06 Standardisation de Principios Activos

Qualit des phytomdicaments
Cours de Pharmacognosie et Phytochimie
Standardisation
Phytomdicaments: critres de qualit
PHYTOMDICAMENT
Pour tre enregistr, il doit satisfaire, comme tout mdicament, certaines exigences
Efficacit Scurit - Tests cliniques - Tests pharmacologiques - Suivi mdical Qualit - Identification du matriel vgtal - Identification des constituants - Standardisation - Toxicologie - Rsidus de pesticides,etc.
Standardisation
Phytomdicaments: critres de qualit
Efficacit
Qualit
Scurit
tests cliniques tests pharmacologiques suivi mdical
identification du matriel vgtal identification des constituants standardisation
composs toxiques teneur en pesticides contamination microbiennes teneur en mtaux lourds
Standardisation
Qualit des drogues vgtales Variabilit de la teneur en principes actifs Choix de la varit Conditions de culture Nutriments, Moment de la cueillette Variabilit saisonnire Dfis pour la production dextraits standardiss tablir des stratgies pour faire face linhomognit et assurer une dose thrapeutique constante ainsi quun bonne scurit demploi
Standardisation
Qualit des drogues vgtales Garantir leffet assurer une dose de principes actifs constante
Dmarche: qualit leve et reproductible de la drogue vgtale extraction par un procd standardis (reproductible), dans des conditions de GMP contrle strict des paramtres variables standardisation de lextrait contrles de qualit de tous les composants de la formulation (standards pharmaceutiques) fabrication du produit mdicinal dans des conditions de GMP (procd reproductible et valid) tudes de stabilit long terme essais cliniques bass sur un profil constant de lextrait et sur une qualit constante du produit mdicinal
Standardisation
Standardisation des extraits
But
La standardisation d'un extrait permet d'assurer sa qualit son efficacit sa reproductibilit
Exigences
Standardisation exige par les offices d'enregistrement des mdicaments
Standardisation
Standardisation: dmarche
Identification du ou des principe(s) actif(s) oui Quantification du ou des principe(s) actif(s) non Quantification d'un compos traceur Quantification de tous composs principaux (profilage) Quantification de l'activit biologique
Niveau des composs responsables des effets secondaires Approche complexe Matrice complique Dosage de plusieurs composs
Techniques analytiques performantes
Exemple de standardisation partir dun test biologique
A minimum of 20% reduction of blood glucose level measured 30 min after glucose loading (2g/kg) in rats has been considered the level for the approval of each industrial batch of MADEGLUCYL MADEGLUCYL.
Standardisation
Standardisation des extraits
Techniques

Dosage directs (UV, Titrimtrie, ) CCM CCM haute performance (HPTLC) HPLC-UV HPLC-UV/DAD HPLC-MS GC GC-MS
Standardisation
Panax Ginseng C.A. Meyer (Araliaceae)

OR1 OH
HO O-R2
Extrait standardis: teneur de plus de 3% ginsnosides Re, Rf, Rg1, Rg2, Rb1, Rb2, Rc, et Rd. Composition variable: - Ginseng de Core - Ginseng du Japon - Ginseng de lhimalaya
Re Rf Rg1 Rg2
R1 R2 glc glc-rha H glc-glc glc glc H glc-rha

OR1 OH
R2-O
R1 R2 Rb1 glc-glc glc-glc Rb2 glc-arap glc-glc Rc glc-araf glc-glc glc glc-glc Rd
Effets pharmacologiques de certains ginsnosides:- Rg1 augmente la tension artrielle et stimule le SNC -Rb1 abaisse la pression artrielle et dprime le SNC - rapport Rg1/Rb1 - haut ->Ginseng asiatique > stimulant - Bas ->Ginseng amricain > stimulant
Standardisation
Dosage des ginsnosides pharmacope EU
Pharmacope EU
Pharmacope USP Asian Ginseng consists of the dried roots of Panax ginseng C.A. Meyer (Fam. Araliaceae). It contains not less than 0.2% of ginsenoside Rg1 and not less than 0.1% of ginsenoside Rb1, both calculated on the dried basis.
Standardisation
Standardisation ginseng: Quantits dclares
Standardisation
Dosage des ginsnosides selon pharmacope EU

Colonne : dimensions : l = 0,125 m, = 4,6 mm, phase stationnaire : gel de silice octadcylsilyl pour chromatographie R (5 m), temprature : 35 C. Phase mobile : phase mobile A : eau R ajuste pH 2 avec de lacide phosphorique R, phase mobile B : actonitrile R, Dbit : 1,0 ml/min. Dtection : spectrophotomtre 203 nm. Dure dquilibrage : 20 min. Injection : 20 l.
Rb1 Rsolution 1.0
Rg1
Ordre dlution : ordre donn pour la prparation de la solution tmoin ; notez les temps de rtention de ces substances. Conformit du systme : solution tmoin : rsolution : minimum 1,0 entre les pics dus au ginsnoside Rg1 et au ginsnoside Re.
Standardisation
Dosage des ginsnosides selon pharmacope EU
Standardisation
Rapport de Rb1/Rg1 dans diffrents extraits de ginseng

Rg1 Rb1 Ginseng amricain sauvage
Ginseng amricain cultiv Rb1
Rg1
Effets pharmacologiques de certains ginsnosides:- Rg1 augmente la tension artrielle et stimule le SNC -Rb1 abaisse la pression artrielle et dprime le SNC - rapport Rg1/Rb1 - haut ->Ginseng asiatique > stimulant - Bas ->Ginseng amricain > stimulant
Rb1 Ginseng asiatique Rg1
Standardisation
Analyse LC/UV dun mlange de ginsnosides

OR1 OH
mAU 30
UV trace (203 nm)

Rf
Rg2 Rb1Rc Rb2 Rd
2 g each
HO O-R2
25
Rg1
Re Rf Rg1 Rg2
20

OR1 OH
15 Re
10 5
Total analysis time: 80 min
R2-O
MeCN-water gradient (RP-18 col. 150 mm) 10 20 30 40 50 60 70 min
R1 R2 Rb1 glc-glc glc-glc Rb2 glc-arap glc-glc Rc glc-araf glc-glc glc glc-glc Rd
Standardisation
Nouvelle mthode: analyse LC/ES-MS dun mlange de ginsnosides

m/z 799 Rg1 Rf
m/z 945
Rd Re Rb1
m/z 1107
m/z 1077
Rc Rb2 Rg2
Spectre LC/ES-MS de Rd
glc O OH
m/z 783
[M-H]945
UV trace (203 nm)
glc
glc
Base peak trace

400 600 800 1000
10
20
30
40
50
60
MeCN-water gradient (RP-18 col. 150 mm)
Standardisation
Nouvelle mthode: analyse LC/ES-MS dun mlange de ginsnosides

m/z 799 Rg1 Rf
Gradient 80 min
Rd
Rg1 Rf
Isocratic 12 min
Rd
HO
OR1 OH
m/z 945
Re
Re Rb1 Rb1
O-R2
m/z 1107
m/z 1077
Rc Rb2 Rg2
Rc
Rb2
Re Rf Rg1 Rg2

OR1 OH
m/z 783
Rg2
UV trace (203 nm)
R2-O
Base peak trace
R1 R2 Rb1 glc-glc glc-glc Rb2 glc-arap glc-glc Rc glc-araf glc-glc glc glc-glc Rd 30 40 50 60
(RP-18 col. 70 mm)
10
20
10
MeCN-water gradient (RP-18 col. 150 mm)
min
Standardisation
Saponines de Panax ginseng

Ginsenosides R1 Ra1 Ra2 Ra3 Rb1 Rb2 Rb3 Rc Rd Rg3 F2 Rh2 R1 RS1 RS2 Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc-Glc Glc Glc Glc-Glc-Ac Glc-Glc-Ac Glc-Glc-Ac R2 Glc-Ara(pyr)-xyl Glc-Ara(fur)-xyl Glc-Glc-xyl Glc-Glc Glc-Ara(pyr) Glc-xyl Glc-Ara(fur) Glc H Glc H Glc-Glc Glc-Ara(pyr) Glc-Ara(fur) R2 Glc-Glc Glc-Ara(pyr) Glc-Ara(fur) Glc R2 Glc-Glc-xyl Glc-Glc Glc-xyl Glc-Ara(fur) R3 H H H H H H H H H H H H H H R3 Ginsenosides R1 Re Rf Rg1 Rg2 Rh1 F1 F3 H H H H H H H R2 Glc H Glc H H Glc Glc-Ara(pyr) R2 Glc H Glc-Glc Glc-Glc R2 Glc R3 O-Glc-Rha O-Glc-Glc O-Glc O-Glc-Rha O-Glc H H R3 O-Glc-xyl O-Glc-xyl Glc Glc R3 Xyl H H R3 O-Glc
OR2 OH
Notoginsenosides R1 R1 R2 R3 R6 H H H H
Malonylginsenosides R1
R1-O R3
Rb1 Rb2 Rc Rd
Glc-Glc-Ma Glc-Glc-Ma Glc-Glc-Ma Glc-Glc-Ma
H H Pseudoginsenosides R1 H H RT3 H R3 H RC1 F8 R1 H
Notoginsenosides R1 R4 Fa Fc Fe Glc-Glc Glc-Glc-xyl Glc-Glc-xyl Glc
Glc Glc Glc-xyl R2 Glc
20-GlucoH ginsenoside H Rf H
Standardisation
Dosage des ginsnosides par LC/ELSD

ELSD
OR1 OH
HO O-R2
chromophore faible dtection UV difficile UV 203 nm
Standardisation
Comparaison de la composition avant et aprs chauffage par LC/ELSD

P. ginseng ginseng blanc ELSD
Les ginsnosides Rg3 et Rg5 notamment napparaissent quaprs traitement 120 C P. ginseng ap chauffage 120C ginseng rouge Rg3 Rg5 Rg3 proprits vasorelaxantes et anit-PAF importantes Rg5 activit anticancreuses
Standardisation
Ginsnosides totaux dans diffrentes phytoprparations du march suisse
50.00 45.00 40.00 35.00 Preparations containing Ginseng only
Mg/day
30.00 25.00 20.00 15.00 10.00 5.00 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Preparation No
Standardisation
Ginsnosides totaux dans diffrentes phytoprparations du march suisse
9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 15 16 17
Preparations containing Ginseng is association
Mg/day
18
19
20
Preparation No
Standardisation
Pharmacope Panax Ginseng : scurit

Pharmacope USP
Microbial enumeration 2021The total aerobic microbial count does not exceed 104 cfu per g. The total combined molds and yeasts count does not exceed 100 cfu per g, and it meets the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus. Loss on drying 731Dry 1.0 g of finely powdered Asian Ginseng at 105 for 2 hours: it loses not more than 12.0% of its weight. Foreign organic matter 561: not more than 2.0%. Total ash 561: not more than 8.0%, determined on 1.0 g of finely powdered Asian Ginseng . Acid-insoluble ash 561: not more than 1.0%. Alcohol-soluble extractives, Method 2 561: not less than 14.0%. Pesticide residues 561: meets the requirements. Heavy metals, Method III 231: 20 g per g.
Pharmacope EU
TESTS Foreign matter (2.8.2). It complies with the test for foreign matter. Panax quinquefolium. Examine the chromatograms obtained in the assay. The chromatogram obtained with the test solution shows the peak corresponding to ginsenoside Rf. In the case of a substitution by Panax quinquefolium no peak corresponding to ginsenoside Rf is present. Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100 C to 105 C. Total ash (2.4.16). Not more than 7.0 per cent. Ash insoluble in hydrochloric acid (2.8.1). Not more than 1.0 per cent.
Standardisation
Dtection des pesticides: pharmacope USP

GENERAL METHOD FOR PESTICIDE RESIDUES ANALYSIS Definition Where used in this Pharmacopeia, the designation pesticide applies to any substance or mixture of substances intended to prevent, destroy, or control any unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport, or marketing of pure articles. The designation includes substances intended for use as growth regulators, defoliants, or desiccants, and any substance applied to crops before or after harvest to protect the product from deterioration during storage and transport. LimitsWithin the United States, many botanicals are treated as dietary supplements and are subject to the statutory provisions of the Federal Food, Drug, and Cosmetic Act that governs foods but not drugs. Limits for pesticides for foods are determined by the Environmental Protection Agency (EPA), and where no limit is set, the limit is zero. The limits contained herein, therefore, are not applicable in the United States when articles of botanical origins are labeled for food purposes. The limits, however, may be applicable in other countries where the presence of pesticide residues is permitted. Unless otherwise specified in the individual monograph, the article under test contains not more than the amount of any pesticide indicated in Table 3. The limits applying to pesticides not listed in Table 3 and whose presence is suspected for any reason comply with the regulations of the EPA. Where a pesticide is not listed in Table 3 or in EPA regulations, calculate the limit by the formula:
AM / 100B
in which A is the acceptable daily intake (ADI), in mg per kg of body weight; M is the body weight, in kg; and B is the daily dose of the article, in kg.
Standardisation
Rsidus de pesticides: Panax ginseng
Analyse des pesticides par GC/ECD (ppb)

10000 9000 8000 7000 6000 ppb 5000 4000 3000 2000 1000
37 3 Li 8 m ite s
Total (PCNB + isomers) Total HCH HCB
0
27 10 1 10 2 21 22 23 35 31 20 42 39 24 25 26 28 30 33 32 34
phytoprparations
Method CG ECD EPA 505 Method GC/MS EPA 608 Environmental Protection Agency
Standardisation
Rsidus de pesticides: Panax ginseng
Cl Cl Cl Cl3 Cl Cl
Cl Cl Cl
DDT (insecticide)
Cl Cl Cl
HCH (insecticide)
NO2 Cl Cl
Cl Cl
Cl
Cl Cl
Cl
HCB (fongicide)
PCNB (fongicide)
Camponovo, F. and Hostettmann, K., Schweiz. Apoth. Ztg 16, 524-527 (1999) .
Standardisation
Amlioration de la qualit des prparations de ginseng
Plante cultive dans des conditions de GAP Racines rcoltes aprs 6-7 ans, nettoyes et sches lair Racines sches sont slectionnes et contrles pour leur conformit Racines acceptes utilises pour lextraction
Standardisation
Ginkgo biloba
feuilles: - flavonodes: principalement des mono-, di- et triglycosides de flavonols (querctine, kaempfrol, isorhamntine, myrictine) et de flavones (apignine, lutoline) - biflavones - catchine, proanthocyanidine, alkylphnols, hydrocarbures et drivs - diterpnes (ginkgolides A, B, C, J, M) - sesquiterpnes (bilobalide)
OR2 R1
- glycosides acyls
R2O O
OH
R1= H ou OH R2= H ou Glc

O
OH HO
O O HO O HO O OH
O OH
- biflavones
R1O O HO
OCH3 OR2 O
R1 sciadopitysine ginkgtine isoginkgtine bilobtine CH3 CH3 H H
R2 CH3 H CH3 H
OH
O OH O
Standardisation
Ginkgo biloba
O
- terpnes
O O HO O O H3C O O O O OH R1 O
HO R2
O O R3
ginkgolide A B C J M
R1 OH OH OH OH H
R2 H OH OH H OH
R3 H H OH OH OH
bilobalide
- alkylphnols
- acides
COOH
(CH2)9 CH=CH(CH2) 5CH3
COOH HO
acide caproque
COOH
acide ginkgoque
acide butyrique
Standardisation
Standardisation ginkgo
Standardisation
Analyse HPLC des flavonodes glycosides du Ginkgo
crude ethanolic extract
reference compounds
Standardisation
Ginkgo flavonols
> 24 flavonols dcrits
free radicalscavenging and antioxidant activities
Standardisation
Dosage des flavonodes selon la pharmacope 5
Standardisation
Standardisation d'un extrait: Ginkgo biloba

OH O-sucres OH
Sucres-OHO
O OH
Hydrolyse
HO
O OH OH OH O
O-sucres OH Sucres-O
OH O
HPLC-UV
Profil des aglycones de flavonodes
quercetine
Profil des glycosides de flavonoides

HPLC: Nucleosil 100-C18 3m, gradient: A isopropanol-THF (25:65), B Hasler, A., Sticher, O. and Meier, B., J. Chromatogr. 605, 41-48 (1992). actonitrile, C 0.5% acide phosphorique (diffrentes proportions)
Standardisation
Spectroscopie UV des flavonodes

Spectres UV
OH
isoflavone
Bande II
HO O OH
flavanone ou dihydroflavonol
OH
Bande I
Bande II
Bande I
flavone flavonol
250-280 nm 330-350 nm
250
300
350
400 nm
Standardisation
Dosage des flavonodes

OH
Bande I
HO O OH
250-280 nm
330-350 nm
HO O OH Al3+
AlCl3 + HCl
extraction hydrolyse extraction AlCl3
des glycosides acide des aglycones
+ AcOH -> mesure de labsorption 425 nm bande I
attention les flavonodes qui n'ont pas un OH en 5 ne sont pas doss par cette mthode mais ils sont trs rares
Attention il faudra adapter la mthode si des C-glycosides doivent tre doss
Standardisation

O HO O
HPLC
Reference compounds UV trace (220 nm) GC Bi
Terpnes
O
O O O
OH
GA GB
Bi
O
Bilobalide MW 326
O HO OH O
GC
OH O
OH O
Ginkgolide C MW432 0 10 20 30 min

O
Prepurified leaf extract UV trace (220 nm)
GA
O O
HO
O OH O
GB
Ginkgolide A MW 408
O O
10
20
30 min
O O
HO OH
Conditions:Nucleosil RP-18 (7 m, 250 x 4.0 mm i.d.), MeOH-H2O (25:75 - > 50:50, 30 min), 1 ml/min.
O OH O O
Ginkgolide B MW 424
Standardisation

GC
TMS-O
Bi
O O
Prparation d'chantillon
TMS-O O O
1) MeOH 2%
MeOH 10% Extract
TMS-O O
GC GB
O-TMS O
2) MeOH 5%
Chromabond polyamide
Chromabond C18
5) Hexane/EtOAc (60:40)
4) Hexane
3) Water
GA GJ
Chromabond C18
a) DMF b) BSTFA 1% TMCS c) 115C (50 min)
Injection
10
15
min.
Conditions : Supelco SPB-1 (0.25 m, 30 m x 32 mm i. d.), det 350 C, inj: 250 C, col.: 230-320 C (6 C/min).
Standardisation

LC/MS
HO O O O OH O O O O
100
HO
O O
426 [M+ NH4] +
Ginkgolide A MW 408
m/z 344 ion trace
80 60
O O OH O O
O HO O
100 50
OH
Bi
40 20
O O O O
100
Bilobalide MW 326
O O
GA
50 100 50
m/z 426 ion trace
HO O O
GJ
GB
m/z 442 ion trace
300
350
400
450
scans
O OH O OH O
100 50 3 2 1 10
GC
m/z 458 ion trace

O
O HO OH O
Ginkgolide J MW 424
O O
UV trace (220 nm))
O OH O O
HO OH
O OH O OH O
TIC trace
5 200 400 600 800 scans
Ginkgolide B MW 424
Ginkgolide C MW432
Camponovo, F., Wolfender, J. -L., Maillard, M., Potterat, O. and Hostettmann, K., Phytochem. Anal. 6, 141-148 (1995).
Standardisation
Dose journalire [mg/jour]
7.00 5.00 3.00 1.00 A1 B1 C1 D1 E1 F1 G1 Bi GA G B GC GJ
Phytomdicaments
C1 = extrait standardis Egb 761, sur lequel sont bass les essais cliniques reports dans la littrature
Camponovo, F. and Hostettmann, K., Schweiz. Apoth. Ztg 14, 461-465 (1999).
Te r
Standardisation
Teneur en composs toxiques: Ginkgo biloba
Anacardic acids and alkylphenols present in Ginkgo biloba

Ginkgo biloba leaves and fruits can contain anacardic acids and related alkylphenols. Among these substances, two are potential allergens: ginkgoic acid (I) and ginkgol (II).
COOH HO (CH2)9CH=CH(CH2)5CH3
HO
(CH2)9CH=CH(CH2)5CH3
II
The level of ginkgoic acid for registered preparations available on the market must be below 5 ppm.
p ne s
Standardisation
Teneur en composs toxiques: Ginkgo biloba
LC/ESI-MS analysis of ginkgoic acid in a CHCl3 extract of Ginkgo biloba fruits

100 80 60 40 20 100 80 60 40 20 100 80 60 40 20
m/z= 373.4
100
373.4 [M-H]COOH HO (CH2)9CH=CH(CH 2)5CH 3
80
UV trace
60
mAu
40
Total ion current trace

20
200
300
nm
UV spectrum of ginkgoic acid
10
15
20
25
min
200
300
400
500
600
700
800m/z
HPLC: col: C-18 Symmetry (150 x 3.9 mm i.d., 5 m); gradient: MeCN-H2O 10:90->100:0 in 30 min (0.05% TFA) 1ml/min ESI: capillary temp. 200C, capillary voltage 3.0 V, spray voltage 4.0 kV 14% NH4OH (5 l/min).
Ndjoko, K., Wolfender, J. -L. and Hostettmann, K., J. Chromatogr. B 744, 249-255 (2000).
Standardisation
Allergnes de la familles des Anacardiace
Standardisation
Quantification des acides phnoliques de G. biloba par GC/MS

acides ginkgoliques
cardanols
hydroxycardanol
urushiols et isourushiols
Schoetz K., Phytochem. Anal. (2004) 15, 1-8
Standardisation
Contenu en acides phnols de diffrents lots de feuilles
Standardisation
Ginkgo biloba: prparation des extraits standardiss
Elimination des alkylphnols durant le procd de fabrication
Standardisation
limination des urushiols
Feuilles
Produit fini
Standardisation
Phytoquivalence : dfinitions
Produits quivalents On parle d'quivalence pharmaceutique de deux prparations : si elles contiennent le mme principe actif, dans la mme quantit si elles se prsentent dans la mme forme galnique ayant des spcifications standards identiques ou quivalentes Produits bioquivalents Deux produits sont bioquivalents : si leur quivalence thrapeutique est prouve si les rsultats des essais de biodisponibilit dmontrent qu'ils s'quivalent Rfrences Note for guidance on the investigation of bioavailability and bioequivalence CPMP/EWP/QWP/1401/98 (janvier 2002)
Standardisation
Phytoquivalence : dfinitions
Phytoquivalence - extrait Deux extraits sont phytoquivalents s'il est possible de dmontrer ou de garantir: l'quivalence de la matire premire l'quivalence de l'agent d'extraction l'quivalence du procd d'extraction la reproductibilit des caractristiques de l'extrait (profil chromatographique) Phytoquivalence - produits Deux produits sont phytoquivalents s'il est possible de dmontrer ou de garantir: l'quivalence de la quantit d'extrait contenue dans chaque dose l'quivalence de la quantit de mdicament qui a le mme contenu en substance active (extrait)
seulement dans les cas o il est possible de dmontrer et de soutenir que l'on a une phytoquivalence: les rsultats des tudes cliniques obtenus avec un extrait seront valables aussi pour le deuxime
Standardisation
Phytoquivalence: exemple Produit A: 40 mg extrait de la plante X (thanol 70%) Produit B: 40 mg extrait de la plante X (thanol 90%) Produit C: 40 mg extrait de la plante X (thanol 50%) on peut dmontrer que A et C sont phytoquivalents (caractristiques de l'agent d'extraction similaires, polarit moyenne et profils chromatographiques pareils) alors que dans le cas de B, l'agent d'extraction (plus lipophile) donne des profils chromatographiques diffrents de A et C
Standardisation
Influence de la nature du solvant dextraction sur la composition Contenu en flavonoides de Ginkgo biloba Spcifications pour la standardisation: glycosides de ginkgoflavonols : 24% (22- 27 %)
Standardisation
Influence de la nature du solvant dextraction sur la composition Contenu en terpnoides de Ginkgo biloba Spcifications pour la standardisation: terpene lactones : 6% (5 7 %)
Standardisation
Diffrence de biodisponibilit entre extraits phytoquivalents

All subjects received an oral dose of 120 mg extract under fasted conditions. There was a washout period of at least 1 week between periods 1 and 2. The reference product contains the standardized Ginkgo biloba extract EGb 761, and the test product another standardized extract. As labelled, both are characterized by a content of 24% flavone glycosides and 6% terpene lactones. Both formulations exhibited minor differences in the content of ginkgolide A, ginkgolide B and bilobalide, which were characterized by a method described in an official pharmacopoeia, DAB 2000. The mean plasma levels of ginkgolide A, ginkgolide B and bilobalide shown in Figure1 were obtained after administration of two capsules each of the test and reference product, corresponding to120 mg Ginkgo biloba extract, to twelve volunteers. There were clear differences in terms of extent of bioavailability (as AUC) for the test vs reference product.
Kressmann et al., J. Pharm. Pharmac., 54 (2002)
Standardisation
Diffrence de biodisponibilit entre extraits phytoquivalents
Lquivalence seule de composition entre deux extraits ne suffit pas toujours garantir un effet, la formulation peut galement jouer un rle important sur la biodisponibilit De plus la mtabolisation des ginkgolides na pas t tudie en dtail et la prsence dautres mtabolites actifs nest pas exclure.
Kressmann et al., J. Pharm. Pharmac., 54 (2002)
Standardisation
Hypericum perforatum L. Hypericaceae

- naphthodianthrones (0.1-0.3%)
OH O OH
- flavonols
OH OH
- xanthones (traces)
HO
HO
HO HO CH3 R
OH
OH
OR OH O
OH
OH
OH
R = CH3 hypericine R = CH2OH pseudohyricine
querctine quercitrine isoquercitirine rutine hyproside
R H Rha Glc Rha-Glc Gal
1,3,6,7-tetrahydroxyxanthone
- biflavonodes
HO O
OH
-phloroglucinols
OH O HO O
HO
OH
O
OH O
R= H hyperforine R= CH3 adhyperforine
I3,II8-biapigenin (0.1-0.5%)
Standardisation
Standardisation des phytoprparations de millepertuis
Standardisation
Dosage de lhypricine selon la pharmacope europenne 5: UV simple

UV pseudohypericine
OH O OH
590nm
HO HO
CH3 R
UV hypericine
OH O OH
R = CH3 hypericine R = CH2OH pseudohyricine
DOSAGE Mesurez labsorbance (2.2.25) de la solution examiner 590 nm par rapport au liquide de compensation. Calculez la teneur pour cent en hypricines totales, exprimes en hypricine, laide de lexpression : en prenant 870 comme valeur de labsorbance spcifique de lhypricine. A= absorbance 590 nm, m= masse de la prise dessai, en grammes. CONSERVATION A labri de la lumire.
Standardisation
Profil HPLC des constituants de H. perforatum
270 nm
rutine rfrence
pseudohypricine hypricine
hyperforine
Brolis et al., J. Chromatogr. A 825, 9 (1998)
Standardisation
Standardisation de H. perforatum selon la pharmacope usp 5

Contenu en hypericine pseudohypericine et hyperforine C is the concentration, in mg per mLof USP Oxybenzone RS in Standard solution 1 F is the response factor of the relevant analyte relative to that of oxybenzone and is 1.30 for hypericin (2) and 1.24 for pseudohypericin (1) 0.46 for hyperforin (3) W is the quantity, in g, of St. John's Wort taken to prepare the Test solution rU is the peak area of the relevant analyte obtained from the Test solution rS is the peak area of oxybenzone obtained from Standard solution 1.
5(C/FW)(rU / rS)
Trace UV (270 nm) mAU 250 200 150 100 0
standardisation
3 12
0 20 40
60
Standardisation
Dosage de lhypricine et de lhyperforine par CCM/densitomtrie

Lipophilic constituents Tablets were extracted with 10 mL methanol, filtered and diluted with methanol to 200mL. Hyperforin was used as standard in a concentration of 0.025mg/mL in methanol. Derivatization was performed with a modified Natural Products reagent, which resulted in reproducible visualization of the compounds. . HPTLC plates DIOL F 254 (Merck) 10x10 cm or 20x10cm Toluene - dichloromethane 4:1 Quantities from 50 to 120 ng were precisely analyzed by absorption measurement at 310 nm without derivatization. . .
Sparation de diffrents extraits de H. perforatum sur HPTLC diol
Standardisation
Variation de la composition des fleurs dans 50 spcimens g/flowers min max 3 23 11 64 206 607 11 71 19 61 44 140 15 107 21 112
constituents hypericin pseudohypericin hyperforin I3',II8-biapigenin rutin hyperosid isoquercitrin quercitrin
Balansard et al., J. Pharm. Bel (1985) 40 : 173-177
Standardisation
Stabilit des extraits de Hypericum perforatum par HPTLC
Stabilit Dosage dune substance traceur insuffisante pour valuer la qualit de lextrait. Mesure de lensemble des constituants dans des conditions chormatographiques valides ncessaires
KOLL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Standardisation
Diffrents dosages dhypericine
348 patients, six semaines 3 extraits standardiss 0.17 mg, 0.33 mg et 1 mg d'hypricine extrait utilis: Hyperiforce (Bioforce) effet comparable avec les 3 dosages pas de relation dose-rponse
Hypricine n'est pas le constituant principal responsable de l'effet antidpressif

Lenoir S et al. (1999) Phytomedicine 6, 141-146.
Standardisation
Controverse
importance de l'hyperforine dans l'effet antidpresseur des prparations base de millepertuis
Extrait: Li 160
hyperforine: 2.2-3%
- activit lie l'hyperforine principalement - risque d'interactions mdicamenteuses
HO
Extrait: Ze117
hyperforine: <0.2%
activit lie des composs non identifis pas de problme d'interaction
O
O O
hyperforine
Standardisation
Teneurs en hyperforine de phytomdicaments base de millepertuis
Remotiv Jarsin 300 Neuroplant 300 Texx 300 Felis 425 Futuran Helarium 425 Laif 600
< 0.2 % 2.22 - 3.07 % 3.90 - 4.29 % 1.71 - 3.17 % 1.89 - 3.47 % 0.95 - 1.86 % 0.91 - 3.89 % 1.51 - 2.38 %
Wurglics M et al. (2000) DAZ 140, 3904-3910.
Standardisation
Echinacea angustifolia L. (Asteraceae)

OH O OH Glc O
O
HOOC
O HO C O O HO
O
OH
OH HO
O O
COOH
Rha
OH HO
Acide chicorique
chinacoside
- polysaccharides: homognes et htrognes - polyactylnes - isobutylamides - flavonodes, en particulier glycosides de la querctine et du kaempfrol - acides gras - strols: bta-sitostrol
polyactylnes
O N
isobutylamides
H O N H
- huiles essentielles: humulne, caryophyllne, caryophyllne poxide echinolone

OH
Standardisation
E. pallida : Pharmacope usp

Content of total phenols Solvent, Solution A, Solution B, Mobile phase, and Standard solution 2Prepare as directed for Content of total phenols under Echinacea angustifolia. Standard solution 1Proceed as directed for Content of total phenols under Echinacea angustifolia, except to use USP Powdered Echinacea pallida Extract RS instead of USP Powdered Echinacea angustifolia Extract RS. Test solutionProceed as directed for Content of total phenols under Echinacea angustifolia, except to use finely powdered Echinacea pallida instead of Echinacea angustifolia. Chromatographic system (see Chromatography 621)Prepare as directed for Content of total phenols under Echinacea angustifolia. Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the chromatogram obtained is similar to the Reference Chromatogram for total phenols provided with USP Powdered Echinacea pallida Extract RS. Chromatograph Standard solution 2, and record the peak responses as directed for Procedure: the capacity factor, k, is not less than 3.0; USP28 the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.5%. ProcedureProceed as directed for Content of total phenols under Echinacea angustifolia. Separately calculate the percentages of caftaric acid (C13H12O9), chicoric acid (C22H18O12), chlorogenic acid (C16H18O9), and echinacoside (C35H46O20) in the portion of Echinacea pallida taken by the formula: 2500F(C/W)(ri / rS), in which F is the response factor and is equal to 0.695 for chicoric acid, 0.881 for caftaric acid, 1.000 for chlorogenic acid, and 2.220 for echinacoside; C is the concentration, in mg per mL, of USP Chlorogenic Acid RS in Standard solution 2; W is the weight, in mg, of Echinacea pallida taken; and ri and rS are the peak responses for the relevant analyte obtained from the Test solution and Standard solution 2, respectively. Calculate the percentage of total phenols in the portion of Echinacea pallida taken by adding the individual percentages calculated. Pas de monographie dans Pharmacope Europenne
Standardisation
Standardisation d'un extrait: Echinacea purpurea

Content of total phenols (USP) ProcedureSeparately calculate the percentages of caftaric acid (C13H12O9), chicoric acid (C22H18O12), chlorogenic acid (C16H18O9), and echinacoside (C35H46O20) in the portion by the formula:
OH HO HO HO CH CH O O OH HO COO O O OH O O OH CH2 CH2 OH OH
Compos traceur
H3C HO HO
chinacoside
2500F(C/W)(ri / rS)
F : response factor 0.695 for chicoric acid, 0.881 for caftaric acid, 1.000 for chlorogenic acid, 2.220 for echinacoside; C: concentration of chlorogenic acid (mg/ml) W: weight, in mg, of Echinacea ri and rS are the peak responses for the relevant analyte
Standardisation
Echinacea: validit du compos traceur Composs traceur : Echinacoside Spcificit du traceur : Echinacoside (6 espces dEchinaceae et Cistanche salsa) Polysaccharides (ubiquitaires dans de nombreuses plantes) Acide chicorique (plusieurs espces dAsteraceae, Posidoniaceae, et Pteridaceae)
Crockett SL, Khan IA. Challenges of standardization: marker compounds in plant species related and unrelated to top-selling herbs. Journal of Herbs, Spices & Medicinal Plants. 2003;10(3):1324.
Standardisation
Echinacea: problmatique du jus frais

Dosage de lacide chicorique
UV 330 n m Mg/100 ml
chantillons de jus
La dgradation de lacide chicorique dans les jus frais est lie la prsence de phnoloxydase Les prparations qui prsentent un contenu constant incluent dans le procd de fabrication une procdure de dsactivation des enzymes Ces compos stimule la phagocytose
Bauer, R. J. Herbs Spices Med.Plant.(1999) 6 :51-62
Standardisation
Echinacea: problmatique du jus frais

Dosage des isobutylamides
UV 254 n m Mg/100 ml
chantillons de jus
La concentration dalkylamides dans le jus frais est plus basse que dans la plante sches tant donn leurs caractres lipophiles Ces composs stimulent la phagocytose
Bauer, R. J. Herbs Spices Med.Plant.(1999) 6 :51-62
Dosage des polysaccharides ?
Standardisation
Echinacea: validit du compos traceur
Aucune mthode ne permet la quantification des polysaccharides qui semblent jouer un rle important comme modulateur de la dfense immunitaire Les alkylamides et lacide chicorique ont dmontr des effets pharmacologiques lis la stimulation du systme immunitaire. Ces composs sont sensibles aux changements des conditions dextraction. Lchinacoside lui na dmontr aucun effet. Dans ltat actuel des connaissance une standardisation sur les alkylamides et lacide chicorique semble raisonnable. Une grande prudence quand la stabilit de la composition des jus frais est de mise.
Standardisation
Qualits des phytoprparations: conclusion

Lassurance
de qualit dune phytoprparation est une procdure complexe qui pour tre bien mene devrait idalement tenir compte des paramtre suivants: La slection dune drogue vgtale de qualit Lextraction dans des conditions reproductibles La vrification de labsence de composs toxiques La mesure de la concentration des principes actifs, ainsi que le rapport idal qui doit tre maintenu entre eux (standardisation) La stabilit de la prparation La biodisponibilit des principes actifs La mtabolisation des principes actifs
Seul
un contrle de tous ces paramtres pourra permettre de garantir lefficacit dune prparation en conformit avec les essais cliniques qui ont t effectus.

06 Standardisation de Principios Activos

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06 Standardisation de Principios Activos

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Qualit des phytomdicaments

Cours de Pharmacognosie et Phytochimie

Phytomdicaments: critres de qualit

Cours de Pharmacognosie et Phytochimie

Phytomdicaments: critres de qualit

tests cliniques tests pharmacologiques suivi mdical

identification du matriel vgtal identification des constituants standardisation

composs toxiques teneur en pesticides contamination microbiennes teneur en mtaux lourds

Cours de Pharmacognosie et Phytochimie

Cours de Pharmacognosie et Phytochimie

Cours de Pharmacognosie et Phytochimie

Standardisation des extraits

La standardisation d'un extrait permet d'assurer sa qualit son efficacit sa reproductibilit

Standardisation exige par les offices d'enregistrement des mdicaments

Cours de Pharmacognosie et Phytochimie

Techniques analytiques performantes

Exemple de standardisation partir dun test biologique

Cours de Pharmacognosie et Phytochimie

Standardisation des extraits

Cours de Pharmacognosie et Phytochimie

Panax Ginseng C.A. Meyer (Araliaceae)

R1 R2 glc glc-rha H glc-glc glc glc H glc-rha

Cours de Pharmacognosie et Phytochimie

Dosage des ginsnosides pharmacope EU

Cours de Pharmacognosie et Phytochimie

Standardisation ginseng: Quantits dclares

Cours de Pharmacognosie et Phytochimie

Dosage des ginsnosides selon pharmacope EU

Rb1 Rsolution 1.0

Cours de Pharmacognosie et Phytochimie

Dosage des ginsnosides selon pharmacope EU

Cours de Pharmacognosie et Phytochimie

Rapport de Rb1/Rg1 dans diffrents extraits de ginseng

Ginseng amricain cultiv Rb1

Rb1 Ginseng asiatique Rg1

Cours de Pharmacognosie et Phytochimie

Analyse LC/UV dun mlange de ginsnosides

UV trace (203 nm)

Rg2 Rb1Rc Rb2 Rd

R1 R2 glc glc-rha H glc-glc glc glc H glc-rha

Total analysis time: 80 min

MeCN-water gradient (RP-18 col. 150 mm) 10 20 30 40 50 60 70 min

Cours de Pharmacognosie et Phytochimie

Nouvelle mthode: analyse LC/ES-MS dun mlange de ginsnosides

UV trace (203 nm)

Base peak trace

MeCN-water gradient (RP-18 col. 150 mm)

Cours de Pharmacognosie et Phytochimie

Nouvelle mthode: analyse LC/ES-MS dun mlange de ginsnosides

R1 R2 glc glc-rha H glc-glc glc glc H glc-rha

UV trace (203 nm)

Base peak trace

MeCN-water gradient (RP-18 col. 150 mm)

Cours de Pharmacognosie et Phytochimie

Saponines de Panax ginseng

Glc-Glc-Ma Glc-Glc-Ma Glc-Glc-Ma Glc-Glc-Ma

H H Pseudoginsenosides R1 H H RT3 H R3 H RC1 F8 R1 H

Notoginsenosides R1 R4 Fa Fc Fe Glc-Glc Glc-Glc-xyl Glc-Glc-xyl Glc

Glc Glc Glc-xyl R2 Glc

Cours de Pharmacognosie et Phytochimie

Dosage des ginsnosides par LC/ELSD

chromophore faible dtection UV difficile UV 203 nm

Cours de Pharmacognosie et Phytochimie