IKHLAS A. KHAN
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School
of Pharmacy, The University of Mississippi
Herbal Medicines - Main issuesHerbal Medicines - Main issues
•SafetySafety
•Quality (Authenticity ?)Quality (Authenticity ?)
•EfficacyEfficacy
Biological Biological
EnvironmentalEnvironmental
ChemicalChemical
StandardizationStandardization
Selection of marker compoundsSelection of marker compounds
Adulteration/misidentificationAdulteration/misidentification
Biological contaminantsBiological contaminants
Environmental ContaminantsEnvironmental Contaminants
Heavy metalsHeavy metalsHeavy metalsHeavy metals
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
A B C I L Q T W
NiCdPb
Ni
Cd
Pb
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
A B C I L Q T W
NiCdPb
Ni
Cd
Pb
Pesticides (quintozene) Pesticides (quintozene) Pesticides (quintozene) Pesticides (quintozene) 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
ppm
A B C D E F G H I J K L M N O P Q R S T U V W X
In ginsengIn ginsengproductsproducts
In ginsengIn ginsengproductsproducts
Concentration Range (ng/g) of DDT and its Concentration Range (ng/g) of DDT and its Metabolites in Select Botanical SupplementsMetabolites in Select Botanical Supplements
Valarian Hypericum P. Flower Echinacea
O,P DDT ND ND-19.3 ND-31.2 ND
P,P DDT ND-12.9 ND-7.0 ND-8.9 ND
O,P DDE ND-5.1 ND-9.6 ND-4.1 ND
P,P DDE ND-4.9 ND-8.5 ND-3.6 ND-3.4
O,P DDD ND ND-4.7 ND ND
P,P DDD ND-4.4 ND-10.9 ND ND-17.1
Heavy Metals - ComparisonHeavy Metals - Comparison
NO P Q R S T
W X
Ni
Cd
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
NipbCd
USA Europe China
Herbal Sexual EnhancersNot So Herbal!
0
10
20
30
40
50
60
70
80
90
100
mg
H4 H5 H6 H11 H12 H15 H16 H25 H26 H27 H28 H30 H31 H32 H33 V
Product
Sildenafil Quantity per Capsule or Tablet
Chemical SafetyChemical Safety
Aristolochia Aristolochia macrophyllamacrophylla
•Aristolochic acid I and II present in Aristolochic acid I and II present in AristolochiaAristolochia appear to be responsible for appear to be responsible for Chinese herbs nephropathy (CHN).Chinese herbs nephropathy (CHN).
•Renal interstitium, mutagenic, and Renal interstitium, mutagenic, and carcinogenic activitycarcinogenic activity
•Snakebites, rheumatism, as a diuretic Snakebites, rheumatism, as a diuretic and analgesic.and analgesic.
Aristolochic AcidAristolochic Acid
AristolochiaAristolochiaA. acuminataA. argentinaA. baeticaA. bracteataA. chilensisA. cinnabarinaA. clematitisA. contortaA. cymbiferaA. debilis3
A. elegansA. esperanzaeA. fangchi3
A. fimbriataA. indica
A. kaempferiA. kwangsiensisA. macrophyllaA. manschuriensis3
A. maurorumA. maximaA. mollissimaA. pistolochiaA. rigidaA. rotundaA. serpentariaA. watsoni(i)A. westlandi(i)A. zollingeriana
AsarumAsarumA. canadense2
A. himalai(y)cum3
A. splendens3
A. forbesii3
A. heterotropoides3
A. sieboldii3
AkebiaA. quinata3
A. trifoliata
ClematisC. armandiiC. chinensisC. hexapetalaC. montanaC. uncinata
CocculusC. carolinusC. diversifoliusC. hirsutusC. indicusC. laurifoliusC. leaebeC. madagascariensisC. orbiculatusC. palmatusC. pendulusC. thunbergii
Bragantia wallichiiDiploclisia affinisDiploclisia chinensisMenispernum dauricumSaussurea lappaSinomenium acutum3
Stephania tetrandra3
Vladimiria souliei3
Other species listed
FDA Listing of plants known or suspected of containing aristolochic acids(updated April 9, 2001)1
1. http://www.cfsan.fda.gov/~dms/ds-bot2.html2. Doskotch, R.W. and Vanevenhoven, P.R. Lloydia 1967, 30(2), 141.3. Hashimoto, K.; Higuchi, M.; Makino, B.; Sakakibara,I.; Kubo, M.; Komatsu, Y.; Maruno, M.; Okada, M. J. of Ethnopharmacology 1999, 64, 185.
AAI or A R1, R2, R3, R5 = H; R4 = OCH3; R6 = OH 341AAIa R1, R2, R3, R5 = H; R4 = OH; R6 = OH 327AAII or B R1, R2, R3, R4, R5 = H; R6 = OH 311AAIII R1, R3, R4, R5 = H; R2 = OCH3; R6 = OH 341AAIIIa or C R1, R3, R4, R5 = H; R2 = OH; R6 = OH 327AAIV R1, R3, R5 = H; R2 = OCH3; R4 = OCH3; R6 = OH 371AAIVa or D R1, R3, R5 = H; R2 = OH; R4 = OH; R6 = OH 357AAVa R1, R4, R5 = H; R2 = OH; R3 = OCH3; R6 = OH 357AAVIa R1, R2, R3 = H; R4 = OCH3; R5 = OH; R6 = OH 357AAVIIa R1, R2, R5 = H; R3 = OH; R4 = OCH3; R6 = OH 357AA E R1, R2, R5 = H; R3 = OCH3; R4 = OH; R6 = OH 357AAI methyl ester R1, R2, R3, R5 = H; R4 = OCH3; R6 = OCH3 355AAIa methyl ester R1, R2, R3, R5 = H; R4 = OH; R6 = OCH3 384AAII methyl ester R1, R2, R3, R4, R5 = H; R6 = OCH3 325AAIII methyl ester R1, R3, R4, R5 = H; R2 = OCH3; R6 = OCH3 355AAIIIa-6-O--O-glucoside R1, R3, R4, R5 = H; R2 = Ogluc; R6 = OH 488AAVII methyl ester R1, R2, R5 = H; R3 = OCH3; R4 = OCH3; R6= OCH3 386
O
O
R1
R2
R3
R4
COR6
R5
NO2
O
O
R1
R2
R3
R4
COR6
R5
NO2
MWAristolochic Acids
Chromatogram Comparison of Purchased Chromatogram Comparison of Purchased Aristolochic Acid StandardsAristolochic Acid Standards
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.000
0.010
0.020
0.030
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.000
0.010
0.020
0.030
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.00
0.05
0.10
0.15
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU0.00
0.02
0.04
0.06
0.08
0.10
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
1
2
3
4a*
4b*
*Same company, different batch number
20%70%
25%72%
24%73%
2%
97%
65%
28%
Chromatograms of Chromatograms of Asarum canadenseAsarum canadense Populations Collected Populations CollectedAU
0.00
0.10
0.20
0.30
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.10
0.20
0.30
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
59 IN: Perry Co.59 IN: Perry Co.
AU
0.00
0.50
1.00
1.50
2.00
2.50
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.50
1.00
1.50
2.00
2.50
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
62 IN: Crawford Co.62 IN: Crawford Co.
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
68 NC: Swain Co.68 NC: Swain Co.
AU
0.00
0.10
0.20
0.30
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.10
0.20
0.30
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
77 SC: Berkeley Co.77 SC: Berkeley Co.
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
88 NY: Cattaraugas Co.88 NY: Cattaraugas Co.
AU
0.00
0.05
0.10
0.15
0.20
0.25
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
AU
0.00
0.05
0.10
0.15
0.20
0.25
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
89 OH: Morgan Co.89 OH: Morgan Co.
•Standardization
•Selection of maker compound/fingerprinting
•Adulteration /misidentification
StandardizationStandardization
• ACTIVE COMPOUNDSACTIVE COMPOUNDS
• MARKER/REFERENCE COMPOUNDSMARKER/REFERENCE COMPOUNDS
Basic needsBasic needs
Standardization Standardization
DIRECT APPLICATIONSDIRECT APPLICATIONS
FUTURE DEVELOPMENTSFUTURE DEVELOPMENTS
• BIO-ANALYSISBIO-ANALYSIS• CULTIVATIONCULTIVATION• GENETICSGENETICS• ENVIRONMENTALENVIRONMENTAL
ANALYTICSANALYTICS
StandardizationStandardization
• TO DEVELOP ANALYTICAL METHODSTO DEVELOP ANALYTICAL METHODS PRECISION, ACCURACY, SPECIFICITY, LINEARITY, PRECISION, ACCURACY, SPECIFICITY, LINEARITY,
RUGGEDNESS, ROBUSTNESSRUGGEDNESS, ROBUSTNESS
• TO DEVELOP FINGERPRINTSTO DEVELOP FINGERPRINTS
• SEMIPREP AND PREPARATIVE ISOLATIONSEMIPREP AND PREPARATIVE ISOLATION
DIRECT APPLICATIONSDIRECT APPLICATIONS
StandardizationStandardization
We would like to have a methodWe would like to have a method
Applicable to all sort of preparationsincludes all the desirable markerscan be used for quality controlcan be used for efficacy measurement
BUTBUT
Don’t know the nature of a preparationDon’t know the marker compoundsDon’t know the active compoundDon’t know about the variability of the extract.
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Minutes
5.00 10.00 15.00 20.00 25.00 30.00 35.00
8.3
85
rutin -
11.7
36
hy
pero
sid
e -
12.4
55
iso
querc
itrin
- 1
3.0
98
querc
itrin
- 1
5.2
21
querc
etin
- 1
8.2
99
I3, II8
bia
pig
en
in -
20.3
59
am
ento
fla
vone
- 2
0.7
19
pseud
ohy
peric
in -
26.7
75
hy
peric
in -
31.3
53
hyp
erf
orin
- 3
2.2
43
200 nm
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
GinsengGinseng
•Panax ginsengPanax ginseng and and P. P. quinquefoliumquinquefolium (Araliaceae) (Araliaceae)
•Saponins (ginsenosides/ Saponins (ginsenosides/ panaxosides)panaxosides)
•ADAPTOGENADAPTOGEN: tonic, : tonic, immunostimulant, immunostimulant, aphrodisiac. Also in high aphrodisiac. Also in high cholesterol, heart attacks, cholesterol, heart attacks, diabetes, cancer, liver diabetes, cancer, liver protection, radiation therapy, protection, radiation therapy, and loss of appetitecand loss of appetitec
Nomenclature of GinsengNomenclature of Ginseng
SCIENTIFICSCIENTIFIC COMMONCOMMON (Ginseng) (Ginseng)
Panax quinquefoliumPanax quinquefolium American, Occidental, CanadianAmerican, Occidental, Canadian
Panax trifoliusPanax trifolius Dwarf Ginseng, GroundnutDwarf Ginseng, Groundnut
Panax GinsengPanax Ginseng Oriental, Chinese, Korean, Red, Oriental, Chinese, Korean, Red,
WhiteWhite
Tartary, KirinTartary, Kirin
Panax notoginsengPanax notoginseng Sanchi, Tienchi, SanchuSanchi, Tienchi, Sanchu
Panax japonicusPanax japonicus Japanese, ChikuJapanese, Chiku
Panax pseudoginsengPanax pseudoginseng HimalayanHimalayan
Panax elegantiorPanax elegantior Pearl Pearl
Eleutherococcus Eleutherococcus Siberian, Russian, EleuthroSiberian, Russian, Eleuthro
sentocosussentocosus Touch-me-notTouch-me-not
Devil’s BushDevil’s Bush
Misnamed GinsengsMisnamed Ginsengs
BRAZILIAN GINSENGBRAZILIAN GINSENG SUMA (SUMA (Pfaffia paniculataPfaffia paniculata))
INDIAN GINSENGINDIAN GINSENG ASHWAGANDA (ASHWAGANDA (Withiana somniferaWithiana somnifera))
ALASKAN GINSENGALASKAN GINSENG DEVIL’S CLUB (DEVIL’S CLUB (Oplopanax horridumOplopanax horridum))
AMERICAN RED AMERICAN RED CANIAGRE (CANIAGRE (Rumex hymenosepalusRumex hymenosepalus))DESERTGINSENGDESERTGINSENG
St. John’s WortSt. John’s Wort
•Hypericum perforatum Hypericum perforatum (Hypericaceae)(Hypericaceae)
•Naphthodianthrones Naphthodianthrones (hypericin, pseudohypericin, (hypericin, pseudohypericin, hyperforin), flavonoidshyperforin), flavonoids
•In mild depression In mild depression
Analysis of rutin (1), hyperoside (2), isoquercitrin (3), quercitrin (4), quercetin (5), I3,II8 biapigenin (6), pseudohypericin (7), hypericin (8) and hyperforin (9)
in different H. perforatum products
0
2000000
4000000
6000000
8000000
10000000
12000000
Methanol Methanol + 1%Pyridin
Methanol + 3 %Pyridin
Methanol + 20 %H2O
Methanol + 40 %H2O
area
1 2 3 4 5 6 7 8 9
Variations in the peak area of 1-9, if one H. perforatum sample (P-N-6) is extracted with different solvents
ExtractionExtraction
Products AnalysisProducts Analysis
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
P-N-1 P-N-2 P-N-3 P-N-4 P-N-5 P-N-6 P-N-7 P-N-8 P-N-9 P-N-10 P-N-11 P-N-12
perc
enta
ge
1 2 3 4 5 6 7 8 9
Percentage (g/100 g) of flavonoids (1-6), naphthodianthrones (7,8) and hyperforin (9) in products
TribulusTribulus
O
O
O
O
OOHO
HO OH
OO O
HOOH
OHO OH
HOOH
O
HO OH
HO
1
3 5 7
1 9 1 1
1 4
1 7
1 8
3
O
O
O
OOHO
HO O
OO O
HOOH
OHO OH
HO
O
HO OH
HO
OHO
HO OH
2 1
1
3 5 7
1 9 1 1
1 4
1 7
1 8
2 1
4
O
OO
OH
OHO
OOH
OH OH
OOH
OH
OHOH
O
OO
OHOH OH
OH
1
3 7
1 9 1 1
1 4
1 7
1 8
2 1
2 5
1 '1 ' ' '
1 ' '
1 '' ' '
2 7
1
O
O
O
OOHO
HO O
OO O
HOOH
OHO OH
HO
OHO OH
HO
OHO
HO OH
OHO
OH
OHOH
O
1
3 5 7
1 9 1 1
1 4
1 7
1 8
2 1
2
mV
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
1
2
3
4
Indian sample
mV
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
mV
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
mV
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
1
1
0
1
2
3
4
5
6
7
percent
Bulgaria- leave
Bulgaria- fruit
China -fruit 1
China -fruit 3
product2
1
Market products
mV
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
mV
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
mV
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
1
1
1
leave
fruit
stemm
V
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
mV
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
1T. terrestris(Utah)
T. cistoides(Florida)
Bulgarian sampledifferent plant parts
shoots of 2 species
ON
NH
O
O OCH3
CH3
7 3 15
19
20
Speciophylline (1) 3R, 7S, 15S, 19S, 20S
Uncarine F (2) 3R, 7R, 15S, 19S, 20S
Mitraphylline (3) 3S, 7R, 15S, 19S, 20R
Isomitraphylline (4) 3S, 7S, 15S, 19S, 20R
Pteropodine (5) 3S, 7R, 15S, 19S, 20S
Isopteropodine (6) 3S, 7S, 15S, 19S, 20S
AU0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Uncaria tomentosa
Selection of Marker Compounds Selection of Marker Compounds
• Tanacetum partheniumTanacetum parthenium L., L., AsteraceaeAsteraceae
• For migraine prophylaxisFor migraine prophylaxis
• ParthenolideParthenolide is the major is the major component (active constituent ?)component (active constituent ?)
O
O
OO
O
O
ParthenolideParthenolide
Abourashed & Khan, JAOAC, 2000.
AU
0.00
0.02
0.04
0.06
0.08
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
1.1
97
1.4
31
1.7
65
2.6
37
3.1
15
3.8
67
4.1
24
4.4
51
part
henolid
e -
7.9
42
10.3
22
14.6
50
16.9
15
17.1
93
17.6
81
18.3
47
19.3
52
19.8
67
leaf In MagnoliaIn Magnolia
In FeverfewIn Feverfew
ParthenolideParthenolide
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
FF1 FF2 FF3 FF4 FF5 FF6 FF7 FF8
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
FF1 FF2 FF3 FF4 FF5 FF6 FF7 FF8
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
FF1 FF2 FF3 FF4 FF5 FF6 FF7 FF8
Cost: Dollars/day
Parthenolide: mg/day
Quality Plant Material
Well-Characterized:BotanicallyChemically
Pharmacologically
Toxicological Evaluation
Clinical Studies
Drug Interactions
FormulatedProducts
Stability
New Crops
New Indications