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Standardization, Bioavailability,Standardization, Bioavailability,Pharmacodynamics, Pharmacodynamics,

Pharmacokinetics, etc.Pharmacokinetics, etc.

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Plant material

Directly Extract

Fresh Dried

IngestedTopically

Powdered Comminuted

jarboxtisan

Herbal teaStandardpowder

Coarse powder

Herbal tea

capsuletablet

EtOH H2OH2O/EtOH

Aceton

concd.

Standardizeddried extract

TeintureExtraitfluide

tablet/capsule

instant tea

ointment

injection

Syrup

Drops

Solution

Only “Standardized vegetable

drugs or extracts” can find a place in Current drug concept

I. Analytical StandardizationII. Clinical Standardization

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Standardization

1. Bioequivalence Biological effects of two plant

extracts/formulations are clinically found identical in pharmacologic and biochemical analysis.

Bioequivalence is necessary for similar pharmacodynamic, pharmacokinetic and bioavailability features of a medicine.

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The results from pharmacological and clinical trials with one HMP could thus not automatically be transferred to another preparation.

2. Phyto-equivalence: Analytical composition of two plant extracts are

chemically found identical in quantitative analysis.

The use of different conditions, extraction solvents, and purification steps resulted in extracts of different quality.

Phyto-equivalence is not a satisfactory proof for biological/pharmacological effects; needs further bioequivalence studies in small groups.

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Active component(s) is used for analytical

standardization Ginkgo biloba (Ginkgo) prep.

Compound for standardization: Ginkgolides or flavonoids(Tebokan®, Ginkgobil®, etc.)

Senna leaves

Compound for standardization: Sennoside B

(Pursenid®, Roha®, Bekunis®, Senekod®, XM® etc.)E. Yeşilada 7

Standardization: A. If the Active component(s) is

known

a. “Marker component(s)”

Standardization may be achived by using any plant constituent(s), even the role on the claimed activity is unknown: Valeriana (Valerian; kediotu) root

valerianic acid (a sesquiterpene)

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Standardization:

B. Unknown active constituents

”Chromatographic identity”

of a certain extract is determined by using: Thin-layer chromatography (TLC) Gas-liquid chromatography (GLC); for volatile

compounds High performance liquid chromatography (HPLC) High performance thin-layer chromatography (HP-

TLC)

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C. “chromato-fingerprinting”

10

TLC or HP-TLC fingerprinting of Hawthorn

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Saw Palmetto (Serenoa repens)Benign Prostate Hyperplasia (BPH)

treatment

–Gas-liquid chromatography fingerprinting of Saw Palmetto

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High performance liquid chromatography fingerprinting of EGb761 (Ginkgo biloba)

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BioavailabilityBioavailabilityPharmacokineticPharmacokinetic

PharmacodynamicPharmacodynamic

Number of Clinical evidences are few! The evidence has been mainly from

Ethnomedical knowledge, Anecdotal or hearsay.

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Evidence of efficiency for HMPs

Phytochemicals may interfere with meals;

Polyphenolics should be taken away from meals because of their interaction with proteins.

Components relying on gastric acid hydrolysis should be taken with meals,

Components damaged by gastric acid should be taken away from meals.

Some foods may interfere the metabolism of drugs; e.g. Grapefruit juice, etc.

The frequency of dosage should be adjusted based on the bioavailability and metabolism data of the drug.

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Optimizing Efficacy

HMPs are not directly introduced into the

bloodstream by parenteral injection & Oral or topical routes of administration are

preferred for HMPs; subjected to a series of metabolic processing

before absorption from GI system. Conventional drugs are designed to have

good bioavailability on oral application, HMPs may exhibit unusual and poor

bioavailability,E. Yeşilada 16

Bioavailability of HMPs

Protecting the active components from

structural transformation to inactive metabolite,

Increasing the absorption of the active compound(s) from digestive system,

Sharing the activity: “Synergy” Reducing the unwanted effects

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Some components in the blend may potentiate the Bioavailability of the drug

In plant material/extract the biological

effect may be achieved by synergistic interactions;

Separation of the active ingredients may lead to reduced efficiency or lost in activity.

e.g. Ginkgo, Kava

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1.“Synergistic interaction” of several components in a herbal extract:

A mixture of pure Ginkgolides A, B and C at a

dose of 100-240 mg can generate a PAF-antagonizing effect in humans.

Same efficiency may be provided with a dose of 120 mg of a “Standardized Ginkgo extract” containing only 6-7 mg of ginkgolides together with bilobalide and flavonol glycosides.

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Ginkgo biloba

Active fraction contain;“

a-zingiberen, b-sesquiphellandrene, bisabolene and curcumene,

Constitutes only a small portion of the active fraction;

The effect was x6 higher than calculated from a summation of the individual ingredients.

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Ginger, Zingiber officinalis,as antiulcer

Product: Ginseng/Ginkgo combination Study: A double-blind crossover trial in 20 young

healthy volunteers, Effect: Improving cognitive function Result: More effective than either alone

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“Synergistic interaction ”: Combination of herbal extracts may potentiate the

efficiency

Scholey AB, Kennedy DO, 2001: Acute, dose-dependent cognitive effects of Panax Scholey AB, Kennedy DO, 2001: Acute, dose-dependent cognitive effects of Panax ginseng and Ginkgo biloba and their combination in healthy young volunteers: ginseng and Ginkgo biloba and their combination in healthy young volunteers: differential interactions with cognitivedemand. Human Pyschopharmacology.differential interactions with cognitivedemand. Human Pyschopharmacology.

Removal of some pharmacologically inactive constituents, but involving in pharmacokinetic and/or pharmacodynamic interactions may lead to

LOST IN ACTIVITYdue to diminishing rate of absorption of the active constituents.

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2.Some constituents may regulate absorption or metabolism rate

Higher

Bioavailabilityis observed for

Ascorbic acidin citrus fruits

(orange,lemon, mandarin, etc.) then in formulations

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Postmenaposal Syndrome estrogenic activity

isoflavon glycosides “daidzin”’

In extract > pure daidzin

Higher plasma concentration

Saponins (a type of plant constituents);

improve absorption and/or solubilization due to higher surface activity characteristics,

St.John’s wort (Hypericum) an antidepresant HMP; procyanidins/flavonoids increase the absorption of active constituents (hypericin/hyperforin):

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Antibacterial components;

geranial and neralAnother component myrsene’ possesses

no antibacterial activity, but increase the effect of other two components

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Lemongrass volatile oil (Cymbopogon sp.)

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Antioxidants, may protect the active ingredients from oxidative decomposition: Pure Hyperforin (active principle of St.John’s

wort) easily decomposed by oxidation when isolated,

Antioxidant “procyanidins” in the crude extract protect the compound from oxidative decomposition.

Therefore “St.John’s wort” crude extract is used in HMP formulations.

3. Some constituents may help protection of the unstable active constituents from

chemical degradation to inactive metabolites:

4. “Preactivation” may be required to activate the

constituent before administration:

Some plant ingredients are in “PRODRUG” form and they should be converted into ACTIVE FORMS before administration.

In “Garlic” -“Alliin” is an inactive precursor or “prodrug” - Enzymatically (allinase) converted to active metabolite “allicin” when the cells are crushed.

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Activity may be lost due to heat or sunshine: Volatile components may be lost (volatile

oils). Volatile constituents of Mint leaves

evaporated if dried under sunshine or in an oven.

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5. Activity may be lost during processing

or storage

Activity may have lost critically due to structural decomposition: Heat decompose proteic

components/enzymes /sulfur-containing: In Broccoli, heat decomposed the enzyme

(myrosinase) needs to convert glucoraphanine into active metabolite sulfurophane.

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5. Activity may be lost during processing

or storage

Activity may be lost due to moisture: Moisture may initiate the enzymatic

reactions to cause inactivation of the active molecule.

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5. Activity may be lost during processing or storage

Removal of “Buffer constituents” may

increase the risk of HMP:

Advers effects/or Contrindications/or Toxicity.

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6. Buffer constituents may prevent the undesirable

effects

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Chemical structure of the ingredients may

be subjected to change By saliva By gastric juice (pepsin/gastric acid) By bile acids By pancreatic juice By intestinal flora

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Changes induced by hepatic drug-metabolizing

enzymes Binding of active ingredients to their receptors

and to plasma albumin Changes in the chemical structure and

concentration of active ingredients in the blood and excretion into the urine, bile and feces.

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On oral administration: I. After ingestion components

Pass through the GI tract Metabolized by the enzymatic action of

intestinal bacterial flora Absorbed into the blood.

II. Some components are directly absorbed Detoxified in the liver Excreted in the bile to interact with intestinal

flora for biotransformation, Then reabsorbed

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Plants have complex chemical composition;

Potential interactions between the plant constituents: synergistic, antagonistic,

Mixture of different compounds in the HMP with different bioavailability,

Active components are often not known or activity is shared by several components, therefore the component(s) in the extract can not be targeted,

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“Herbal Pharmacokinetics“ extraordinarily complex to study

Prodrug;

Natural compounds are often metabolized in the digestive tract;

Thus pharmacokinetic characteristics are not predictable,

Large molecules are often involved, which might be expected to have poor and unpredictable bioavailability.

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“Herbal Pharmacokinetics“ extraordinarily complex to study

Solubility characteristics of the components:

Liposolubility of the molecule increase the bioavailability,

H2O-soluble molecules can be expected to have poor bioavailability,

Ionization of the molecule usualy means poor bioavailability,

A molecule possessing both H2O and fat soluble parts will exhibit very good bioavailability; Dissolve in digestive juices and then cross lipid

membranes.E. Yeşilada 39

Key issues pertaining the bioavailability

Effect of liposolubility on bioavailability

Cardiac glycoside Partition between H2O and octanol

Bioavailability (%)

g-Strophanthin 0.01 6.6

Convallatoxin 0.33 13.6

Digoxin 18.2 26.4

Digitoxin 70 74.9

Oleandrin 338 86.0

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Size of the molecule; A small portion of relatively large

molecules, i.e. Saponins, tannins, polysaccharides, proteins, will be absorbed.

Even very large molecules may still have some bioavailability (<1%); pinocytosis.

Specific factors for crossing the gut wall; active transportation,

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Key issues pertaining the bioavailabilityKey issues pertaining the bioavailability

Factors involved within the gut;

Interaction with food, Interaction with the pharmacotherapy, Stability in the gut, Gastric emptying rate,

Individual factors of the patient: Influence of personal pathological

factors. Genetical factors: absence of

particular drug metabolizing enzymes,

Age and gender. E. Yeşilada 42

Key issues pertaining the bioavailabilityKey issues pertaining the bioavailability

The type of pharmaceutical preparation: Infusions and decoctions extract H2O-soluble

components, Many of these polar components will have poor

bioavailability, They are inferior preparations for extraction and

delivery of herbal constituents. HMPs rely heavily on aqueous preparations; Infusion of VO-containing plant;

VO will be collected on the surface of the hot H2O.

Addition of saponin-containing herbs to the mixture will increase the solubility of H2O-insoluble compounds, which may then have better bioavailability.E. Yeşilada 43

Key issues pertaining the bioavailabilityKey issues pertaining the bioavailability

Metabolism in the gut and first-pass

metabolism by the liver, H2O-soluble components may subjected to

structural changes in the GI tract; Glycosides are converted to aglycones in

the caecum and large bowel, will render them more liposoluble and increase bioavailability.

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Key issues pertaining the bioavailabilityKey issues pertaining the bioavailability

A number of plant glycosides are modified

By the action of gastric acid or By the alkaline conditions of duodenum. By the intestinal flora of bowel.

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GLYCOSIDES and Gastric modification

Glycosides:

H2O- soluble Poorly absorbed in the intestines Low bioavailability; it does not mean low activity????

Metabolic transformation of glycosides In the Stomach:

If resistant to gastric acid and digestive enzymes, pass unabsorbed through the upper intestinal tract.

In the intestines: Retained in the lower GI tract Enzymatic transformation: Mostly hydrolyzed to the

corresponding aglycones by intestinal anaerob bacterial glycosidases

Then absorbed slowly and continuously to exhibit pharmacological activities.

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Pharmacodynamics of glycosides

Present in many phytomedicines and foods

They may have a role to play in increasing the biological activity of other compounds by synergistic or other mechanisms:

Antimalarial compound Artemisinin (sesquiterpenoid), from Artemisia annua, activity enhanced by the presence of the flavonoids “armetin” and “casticin”.

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GLYCOSIDES: Flavonoids

Only <20% of the administered glycoside will

generally be absorbed as the intact aglycone. Levels of a flavonoid aglycone in the blood

stream will vary depending on; Flavonoid form; aglycone or glycoside,

Flavonoid glycosides are prodrug form The nature of the individual bowel flora;

Partialy dependent on the individual diet.

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Flavonoids

O

OH O

OH

A C

B

Flavonoid

HO

O

OH

B

m-hydroxyphenylpropionic acid

C-FISSION

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Oral doses of flavonoid aglycones are less bioavailable than their glycosides,

They are more susceptible to “C-ring fission” by the bowel flora,

FLAVONOID-O-GLUCOSIDE

FLAVONOID AGLYCONE

Too large and polar to beabsorbed

Hydrolysis of -linkageby bacterial enzymes

in bowel

can be absorbed

ring fission by bacterialenzymes

Absorption into the bloodstream Smaller ring fission products readily absorbed

Complete breakdown to CO2

Absorption into the bloodstream

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Pharmacokinetics of flavonoid glycosidesPharmacokinetics of flavonoid glycosides

Quercetin, rutin:

3,4-dihydroxyphenylacetic acid 3-methoxy-4-hydroxyphenylacetic acid, m-hydroxyphenylacetic acid,

Hesperidin, diosmin, eriodictyol: m-hydroxyphenylpropionic acid, m-coumaric acid (rat) 3-hydroxy-4-methoxyphenylhydracrylic acid (human),

(+)-Catechin: -hydroxyphenyl--valerolactones m-hydroxyphenylpropionic acid m-hydroxybenzoic acid m-hydroxyhippuric acid

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Flavonoid ring metabolites after fission

Baicalin

Flavonoid from Scutellaria roots Antiallergic, stimulate bile secretion Baicalin is not directly absorbed Circulating conc. of “baicalin” was

found higher than “baicalein glucuronide”

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In particular cases different pharmakokinetic ways may be observed

O

OOH

HO

OO

OOH

HO

HOGlcUA

Baicalein (B)Baicalin (BG)

Intestine Intestinal cell Blood circulation

BG B B BG BG

Intestinal flora -glucuronidase UDPG-glucuronyl

transferase

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Affinity to oestrogen

receptors; Soy beans: Soja hispida

daidzin (daidzein), genistin (genistein)

Red clover: Trifolium pratense formononetin, biochanin A formononetin is converted to

daidzein

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GLYCOSIDES: Isoflavones

O

O

RO

OHR1

R=R1=H Daidzein

R=Glu R1=H DaidzinR=H R1=OH Genistein

R=Glu R1=OH Genistin

ORO

OHR1

EQUOL

bowel flora

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Isoflavones are converted by the bowel flora to “EQUOL” which possesses stronger oestrogenic activity than its precursor.

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Lactococcus garvieae

Erdem Yeşilada

Soy isoflavones are degradated up to 85% in the intestines.

Differences in the fecal flora account for the differing metabolism of soy isoflavones.

Faecal flora could completely degradate isoflavonoid (genistein and daidzein).

Differences in faecal excretion of isoflavones profoundly altered isoflavone bioavailability:

Higher faecal excretion is correlated with higher bioavailability. Such subjects may have fewer bacteria which degrade isoflavones, leaving more intact for absorption.

Bioavailability varied from 13 -35% depending on the individual gut microflora.

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Soy protein: contains isoflavone glycosides;

increase follicular phase length in women,

Miso: contains isoflavone aglycones: Not affect.

Suggests that the glycosidic group delays the degradation of isoflavones, resulting in higher bioavailability of their aglycones or equol.

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Urinary excretion rates of daidzein and 2 metabolites after soya administration over 3 days

Mean excretion (mol)/3 days

Metabolite

Low equol producers< 8 mol equol (n=8)

High equol producers< 25 mol equol (n=4)

Daidzein

23.05 (12.43) 14.95 (6.69)

Equol 1.53 (2.60) 64.89 (59.23)

O-Dma 21.72 (17.93) 6.97 (6.47)

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CH2OH

O-Glu

Salicin

CH2OH

OH

COOH

OH

Salicyl alcohol Salicylic acid

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GLYCOSIDES: Phenolic glycosides Salicin from Salix ssp. Barks

BODY FLUIDSGUT

SalicinSalicortinTremulin

Tremulacin

stomachor small intestine

Salicin

intestinal bacteria(colon or distal column)

Salicyl alcohol(Saligenin)

Salicin

Salicyl alcohol

Salicinsmall intestine excretion

large intestine excretion (4%)Salicyl alcohol

enzymes inblood and tissue ?

oxidation in blood,tissue and liver

excretion (12%) Salicylic acid

Hepatic biotransformation

Salicylic acidconjugates

Salicylic acidconjugates

excretion (65%)

Gentisic acidexcretion (5%)

Gentisic acid

URINE(Over 24 h)

Salicylic acid

E. Yeşilada 62Pharmacokinetics of salicinPharmacokinetics of salicin

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Relative bioavailability of salicin and salicylic acid

salicin has a greater half-life

Devil’s claw: Harpagophytum procumbens, Harpagoside; Oral anti-inflammatory activity, In stomach; gastric acid convert to

“harpagogenin”, possess no anti-inflammatory activity,

Suggested to Use enteric coated tablets or Administer between meals to optimize the bioavailability.

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GLYCOSIDES: GLYCOSIDES:

IridoidsIridoids

Laxative effect Orally: in equivalent doses

only glycosides active, takes 6-8 h to exert laxative effect, effective dose often varied from person to

person, aglycones not active; broken down or absorbed

before reaches the colon

Injection: in equivalent doses aglycone was more active,

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GLYCOSIDES:Anthraquinone Glycosides

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Sinameki (Senna) leaves Active ingredients: Active ingredients:

antraquinonesantraquinones; ; Sennoside ASennoside A andand CC showed showed similar degree of laxative similar degree of laxative effect in mice,effect in mice, In leaves they are in the In leaves they are in the

ratio of ratio of 7:37:3 which shows which shows 2x2x higher activityhigher activityFormulations (Formulations (Pursenid®, Senekot®, Pursenid®, Senekot®,

Bekunis®, Roha®Bekunis®, Roha®) are prepared using the ) are prepared using the standardized extractstandardized extract

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Salvia divinorum Hallucinogenic: fresh leaves are chewed, Salvinorin A (1) [active] is converted to

salvinorin B (2) [inactive] by gastric acid. Chewing; provide absorption from oral

mucosa, without decomposition.

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GLYCOSIDESGLYCOSIDES: : DiterpenoidsDiterpenoids

Saponins are good “prodrugs” Small intestines:

Due to water solubility not absorbed, Pass to large intestines;

Converted by the gut flora to the sapogenins, Sapogenins are lipophylic and absorbed to

some extent.

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SaponinsGLYCOSIDES: GLYCOSIDES: Triterpenes glycosidesTriterpenes glycosides

Saponin type compounds increase the gastro-intestinal absorption rate of other components in the multi-component drugs.

Saponin-containing plants i.e., licorice (meyan kökü), were used in Oriental Medicines in order to increase absorption of components, Co-ordinate the effect of components

in formulations, like a “chorus-conductor” as well as for detoxification.

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Orally administered

Hepatoprotective Antiviral against HIV-1

Pharmacokinetic studies: Glycyrrhizin is converted to Glycrrhetinic acid

(aglycone) in the human intestinal flora, Glycrrhetinic acid is found as the predominant

form in the blood, Only “Glycrrhetinic acid-glucuronide metabolite”

possesses antiviral and antihepatotoxic activity.

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Licorice: Glycyrrhizin (triterpenoid

saponin)

O

O

COOH

O

O O

OH

HO

COOH

COOH

HOHO

O

COOH

OH

OH

Glycyrrhizin

Glycyrrhetic acid

Human feces

Eubacterium sp.(glycyrrhizin -glucuronidase)

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Digitoxin; absorbed quantitatively,

Oral and i.v. doses are same. Digoxin is excreted largely unchanged in the

urine. Ouabain (from Strophanthus sp.)

has poor and erratic oral absorption, only be given by injection.

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GLYCOSIDES: Cardioactive glycosides

Principally low bioavailability Large molecular weight High affinity to bind with proteins Poor lipid solubility

Poor bioavailability of intact tannins is important to avoid from toxic effects; Hepatotoxicity: Hydrolysable tannins absorbed

into the bloodstream cause, Cancerogenicity (on sc.)

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Tannins/Procyanidins

Breakdown products of tannins take place in the large bowel by bowel flora, in the small bowel for hydrolysable tannins,

Metabolites show potent antioxidant activity.

“Pycnogenol”: Oligomeric proanthocyanidin (OPC) [condensed tannin]; antioxidant in pine bark.

Cross the blood-brain barrier (?)E. Yeşilada 75

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Lifecycle of tannin through the gut

CONDENSED TANNINS ELLAGIC ACID

pH 7-8caecum microflora

HYDROLYSABLE TANNINS(TANNIC ACID)

GALLIC ACID

pH 7-8caecum microflora

Antioxidantanticancer

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Polyphenol in Tea Green tea:

EGCG: Epigallocatechin gallate EGC: Epigallocatechin

Black tea: Fermentation; leads to polymerization to more

complex molecules (mw 500 to 3000); Theaflavins, Thearubigens,

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Clinical: Green tea and black tea provide a significant

increase in the antioxidant activity of plasma, Antioxidant activity;

Green tea > black tea in vitro x5; in vivo x2 times

The effect is rapid, peaking at ~ 30 min after green tea, ~ 50 min after black tea consumption.

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EGCG is stable in the upper digestive tract

Possibly tea polyphenols undergo spontaneous decomposition in the gut and the smaller antioxidant molecules are then absorbed.

However 0.2-2.0% of the orally administered EGCG was found to be absorbed in the blood after 90 min without any decomposition.

40% of EGCG at 50 mg dose administered orally to rats was excreted unchanged with the faeces.

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Adding milk completely detroy

antioxidant effect of tea. Causes protein binding would inhibits

decomposition of polyphenolic constituents and thus bioavailability.

NB: the tannin-protein complex later may yield metabolites in the gut and may show activity.

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Role as immune-enhancing agents,

Echinacea Urtica Ginseng Ganoderma (reishi mushroom)

Macromolecules composed of sugar and uronic acid moiety,

In plants is a component of cell wall. H2O-solubl or swelling molecules, EtOH or EtOH/H2O (less than 50%) extracts

generally do not contain polysaccharidesE. Yeşilada 82

Polysaccharides

Polysaccharides possess low

bioavailability, Advised to be administered in sufficient

doses for compensation of the poor bioavailability.

Unabsorbed polysaccharides Pass into the large intestine, Broken down by bowel flora , Also may have a protective role balacing

bowel flora. E. Yeşilada 83