name,
followed by the Latin binominal name and the part of the plant contained in the article.
API reference standards:
API Picroside-I RS and Picroside-II RS
MaμjiÀ¶Å¡ consists of the dried root of Rubia
cordifolia L. (Fam. Rubiaceae), a perennial
herbaceous creeper or climber, with hooked prickles and whorls of four leaves, but without interpetiolar stipules, found throughout the country ascending to 3750 m. It contains not less than 0.04 per cent of rubiadin when assayed.
Synonyms: Yojanavall¢, T¡mravall¢, Vastraraμjin¢, Rakt¡
Other/Regional Language Names: Assamese:
Phuvva; Bengali: Manjishtha, Manjith; English: Indian Madder; Gujarati: Manjitha; Hindi: Manjitha, Manjit; Kannada: Manjustha;
Malayalam: Manjatti, Manchatti; Marathi:
Manjishtha; Punjabi: Manjistha, Manjit; Tamil: Manatte, Manjitti; Telugu: Manjishtha
Description:
a) Macroscopic:
Root - Cylindrical, often surmounted by a knotty
crown of root stock; about 2 to 9 cm in length and 0.2 to 0.6 cm in width; surface smooth finely striated longitudinally and occasionally grooved, often exhibiting lateral root scars; dark reddish brown both externally and internally. Fracture short, taste sweetish, acrid and disagreeable, odour pleasant
b) Microscopic:
TS of root shows a well developed cork, consisting of 3 to 8 layered suberized radially arranged cells, occasionally filled with reddish brown content, followed by a cortex of 3 to 10 cell layers; some cortical cells filled with acicular and sandy crystals of calcium oxalate more towards periphery. Phloem 8 to 12 layers wide, consists of sieve tubes, companion cells and phloem parenchyma. Xylem consists of vessels, fibres, tracheids and xylem parenchyma. Vessels are broader towards the peripheral region of the xylem. The size of vessels vary from 30 to 270
m in length and 18 to 90 m in breadth.
Medullary rays are uni- to multiseriate and oval to circular starch grains present in cortical and phloem parenchyma cells.
c) Powder:
Shows numerous fragments of cork, lignified xylem vessels, tracheids and fibres, raphides, clusters and sandy oxalate crystals, parenchyma with red content and starch grains (Fig. 1)
Fig. 1: Powdered drug of MAØJIâÙéË
(Rubia cordifolia L.)
Identity, Purity and Strength:
Identification:
Thin-layer chromatography:
Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using rubiadin as a reference standard. Test
solution: Extract 2 g of substance by refluxing
with chloroform (25 ml x 3) for a period of 10-15 min each. Filter and concentrate the combined extract to dryness. Dissolve the residue in 2 ml of
Rf
0.0 0.5
1.0
methanol. Standard solution: Dissolve 1 mg of rubiadin RS in about 10 ml of methanol.
254 nm 366 nm
RS T RS T
Fig. 2: Thin-Layer Chromatogram of MaμjiÀ¶h¡
RS: Rubiadin, T: Test solution
Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop upto 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate :
chloroform : glacial acetic acid (10.0 : 5.0 : 1.0 :
2.5). Dry the plate in air and examine under UV 254 nm and under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of standard solution(Fig.2).
Quantitative parameters:
Foreign matter: not more than 2.0 per cent
(Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-
insoluble ash: not more than 0.5 per cent
(Appendix 2.1.7); Alcohol-soluble extractive: not less than 3.0 per cent (Appendix 2.1.8); Water-
soluble extractive: not less than 10.0 per cent
(Appendix 2.1.9)
Other requirements:
Heavy metals: Complies with the prescribed limits,
(Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide
residues: Complies with the prescribed limits,
(Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)
Assay:
Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (50 ml x 3) on water bath for 5-10 min each, cool and Filter. Combine all the filtrates, concentrate to 50 ml and transfer in a 100-ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, rubiadin RS in a 100-ml volumetric flask and dissolve in about 50 ml of
methanol and make up the volume with methanol.
Filter through 0.42 m membrane.
Chromatographic system: High performance
liquid chromatography. Column and stationary
phase: C18(250 mm x 4.6 mm, 5 m). Mobile
phase: Filtered and degassed gradient mixture of
phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric
acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:
Time
(min) Phosphate buffer (per cent) Acetonitrile (per cent)
0.01 65 35 5 50 50 15 20 80 20 15 85 25 65 35 30 65 35
Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 278 nm. Procedure: Inject 20
l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the
response for the analyte peak. Calculate the content of rubiadin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.
Fig. 3: HPLC chromatogram of MaμjiÀ¶h¡ with Rubiadin as RS
Additional requirements:
Storage: Store in well closed container protected
from heat, light, moisture and against attack by insects and rodents.
Labelling: The label states the official name,
followed by the Latin binominal name and the part of the plant contained in the article.