ikhlas a. khan national center for natural products research, research institute of pharmaceutical...

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


• 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


• 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


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


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


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

ikhlas a. khan national center for natural products research, research institute of pharmaceutical...

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