Nattaya Lourith, Ph. D. [13 Feb 10] 1

Nattaya Lourith, Ph.D.13 Feb 10

Herbal extraction & isolation

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Contents: category of herbal useconsumption value & trenddata requirementsforms usedherbal selectionherbal extractionherbal extract’s quality

Herbal extraction & isolation

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Herbal usesFood herbs- garlic, lemon, peppermint, ginger, etc.Medicinal herbs

- aloe, ginkgo, ginseng, etc.Poisonous herbs- some mushrooms, etc.

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Herbal products in each culture

IndiaChinaPakistanMiddle EastJapanOther AsiansEuropean +African

PAMPAMPAMPAMPAMPAM

P

Ayurvedic ChineseIndusynunicIslamic : Unani-TibbKampoOriental

Herbalism/Homeopathy/Botanical

Origin Source Type

P – plants: A – animals: M – minerals

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Current herbal useComplementary and alternative medicine

(CAM)Fast growing over-the-counter products

- US$ 23 billion by 2010Beauty alimentsSkin rejuvenation

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Rx or OTC

Rx or OTC

OTC

OTC, Rx, Grocers

Rx

Vinblastine, taxolor aspirin

CAM

Garlic

Canola oil, golden rice

During clinical trials

Single ingredient: plant derived

Clinically validated and standardized phytochemical mixtures

Plant component with health benefits

Food engineered or supplemented to provide health benefits

Plant isolated proteins

Drug

Botanical drugs

Dietary supplements/nutraceuticals

Functional/medicinal foods

Recombinant proteins

AvailabilityExampleDescriptionTherapeutic

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Topical analgesic

Analgesic, antitussiveLocal anaestheticSmoking cessation therapyCardiac depressant

Antimalarial

Capsicum spp.

Papaversomniferum L.Erythroxylumcoca LamarckNicotiana spp.

Cinchona spp.

Cinchona spp.

Phenylalkyl-amine alkaloidOpium alkaloidCocaine alkaloidPyrrolidinealkaloid

QuinolinealkaloidQuinolinealkaloid

Alkaloids: 4,045 Capsaicin

Codeine, morphine

Cocaine

Nicotine

Quinidine

Qunine

Terpenes and steroids: 12,400Glycosides: 9,230Others: 5,014

Therapeutic useSourceTypeName: US$M (2002)

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General information requirements for herbal cosmetic ingredients

Basic Data RequirementsFantasy & TrendsFunction/Activity

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Basic Data Requirements1. Formulation

- Stability: Freeze-thaw cycles: High & low temperatures/storage

recommendations: Centrifugation (for emulsions)

- Consistency in certain quality parameters- Compatibilities- Manufacturing recommendations

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Basic Data Requirements2. Safety

- Toxicological information: Skin & eye irritation, sensitization: Fragrance allergens: Animal friendly

- Pesticides and heavy metals- Microbiological specifications- Impurities- GMO-free certificate

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Basic Data Requirements3. Environmental Responsibility

Sustainability

4. Traceability, Fair Trade and Social Responsibilityexp. Deal, Labor, etc.

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Fantasy & Trends1. Fantasy

- Natural or as Natural is highly demandSpecific geographical source is requested

e.g. India, China/Japan/Asia, Amazon, Deserts/Alpine regions/Plant from extreme environments- Plants in different parts of the world in traditional herbal medicine- Extraordinary feeling create- A very specific product brief

2. Trends

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Function/Activity1. Plant properties

exp. sebum regulating, anti-bacterial, anti-inflammatory/anti- irritant, soothing/calming, skin healing & regeneration, stimulate skin regeneration, stimulate collagen production or inhibit breakdown, protect against UV & hash conditions, anti-oxidant/free radical scavenger, phytopheromones, moisturising/nourishing/smoothing, circulatory stimulant, anti-swelling, astringent, etc. 14

Function/Activity2. Plant activities

- Identification of plants high in specific actives- Analytical data- Claims substantiation

: chemical measurement: in vitro: in vivo

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Herbal forms used Total extract or crudeSelected active principlesIsolated pure compounds

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Herbs

Crude

Selected active principles

Pure compounds

Primary extraction

Secondary extractions

Extractions

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Herbal Quality Analysis

- Chromatographic Methods- Spectral Methods- Bioassays

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Plant derived ingredientBotanical namePart of usedPreparation methodCharacteristic compositionPhysicochemical specificationMicrobiological qualityAllergenic potential

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Traditionally used Safety Active compoundsClinical proven Literatures

Herbs selection

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Extraction/Isolation/Purification techniques

- Drying- Hot - Cold

- Grinding

Sample preparation for extraction

Bad sample preparation & extraction : loss of actives

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Drying: hot

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Drying: cold

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Drying: cold

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Grinding

powderpulveratapulverized

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Extraction techniques• Cold extraction

– Maceration – Percolation

• Hot extraction– Decoction – Distillation – Infusion – Soxhlet

• Accelerated extraction– Microwave assisted process– Sonication– Supercritical fluid

Proper solvent

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Solvent used

C6H14

Toluene EtOAc

CHCl3

Acetone

AcCN

BuOH

PrOH

EtOH

MeOH

H2O

C6H6 Ether

Apolar Polar

Alkaloids TerpenoidsCoumarinsFatty acids Flavonoids

Terpenoids

Flavonols

Alkaloids, Flavones Polyphenols, Tannins Saponins, Lectins

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Maceration: soaking in solventMacerate with gradient solvent of different

polarity With or without stirring

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Percolation

- Passing solvent through sample

- Continuously flow of solvent

- Large volume of solvent used

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Decoction:extract in boiling water

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Infusion:treat with hot water or EtOH + water

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Soxhlet: reflux temp.

• Cellulose thimble– Organic compounds

• Glass thimble– Inorganic compounds : high

temp. up to 550 ˚C

Thimble

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Microwave assisted process

H2O vibration

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Fluid as solventNo solvent residueNo need to

concentrateFast & effectiveCompounds

selectable High pressure Low temperatureHigh cost

Supercritical fluid extraction

CO2

31 ˚C74 bar

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Comparison of accelerated extraction methods

0.250.050.051Relative energy consumption

150-6501-5AmbientAmbientPress. (atm)

30-60 m30-45 s30 m16 hTime

50, 20040, 70, 100Rt.Boiling point

Temp. (°C)

5-25<100500500-1000Vessel Volume(ml)

5-2510-20300>300Solvent Volume (ml)

CO2Hexane/EtOH

**Solvent

1-100.5-15-305-10Sample weight (g)

Supercritical fluid

MicrowaveSonicationSoxhlet

* CH2Cl2, Acetone, hexane, cyclohexane, toluene, etc.

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Extractions Protocol may be modified

depending on the extracted molecules

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Concentrations Reduced pressure

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Concentrations Evaporation

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Spray dry Concentrations

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Freeze dryConcentrations

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N2 blown down

Concentrations

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• Crystallization• Sublimation • Distillation • Liquid-liquid extraction : Partition• Chromatography

Isolation/ Purification techniques

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Crystallization

Protein Kojic acid

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Sublimation

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Distillation

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Liquid-liquid extractionPolarity + gravity employmentsLike dissolve like

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Chromatography• Stationary phase• Mobile phase

+ partition+ adsorption + dissolution

47Chromatographic extraction 48

Column chromatographic extraction

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Extraction trackingThin layer chromatography : TLC

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TLC tank preparation

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TLC plate preparation

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TLC spotting

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TLC visualizing

UV : 254, 366 or 365 I2 vapourSpraying agents 54

Rf calculation

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High performance liquid chromatography

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determination of selectedflavonols in Ginkgo biloba

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Gas chromatography

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gas chromatographic determination of organochlorinepesticides residue in some Chinese herbs

electron captured detector (ECD) and a split-splitlessinjector. HP-5 Capillary column

1, α-BHC; 2, β-BHC; 3, χ-BHC; 4, δ-BHC; 5, pp’-DDE; 6, pp’-DDD; 7, op’–DDT; 8, pp’-DDT

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Supercritical fluid chromatography

GC & HPLC Hybridization

Thermolabile/High molecular weight compounds

60Supercritical Fluid Chromatogram for achiralscreening in drug discovery compounds

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Fraction collector: HPLC, SFE

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Bioassay-guided fractionation

Total extract or crudeSelected active principlesIsolated pure compounds

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BioassaysBiological target specific

- Lower organism: microorganisms (bacteria, fungi, viruses)- Invertebrates: insects, crustaceans, molluscs- Isolated subcellular systems: enzymes, receptors- Animal or human cell cultures- Isolated organs of vertebrates- Whole animals 64

Biological Activities

• MIC : minimum inhibitory concentration• MLC : minimum lethal concentration• MFC : minimum fungicidal concentration• MBC : minimum bactericidal concentration• LC50, LC90 : Lethal concentrations/doses • 95% Confidential limits

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Biological activity• Antioxidant• Anti-inflammatory • Tyrosinase inhibition• Melanogenesis inhibition• Keratinolysis• Collagen induction• Blood circulation • 5α-reductase inhibition

Cosmetics : Radical scavenger (2,2-diphenyl-1-picrylhydrazyl: DPPH)Tyrosinase inhibitorPropionibacterium acnes

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Antioxidant Exp.• DPPH assay

– 2,2-diphenyl-1-picryl-hydrazyl+ Vitamin C

• TEAC assay– 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic

acid)+ Vitamin C

• β-carotene bleaching assay– β-carotene+ Linoleic acid

• FRAP assay– 2,4,6-tripyridyl-S-triazine+ FeCl3

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Anti-inflammatory• Free radicals• Acne • Hair loss• Keratin

interleukin (IL)-1αtumor necrosis factor (TMF)-αgranulocyte-macrophage colony

stimulating factor (GM-CSF)68

Melanin Biosynthesis pathway

COOH

NH2HO

COOH

NH2HO

HO COOH

NH2

O

O

O

N COOHHO N COOHHO

HO

H

N COOHHO

HO

H

O

O NH

O

O COOHNH

NHO

HO

H

COOH

NH2

HO

HN

SHOOC

Tyrosine DOPA Cysteine/Glutathione

Leuco DOPA chrome

DHI DHICA

Indole-5,6-quinone-2-calboxilic acid

PheomelaninEumelanin

Tyrosinase

TRP-1

TRP-2

Pmel17/silver locus protein

Tyrosinase

Tyrosinase and/or peroxidase

DOPA quinone

DOPA chrome

Indole-5,6-quinone

Dihydrobenzothiazone

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Keratinolysis• Hard keratin

–Hair & nail• Soft keratin

–SkinDisulfide bonds content

Keratinolytic agent = proteolyticenzymes exp. Trypsin, pepsin, papain 70

Collagen induction• Radicals • Skin elasticity • Skin sagging • Skin wrinkling

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Blood circulation

Toxin drainage Inflammatory reduction

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5α-reductase inhibitionAntiandrogenetic• Hair loss treatment• Acne • Sebum hypersecretion• Hirsutism

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Additional benefitSurfactant properties:

– Detergency – Foaming – Chelating – Surface tension– Emulsifying

HumectancyEtc. 74

• Allergy • Toxicity

Adverse effects

Doses & efficacy : Threshold

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Adverse effects

• Skin prick test• Patch test

–Atopic dermatitis–Irritation

Allergen

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• Cytotoxicity• Human skin cell

Adverse effects

Toxicity : positive false result

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Standardized & Qualified extracts

Minimum level of active ingredients assuredQuality consistency Safety confirmatory

Monograph or Pharmacopeia

Identity, purity, content or assay78

Standardized extracts: expGingko extracts:

standardized extracts (dry extracts from dried leaves)acetone + water, drug: extract = 35-67:1

Contain;22-27% flavone glycosides5-7% terpene lactones2.8-3.4% gingkolides A, B, C2.6-3.2% bilobalide

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Standardization prevent

- biological & chemical variations- adulteration- contamination

+ microbial + metal

- toxicity

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Specification of herb- Identification- Purity

- Physical Tests- Chemical Tests- Chromatography- Spectroscopy

Analytical methods

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Identification1. Pharmacognostic charateristics

- Macroscopical description- Microscopical description

2. Chemical characteristics- Preliminary test- Confirmatory test

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Macroscopical descriptionMorphology/anatomyCharacteristic color, fracture, smell, taste

Microscopical description- Histologic characteristics- Description of powered herb

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Microscopical description : Purity

1. Foreign matter2. Volatile & non-volatile3. Moisture4. Microbial contamination5. Ash content6. Pesticide & Fungicide residue contamination7. Metal contamination8. Radioactive contamination

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Microbial contaminationTotal aerobic microbial countTotal fungal countTotal Enterobacteriaceae countProhibited microbes

- Staphylococcus aureus- Escherichia coli- Pseudomonas aeruginosa- Candida albicans- Aspergillus niger

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85Pour plate Disc diffusion

Microbial test

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TLC: bioautography- Contact- immersion- direct

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Test kit

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Ash Total ash = organic + inorganic mattersAcid-insoluble ash = inorganic matters

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Pesticide & Fungicide residue

Toxicity from agricultural practices- spraying- cultivation soil- fumigant storage

ExtractionPartition/adsorption for impure movementAnalyze

- GC- MS- GC/MS 90

Metal contaminationToxic metal : Pd, Cu, Cd, AsAnalyze

AAS- inductive couple plasma; ICP - neutron activation analysis; NAA

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Radioactive contaminationNuclear exposure

– Skin – Hair – Brain – Thyroid – Blood system– Heart – Gastrointestinal track – Reproductive track

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Chemical characteristics: Chemical tests

• Tannins– FeCl3 : blue-black = gallitannins, ellagitannins

: brownish-green = condensed tannins– Gelatin + 10% NaCl: precipitate = pseudotannins– Matchstick + HCl conc. + warm: pink or red = catechin– NH4OH + air: green = chlorogenic acid

• Alkaloids– Mayer’s reagent = cream – Hager’s reagent = yellow– Wagner & Dargendorff reagents = reddish-brown

• Anthraquinones: NH4OH or NaOH = pink, red, violet• Coumarins: NH4OH = blue, blue-green, violet

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GOOD PRACTICE

Herbal extract’s quality+ efficacy+ stability+ safety+ usability

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GOOD PRACTICEThe Five “G’s”

Good Agricultural Practice (GAP)Good Harvesting Practice (GHP)Good Clinical Practice (GCP)Good Laboratory Practice (GLP)Good Manufacturing Practice (GMP)

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ReferencesRaskin I, Ribnicky DM, Komarnytsky S et. al. Plants and human

health in the twenty-first century. Trends Biotechbol. 2002. 20: 522-531.

White IR. Plant products in perfumes and cosmetics. SemDermatol. 1996. 15: 78-82.

Ahmad I, Aqi F, Owais M. (eds.) Modern phytomedicine. Weinheim, Germany. 2006.

Agriculture and Agri-Food Canada. Good practices for plant identification for the herbal industry. British Columbia. Canada. 2004.

World Health Organization. WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants. Geneva. Switzerland. 2003.

Lourith N. Herbal cosmetics. [Lecture Handouts]. Chiang Rai: MFU; 2009. 96

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