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Campo Novel Liquid & Soft-Paste Ceramides

CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 2

INDEX

CLEAR COLORLESS CERAMIDE OIL COMPLEX

MULTI-VITAMINS CERAMIDE RW (OIL)

CAMPO CERAMIDE 6 (VI)

ENZYME: EC 2.3.1.76

ENZYME: EC 3.5.1.23

CAMPO CERAMIDE 3a (IIIa)

CAMPO CERAMIDE 3 (III)

CAMPO CERAMIDE 3 (III) - Wax Granules

TOXICOLOGY PROFILE

CLINICAL TESTS

ANALYTICAL REPORT



CLEAR COLORLESS CERAMIDE OIL COMPLEX (A BIOTECHNOLOGIC HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

BIOTECHNOLOGIC HUMAN SKIN CERAMIDE OIL -

THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION

Ceramide Oil in a novel clear colorless liquid form is edible and has various use as/for

Pharmaceuticals, Cosmetics, and Supplementary Food/Dietary uses.

For cosmetics applications such as Novel Ceramide Lipsticks, Decorative Cosmetics, and any other

conceivable type of Cosmetics or Cosmoceuticals are being now possible with this Novel

Biotechnologic Human Skin Ceramide oil (derivative of the DAG form).

The extraction of Ceramide Oil is from Olive Fruits - totally natural and a Biotechnologic Human

Skin Ceramide form identical to the Human Skin Isolates of Ceramides And Sphingolipids found

both in the Human Skin and as well as in other parts of the Human Anatomy.

STRUCTURE

General structures of DAG and Dihydroceramides (not the wax-like form) but of which are

liquidified (known as dihydroceramides) in the 100% colorless, liquid Ceramide Oil (DAG and

dihydroceramides are bioactive in their own way in the paradigm of signalling within the

intracellular matrix but inactive far as cell-apoptosis (programming cell-death) are concerned and

(DAG & dihydroceramides are not) as is reported in the cases of wax-like, occlusive ceramides

(such as pure natural ceramides isolates from sources of bacterial or yeast or non - Biotechnologic

plants) - of which are known now to and re-considered for inducing apoptosis in both hemopoietic

and nonhemopoietic cell lines, including fibroblasts and fibro - sarcoma cell lines; morphological

changes of apoptosis and suppression of clonogenicity (i.e regrowth) in cancers and retroviral

diseases like AIDS and hepatitis.



Composition of Ceramides and Sphingolipids in Percentage Levels:

Sphingosine, N,N-Dimethyl- > 99%

(a DAG Ceramide derivative)

D-erythro-Sphingosine < 0.0003%

(a dihydroceramide derivative form)

4-Sphingenine (Sphingosine) < 0.0002%

(a dihydroceramide derivative form)

Other Dihydroceramides < 0.5%

SPECIFICATION

Appearance colourless clear liquid

Odour odourless

Refractive index (20C) 1.35 - 1.50

Specific gravity (20C) 0.900 - 0.980

Water content (%) 0.1 - 0.5

Solubility in:

Water < 0.2%

Ethanol soluble

Other cosmetic oils soluble

Fragrance oils soluble

SUGGESTED COSMETIC APPLICATION DOSAGE LEVELS : > 0.001% - > 10%

Bath Oil, Skin Creams, skin moisturizing, nourishing, & conditioning treatments; Shampoos, and

Lotions, hair groom oils, creams and pomades; Sunscreen oils, creams and lotions; After sun -

creams and lotions; Skin oils, lotions, and tonic; Cleansing creams, lotions, and milks; Make-up

creams, sticks, and godets and Baby oils, creams and lotions.

Campo Research, Singapore



CLEAR COLORLESS CERAMIDE OIL COMPLEX (A BIOTECHNOLOGIC HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

BIOTECHNOLOGIC HUMAN SKIN CERAMIDE OIL -


TOXICOLOGICAL & ECOTOXICOLOGICAL DATA

TOLERANCE

As to ensure a good level of innocuity THSC Ceramide Oil was tested in- invitro as follows:

* Irritation potential of the chorio-allantoic membrane of an egg.

When tested on the chorio-allantoic membrane of a chicken egg, according to the technique

developed by LUEPKE** in a 10% active liposoluble solution,THSC Ceramide Oil is classified as

non-irritant.

* Cytotoxicity on human fibroblasts

When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to

both hydrosoluble as well as liposoluble products). THSC Ceramide oil in a 10% active aqueous

phase and/or 10% active oily phase, does not show any signs of toxicity towards fibroblasts in

culture.

* Eytex

According to this technique, in a 10% active solution, THSC Ceramide oil is totally non-irritant.

* Skintex

According to this technique, in a 10% active solution, THSC Ceramide Oil is a non-irritant.

This tolerance data is confirmed by the tests carried out in vivo on health humans.

* Test on healthy humans

When patch tests were carried out at increasing concentrations (0.5%, 1.1%, 2.2%, 4.7 & and 10%

respectively) on 20 healthy subjects, THSC Ceramide Oil did not show any significant irritant

reaction at all. Its tolerance is totaling satisfactory.

COMEDOGENESIS

THSC Ceramide oil was tested in a 10% active solution on human volunteers, according to the usual

protocols has proven to be free of comedogenic effect.

Due to its excellently good level of innocuity. THSC Ceramide oil has proved to be First Class

emulsifier for a large number of formulae where tolerance is imperative (dermatological cream, anti-

acne, baby cream, face cream. etc)

BIODEGRADABILITY

The ultimate aerobic biodegradability of THSC Ceramide oil is measured according to STRUM

TEST (OCDE 301 B, guideline EEC 84/449. Annex V. Method C5).

Under these conditions, a level of biodegradability of THSC Ceramide oil is 100% in 28 days at

20mg/ml.

The level of biodegradability of THSC Ceramide oil is considered to be excellent.

Campo Research. Singapore



CAMPO RESEARCH

MULTI-VITAMINS CERAMIDE RW (OIL) product # 94-18-33 RC

Composition percentage Vitamin A 25%

(As retinol palmitic acid esters)

Vitamin E 3%

(As tocopherol derivatives)

Vitamin F 0.32%

(As essential fatty acids (with high content

> 76% of linoleic acid) partly in

free- form and partly in glycerol ester form)

Ceramide clear, colorless liquid oil 71.68%

Technical specification: Product multivitamins-ceramide rw (oil)

Appearance clear colorless liquid oil

Odor odorless

Clouding point deg.cent. minus - 5 deg.cent

Viscosity < 50 cps

Anti-oxidative agents none,- naturally stable without oxidation

Preservative none

Microbiology < 100 cfu/ml (non - pathogenic)

Pesticide content nil

Toxicology & tolerance test data Ames test non-mutagenic

Skin irritation none

Eye irritation none

Ld 50 mice 75 mg/gram body weight (edible)

Ld 50 rat 88 mg/gram body weight (edible)

Application & dosage levels

Hair shampoo & lotions > 0.3%



RADIOACTIVE TAGGING OF MULTIVITAMINS-CERAMIDE RW (OIL)

The three predominant oil-soluble Vitamins A,E, and F representing approximately 28.32% of the

composition ratio of Multivitamins-Ceramide RW; and Ceramide oil (the clear colorless liquid oil)

were sent to Radiochemicals Div.,of Sigma Chemicals USA, for radio tagging as C14 radioactive

materials.

These tagged materials were mixed in correct ratios, and then added to larger quantities of unlabelled

multivitamins Ceramide RW oil.

HAIR TREATMENT

Virgin, brown hair (ex DeMeo Brothers) was used exclusively. Duplicate swatches-100 mg., were

treated with 10cc portions of the multivitamins Ceramide RW oil solutions for 15 minutes, followed

by three 15 seconds rinses with 10 cc portions of distilled water to remove unbound Vitamin A, E &

F and Ceramide RW oil. The hair swatches were blotted on paper tissue and hydrolyzed in 10 cc of

concentrated hydrochloric acid (SG 1.18) in sealed tubes for 48 hours.

SCINTILLATION COUNTING OF SAMPLES

0.05 cc quantities of hydroslysate were pipetted into 10cc of scintillant and counted automatically on

Hewlett-Packard liquid scintillation counter.

The washing from hair treatment were similarly checked to confirm that the washing procedure had

been successful.

RESULTS

TEST # 1 TEST # 2 AVERAGE

2% MVC* 8.5 mg / g HAIR 9.01 mg/g HAIR 8.70 mg / g HAIR

(MCV*= multivitamins ceramide RW oil)

the results obtained demonstrated that multivitamins - ceramide RW oil is highly substantive to

virgin hair.



FUNCTIONS AND APPLICATIONS

MultiVitamins Ceramide RW oil is a new novel range of oil-soluble bioactives and conditioning

additive recommended for use in hair-care preparations.

MultiVitamins ceramide RW oil, is also suitable for skin-care preparations.

MultiVitamins ceramide RW oil’s small molecules of low molecular weight indicates of the

penetration of the cuticle in the undamaged hair.

The unique properties combined with its moisture binding activities can be expected to produce a

deeper lasting conditioning effect when applied to the hair and as well as these activities are noted in

the skin, too.

The inclusion of MultiVitamins - Ceramide RW oil into Shampoos and conditioners will result in

improvements in manageability, gloss, sheen, feel, and hair’s body texture.

The Multivitamin ceramide RW oil has fine film forming properties.

These are a very important bio-activity for damaged hair since the Multivitamin-ceramide RW oil

will coat the hair shaft and increase moisture retention at the hair surface. In addition, Multivitamin

ceramide RW oil will provide a protective effect on hair and scalp; while helping to combat scalp

chapping (anti - dandruff) and irritation caused by the detergents (detergency properties) in the

shampoos.Multivatimin ceramide RW oil will also complement and assist the natural retention of

moisture at the scalp surface and hair, which are vital for manageability and for the natural healty

sheen and fine body healthy textured look of the hair.

Campo Research, Singapore



CAMPO CERAMIDE 6 (VI) -

TRUE HUMAN SKIN IDENTICAL SPHINGOSINE

Art No. 96/10/0055

uman Skin Identical Ceramide IV ( Sphingosine ) is isolated from biotechnological olive

fruit cells via huddle ( complex modified Huddle Technology ) ; Transcription/Restriction

Enzymes Technique; biotechnological cells mass-culture techniques; and via a novel

proprietary extraction techniques utilizing gaseous mediums such as carbon dioxide, nitrogen and

ozone; and Water as the menstrum/carrier medium in the biotechnology variation as ( of ) the human

skin’s “water of crystallization” liquid. This variation of water molecule(s) is identical to that found

in the human skin moisture/lipids barrier.

The use of skin’s Bio-Aqueous liquid as an identical carrier/menstrum in the extraction of true

human skin identical Ceramide IV from biotechnological olive fruit cells; the end-product Ceramide

IV ( as all other Campo True Human Skin Identical Ceramides in the CAMPO

THSICERAMIDES -INGREDIENT RANGE ) exhibits a high HLB range 17-20 - which is the

true nature and existent state of skin Ceramide(s) 1, 2, 3, 4, 5, 6, 6I and 6 II to exhibit a high HLB,

Not less than a minimum HLB 15 .

Extraction of Ceramides ( from yeast fermentation or from crop plants/herbs by-products ) via

any other methodologies currently till present has yield Ceramide-like structures which are

occlusive, wax-like with negligible HLB properties, and extremely hydrophobic in nature but

pass off as Skin-Identical Ceramides or the like; as without the ever-present water of

crystallization and the Water Related Enzymes ( such as Ceramidase {EC 3.5.1.23 } and

Sphingosine N-Acyltransferase {EC 2.3.1.24 }, which are lacking in the fatty acids tails ( of

Ceramides ) which may contain as many as 30 carbon atoms. Hence, rendering this so-called

Ceramides ( occlusive waxes ) - the extreme hydrophobic , and occlusive wax-like nature.

Therefore, the presence of these Ceramides’ Water Related Enzymes; Acyl-CoA Related Enzymes

and N-Acylsphingosine Related Enzymes form the basis of True Human Skin Identical ( THSI )

Ceramides and these THSI Ceramides exhibits water-solubility ( ie: high HLB range ) and the long-

lasting effect of smooth skin as is the true actual state and functionalities of the Skin Ceramides

existing in the Human Skin.

Campo Ceramide 6 contain All these Related Enzymes and also the presence of another ever-

important “ Retinol O-fatty-acyltransferase Enzyme { EC 2.3.1.76 } ” which is involved

biosynthesis and metabolism of Retinol ( ( vitamin A ); in the skin or skin’s retinol content ) by

acting on the palmitoyl-CoA and other long-chain fatty-Acyl derivatives of CoA.

This Retinol Enzyme’s low content or total lack of presence in the skin ceramides - results as

loss of Skin Ceramides which is inter-related with loss of skin’s Retinol with malfunctions such

as- actinic aging, fine wrinkles, sagging skin and other related problematic skin conditions

begins to set in.

Retinol enzyme { EC 2.3.1.76 } in Campo Ceramide 6 triggers the natural CoA fatty lipids

present in the skin to metabolise the retinol content ( increase in natural content ) and an

increase of this form of natural retinol in the skin is free of -side effects such sunlight

sensitivity, photo caused irritation as experienced in synthetic retinol based ( Tretinoin )

creams application for acne, and fine wrinkles.

H



Commercial Human Skin Identical Ceramides ( as skin’s complex lipids ) without the

enzymatic components in general fail to provide the requisite compatibility with skin’s simple

lipids, and their efficacy is limited by their minimal substantivity to the skin.

By arranging the Ceramidase { EC 3.5.1.23 } and Sphingosine-N-Acyltransferase {EC 2.3.1.24}

enzyme(s) related links ( ie: Transferase Class ) in each CAMPO THSI CERAMIDE(S’)

structure and similarly, by arranging a secondary ( other ) related enzymatic links ( ie: Retinol

O-fatty-acyltransferase or Alpha / Omega -Hydroxy-Acid related enzymes links ); these

CAMPO Versions of THSI Ceramides closely resemble the natural structures and their

respective skin identical physiological functionalities.

The disadvantages of adverse effects suffered by commercially available Ceramides from the

preservative systems used to protect the cosmetic preparations are nil with Campo THSI

Ceramides, with Campo THSI Ceramides while the known natural Ceramides’ potent

antimicrobial (one of the ceramides’ physiological function) activity are intact without

interfering with the preservation systems, displaying complementary potent anti-microbial

activity while remaining completely safe and non-toxic and extremely mild to skin and eyes -

when used in very high concentration ( 45-73 % usage ).

Campo THSI Ceramide 6 ( similarly identical to natural skin ceramide 6 ) maintains an

extremely high degree of substantivity by virtue of having multiple cationic binding sites, as well as

having a amphoteric character, remains completely compatible with virtually all types of cosmetic

ingredients including anionic surfactants and totally non-comedogenic.

CAMPO THSI Ceramide 6 ( VI) extremely high degree of substantivity is non-comedogenic and

skin-pores would not be clogged. It is multi-functional and provides true skin moisturisation and

long-lasting skin smoothing characteristic with high degree of elegance.

The formulation benefits include: Silky Smooth Elegance Afterfeel, True Natural Non-Occlusive

Moisturizer, Effective Skin Conditioner, Low Irritation Potential with High Concentration,

True Natural Compatible with Skin Mantle pH, and Replaces Anionic Emulsifiers.

TOXICOLOGAL PROPERTIES

Dermal Evaluation ( 5% in Water )

48 Hour 50/50 completely non-irritating

Human Patch Test ( non-erythema causing THSI ceramide ) 50 Test Subjects

IN-VITRO OCULAR EVALUATION ( 3% IN Water )

Ropak, Eyetex Eyetex Classification

Rapid Membrane Assay Minimal/ Mild



Available on request are True Human Skin-Identical Ceramides 1, 2, 3, 3a, 4, 5, DAG-Ceramides,, and

DAG-Sphingosines.

BOTANICAL INFORMATION - LATIN Olea europea

- ENGLISH Olive

INCI/CTFA NAME ( PROPOSED ) Sphingosine(AND)CeramidaseEnzyme (AND)

RetinolO-Fatty-AcyltransferaseEnzyme (AND)

Sphingosine N-Acyltransferase Enzyme

PLANT MATERIAL Olive Fruit Cells - biotechnological

ACTIVE COMPONENTS OF PLANT Sphingosine and Transferases Enzymes, and

Hydrolases Enzyme.

PRODUCT ATTRIBUTES Emollient Moisturizers, Long-lasting Skin

Smoothing, Fine Wrinkle Remover,

Effective Skin Conditioner ( anti-aging

Products); Non-comedogenic,

Non-occlusive, Substantive as true nature.

SPECIFICATIONS

SPECIFIC DENSITY (20C) 1.1400 - 1.2550

REFRACTION INDEX (20C) 1.200 - 1.3750

pH-VALUE ( 10% in 50/50 IPA/Aqueous ) 6.5 - 7.5

DRY RESIDUE (METTLER 160C) 40 - 42%

EXTRACTION VEHICLE Water - Purified ( ex-Whey )

PRESERVATION Non

TOTAL MICRO COUNT < NIL cfu/ml

TOTAL YEAST/MOLD COUNT < NIL cfu/ml

PATHOGENIC BACTERIA 0

APPEARANCE Waxy Solid Paste

COLOUR White to White-Buff

ODOUR Characteristic

APPLICATION Shampoos : 3 - 5%

Facial Moisture Creme Gel : 5 - 10%

Day & Night Creme : 5 - 10%

Store in a closed container in a dark place.

CAMPO RESEARCH, SINGAPORE

Level 30, 6 Battery Road

SINGAPORE 049909

Tel: 63833202 / Fax: 63834034



Formulary suggestions - Campo Human Skin-identical Ceramides

Campo Ceramide 6 (VI) – formulation benefits:

Replaces anionic emulsifiers, Nonrriation potential, Smooth Silky After-feel;

Identical skin- compatibility with skin mantel pH, Non-occlusive skin identical natural moisturize

Effective natural skin-identical conditioner

Light Body Crème

A high humectant crème provides a long lasting soothing feel without the greasiness felling inhere

with most other synthetic ceramides.

Phase A Weight by %

Campo Ceramide 6 (VI) 03.00

Steareth –20 00.45

Glycerin 05.00

Water 77.75

Phase B

Steareth-2 00.80

Ceteary Alcohol 03.50

Myristy Myrustate 03.50

Finsolv TN (finetex) 01.50

Isopropyl palmitate 03.00

Dimethicone (100cs) 01.00

Planservative (Lonicera japonica extra.)as preservative 00.50

100.00

Procedure: Heat both phases to 65 Deg. Cent. And homogenize the oil phase into the water phase.

Stir-cool to 40 Deg.Cent. and fragrance, coloring or (preservative) as required.

Deep Moisturizing Crème – Facial

An highly elegant crème with deep-moisturization effect,

With excellent rub-off resistance, suitable for overnight skin treatment care.

Phase A Weight by %


Steareth –20 00.20

Water 81.50

Phase B

Steareth-2 01.30

Ceteary Alcohol 04.00

Myristy Myrustate 04.00

Isopropyl palmitate 04.00

Dimethicone (100cs) 01.00

biotechnological herb Lanolin alcohol (Campo TLA –ex-olive) 00.50

Planservative (Lonicera japonica extra.)as preservative 00.50

100.00

Procedure: Heat both Phases to 65 Deg.Cent. and homogenize the oil phase into the water phase.

Stir-cool to 40 Deg.Cent. and fragrance, coloring or (preservative) as required.

Evening Body Lotion

A lotion to be apply to the whole body in the evening, for a cool, smooth and refreshing elegant feel

that will be lasting many hours. This lotion will remove the harshness of the heat experienced duty

the day from the body.



Phase A Weight by %

Water 51.60

Carbomer940 00.30

Triethanolamine (99%) 00.40

Campo Pearl Extract ws 00.05

Phase B

Campo Pearl powder Extract 00.15

Phenyl Dimethicone 01.50

Phase C

Water 30.00


Alcohol (Denatured) 15.00

Procedure:

Phase A – add Campo Pearl Extracts PWS to water 70 Deg. Cent.(qs) and stilt with agitator totally

dissolved ( filter if required.) Slurry Carbomer 940 with balance of water and add triethanolamine –

agitate until completely dissolved and add the dissolved solution of Campo pearl Extract PWS to the

dissolved Carbomer – Trientanolamine and agitate till totally homogenize.

Phase B - Disperse Campo Pearl Powder extract in phenyl Dimethicone.

Phase C - Heat water to 65 deg. Cent. And add Campo Ceramide 6. Cool to 30 Deg. Cent. And add

ethanol Mix Phase C into Phase A and add Phase B. Add color and Fragrance as required.

Suggested formularies are available on request for other Campo True Human – Skin - Identical

Ceramide.



ENZYME: EC 2.3.1.76

Official Name: RETINOL O-FATTY ACYLTRANSFERASE.

Reaction catalysed: ACYL-COA + RETINOL < = > COA + RETINYL ESTER

Comment(s):

ACTS ON PALMITOYL-COA AND OTHER LONG -CHAIN FATTY-ACYL-

DERIVATIVES OF COA.

Cross-Reference (s)

EMP / PUMA: 3.5.1.23.

KYOTO UNIVERSITY LIGAND CHEMICAL DATABASE : 3.5.1.23



ENZYME: EC 3.5.1.23

Official Name: CERAMIDASE

Alternative Name(s): ACYLSPHINGOSINE DEACYLASE

Reaction catalysed: N-ACYLSPHINGOSINE + H(2)O < = > A FATTY ACID + SPHINGOSINE

Cross-Reference (s)

EMP / PUMA: 3.5.1.23.

KYOTO UNIVERSITY LIGAND CHEMICAL DATABASE : 3.5.1.23

CAMPO RESEARCH

NEW PRODUCT LITERATURE

In-Cosmetics 1996/ICI Surfactants/Bregaglio Stand



CAMPO CERAMIDE 3a (IIIa) -


Art No. 96/10/005502

uman Skin Identical CERAMIDE IIIa ( SPHINGOSINE ) is isolated from either

biotechnological olive fruit cells or Aloe vera leaf cells via huddle ( complex modified

Huddle Technology ) ; Transcription/Restriction Enzymes Technique; biotechnological cells

mass-culture techniques; and via a novel proprietary extraction techniques utilizing gaseous

mediums such as carbon dioxide, nitrogen and ozone; and Water as the menstrum/carrier

medium in the biotechnology variation as ( of ) the human skin’s “water of crystallization” liquid.

This variation of water molecule(s) is identical to that found in the human skin moisture/lipids

barrier.


human skin identical Ceramide IIIa from biotechnological olive fruit cells; the end-product

Ceramide IIIa ( as all other Campo True Human Skin Identical Ceramides in the CAMPO


true nature and existent state of skin Ceramide(s) 1, 2, 3, , 3b, 4, 5, 6, 6I and 6 II to exhibit a high

HLB, Not less than a minimum HLB 15 .

Ceramide III a is an exceptional to the HLB value, ( see water solubility table )








Ceramides ( occlusive waxes ) - the extreme hydrophobic , and occlusive wax-like nature.



Ceramides and these THSI Ceramides exhibits water-solubility ( i.e.: high HLB range ) and the long-



Campo Ceramide IIIa contain All these Related Enzymes and also the presence of another

ever-important “ Retinol O-fatty-acyltransferase Enzyme { EC 2.3.1.76 } ” which is involved

biosynthesis and metabolism of Retinol ( ( vitamin A ); in the skin or skin’s Retinol content )

by acting on the palmitoyl-CoA and other long-chain fatty-Acyl derivatives of CoA.

This Retinol Enzyme’s low content or total lack of presence in the skin Ceramides - results as



begins to set in.


present in the skin to metabolize the Retinol content ( increase in natural content ) and an

increase of this form of natural Retinol in the skin is free of -side effects such sunlight

H



sensitivity, photo caused irritation as experienced in synthetic Retinol based ( Tretinoin )





By arranging the Ceramidase { EC 3.5.1.23 } and Sphingosine-N-Acyltransferase { EC 2.3.1.24

} enzyme(s) related links ( i.e.: Transferase Class ) in each CAMPO THSI CERAMIDE(S’)

structure and similarly, by arranging a secondary ( other ) related enzymatic links ( i.e.:

Retinol O-fatty-acyltransferase or Alpha / Omega -Hydroxy-Acid related enzymes links );

these CAMPO Versions of THSI Ceramides closely resemble the natural structures and their




Ceramides , with Campo THSI Ceramides while the known natural Ceramides’ potent

antimicrobial ( one of the Ceramides’ physiological function ) activity are intact without



when used in very high concentration.

Campo THSI Ceramide 3a ( similarly identical to natural skin ceramide 3 ) maintains an




CAMPO THSI Ceramide 3a ( IIIa ) extremely high degree of substantivity is non-comedogenic

and skin-pores would not be clogged . It is multi-functional and provide true skin moisturization and


The formulation benefits include : Silky Smooth Elegance Afterfeel, True Natural Non-Occlusive



TOXICOLOGICAL PROPERTIES



Human Patch Test ( non-erythema causing THSI ceramide )

50 Test Subjects






BOTANICAL INFORMATION – LATIN Aloe Vera flora callus

- ENGLISH Aloe leaf cells

INCI/CTFA NAME CERAMIDE III ( CERAMIDE 3 )

PLANT MATERIAL Aloe Leaf Cells - biotechnological


Hydrolases Enzyme.

PRODUCT ATTRIBUTES Anti-Acne Fine Wrinkle Remover, Effective

Skin Conditioner ( anti-aging Products);

After-shaves,Anti-perspirants& deodorants;

Non-comedogenic,Non-occlusive,

Substantive as true natural skin ceramides.

SPECIFICATIONS

Assay ( chelatometric, Zn ) 24 -33 %

Assay ( from N, Ceramide IIIa ) 36 - 45 %

pH-VALUE ( 1% in water ) 4.0 - 5.0

Water Content 6 - 9 %

EXTRACTION VEHICLE Liquid Nitrogen at -1,900 Deg C ( minusZero)

PRESERVATION None




Appearance Fine Powder

Sieve Size of Freezed Dried Powder

( max 100 um )

minimum 95%

COLOUR White to White Buff

ODOUR Characteristic minimal

APPLICATION Anti-Acne products : 0.5 - 5 %

After Shave preparation : 0.2 - 03 %

Anti-Perspirant & Deodorant : 0.25 -0.5%

Store in a closed container in a dry &dark

place.



SINGAPORE 049909

Tel: 63833202 / Fax: 63834034



ADDITIONAL INFORMATION ON CERAMIDE IIIa

Solubility

Solvent Temperature Solubility

Water 20 d C approx. 1.3 %

Ethanol ( 50%) 20 d C approx. 0.8 %

Isopropanol ( 50% ) 20 d C approx. 1.0 %

Ethanol ( 96 % ) 20 d C in-soluble

Ether 20 d C in-soluble

Uses

ACNE PREPARATIONS

A concentration of 0.5 - 2 % of Ceramide III a is recommended for Acne preparations.

Ceramide IIIa has a mild astringent effect promotes the drying of oozing skin areas and

pustules.

Ceramide III a has ameroliation properties that decreases infected eruptions, heals the

affected skin portions and provides a soothing effect to the affected parts.

Ceramide III a can be also combined effectively in a 0.5% aqueous alcoholic solution and in

ointments with antimicrobial compounds for control and preventive of acne.

AFTER SHAVE PREPARATIONS

Ceramide IIIa should be in concentrations of 0.2 - 0.25 % in these After-Shave preparations.

Shave-cut injuries and close-shave irritations which often occurs on the facial skin, legs, thighs

and arms during and after shaving are rapidly and instantaneously reduced.

ANTI-PERSPIRANTS AND DEODORANTS

Ceramide IIIa in concentration 0.25 to 0.5 % have proved to be suitable for these type of

preparations. The slight irritations on the skin which are commonly felt and experienced

during the applications of anti-perspirants and deodorants are greatly alleviated by the

addition of Ceramide IIIa into the formulations of these type of preparations.

Aluminum cholorohydroxide should be 99% and Ceramide IIIa at 1% will be an effective

addition

to anti-perspirants and deodorant preparations.



CAMPO CERAMIDE 3 (III) -


Art No. 96/10/00550-100

uman Skin Identical CERAMIDE III ( SPHINGOSINE ) is isolated from either




mediums such as carbon dioxide, nitrogen and ozone; and Water as the menstrum/carrier medium in

the biotechnology variation as ( of ) the human skin’s “water of crystallization” liquid.


barrier.



Ceramide III ( as all other Campo True Human Skin Identical Ceramides in the CAMPO




Ceramide III is an exceptional to the HLB value.








Ceramides ( occlusive waxes ) - the extreme hydrophobic ( water repelling ) , and occlusive wax-

like nature.






Campo Ceramide III contain All these Related Enzymes and also the presence of another ever







begins to set in.



H


























CAMPO THSI Ceramide 3 ( III ) extremely high degree of substantivity is non-comedogenic










50 Test Subjects






BOTANICAL INFORMATION - LATIN Olea europea fruit callus

- ENGLISH Olive Fruit cells




Hydrolases Enzyme.






SPECIFICATIONS


Assay ( from N, Ceramide III ) 36 - 45 %

pH-VALUE ( 1% in water ) 4.0 - 7.5



PRESERVATION None




Appearance Liquid

COLOUR Amber - golden brown




Skin-Care preparations : 0.5 - 2.0%

Hair-Care preparations : 0.5 - 1.5%



place.



SINGAPORE 049909

Tel: 63833202 / Fax: 63834034



CAMPO CERAMIDE 3 (III)-Wax Granules (w/s & o/s -amphilic)


Art No. 96/10/00550-100wax-amphilic

uman Skin Identical CERAMIDE III ( SPHINGOSINE ) is isolated from either




mediums such as carbon dioxide, nitrogen and ozone; and Water as the menstrum/carrier medium in

the biotechnology variation as ( of ) the human skin’s “water of crystallization” liquid.


barrier.



Ceramide III ( as all other Campo True Human Skin Identical Ceramides in the CAMPO




Ceramide III is an exceptional to the HLB value.








Ceramides ( occlusive waxes ) - the extreme hydrophobic ( water repelling ) , and occlusive wax-

like nature.






Campo Ceramide III contain All these Related Enzymes and also the presence of another ever







begins to set in.



H


























CAMPO THSI Ceramide 3 ( III ) extremely high degree of substantivity is non-comedogenic







Dermal Evaluation ( 100% in 10ml of Water )



100 Test Subjects

IN-VITRO OCULAR EVALUATION ( 10 % soluble In 10ml Water )





BOTANICAL INFORMATION - LATIN Olea europea fruit callus

- ENGLISH Olive Fruit cells




Hydrolases Enzyme.






SPECIFICATIONS


Assay ( from N, Ceramide III ) 36 - 45 %



PRESERVATION None

TOTAL MICRO COUNT < - cfu/ml

TOTAL YEAST/MOLD COUNT < - cfu/ml

PATHOGENIC BACTERIA -

Appearance Waxy Hardened Granules

COLOUR Amber - golden brown




Skin-Care preparations : 0.5 - 2.0%

Hair-Care preparations : 0.5 - 1.5%



place.



SINGAPORE 049909

Tel: 63833202 / Fax: 63834034



TOXICOLOGY PROFILE

Ceramide 6 (VI)

Ceramide 3a (IIIa) (powder)

Ceramide 3 (III) (liquid)

Clear Colourless Ceramide Oil Complex



CERAMIDE 6 (VI) (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

Biotechnological HUMAN SKIN CERAMIDE



TOLERANCE

As to ensure a good level of innocuity THS Ceramide 6 was tested in in-vitro as follows:

*Irritation potential of the chorio-allantoic membrane of an egg.


developed by LUEPKE** in a 10% active hydrosoluble solution, THS Ceramide 6 is classified as

non-irritant.

**LUEPKE, N.P., Hen’s egg chorio-allantoic membrane test for irritation potentiation. Fd. Chem.

1986, 24, 6-7, 495-496.

* Cytotoxicity on human fibroblasts.


both hydrosoluble as well as liposoluble products). THS Ceramide 6 in a 10% active aqueous phase

and/or 10% active oily phase, does not show any signs of toxicity towards fibroblasts in culture.

* Eyetex According to this technique, in a 10% active solution, THS Ceramide 6 is totally non-irritant.

* Skintex

According to this technique, in a 10% active solution, THS Ceramide 6 is non-irritant. This

tolerance data is confirmed by the tests carried out in vivo on health humans.


When patch tests were carried out at increasing concentrations (0.5%, 1.1%, 2.2%, 4.7% & 10%

respectively) on 20 healthy subjects, THS Ceramide 6 did not show any significant irritant reaction

at all. Its tolerance is total satisfactory.

COMEDOGENESIS

THS Ceramide 6 was tested in a 10% active solution on human volunteers, according to the usual


Due to its excellently good level of innocuity. THS Ceramide 6 has proved to be first class

emulsifier for a large number of formulae where tolerance is imperative ( dermatological cream,

anti-acne, baby cream, face cream, etc. ).

BIODEGRADABILITY

The ultimate aerobic biodegradability of THS Ceramide 6 is measured according to STRUM TEST

(OCDE 301 B, guideline EEC 84/449, Annex V. Method C5).

Under these conditions, a level of biodegradability of THS Ceramide 6 is 100% in 28 days at 50

mg/ml.

The level of biodegradability of THS Ceramide 6 is considered to be excellent.

Campo Research Singapore

December 21st 1996



CERAMIDE 3a (IIIa) (Powder)

(A BIOTECHNOLOGICALHUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE



TOLERANCE

As to ensure a good level of innocuity THS Ceramide powder was tested in in-vitro as follows:



developed by LUEPKE** in a 20% active hydrosoluble solution, THS Ceramide powder is

classified as non-irritant.


1986, 24, 6-7, 495-496.

*Cytotoxicity on human fibroblasts.


both hydrosoluble as well as liposoluble products). THS Ceramide 3 powder in a 20% active

aqueous phase and/or 20% active oily phase, does not show any signs of toxicity towards fibroblasts

in culture.

*Eyetex

According to this technique, in a 15% active solution, THS Ceramide 3 powder is totally non-

irritant.

*Skintex

According to this technique, in a 15% active solution, THS Ceramide 3 powder is non-irritant. This


*Test on healthy humans


respectively) on 20 healthy subjects, THS Ceramide wax granules did not show any significant

irritant reaction at all. Its tolerance is total satisfactory.

COMEDOGENESIS

THS Ceramide 3 powder was tested in a 15% active solution on human volunteers, according to the

usual protocols has proven to be free of comedogenic effect.

Due to its excellently good level of innocuity. THS Ceramide powder has proved to be first class



BIODEGRADABILITY

The ultimate aerobic biodegradability of THS Ceramide 3 powder is measured according to STRUM

TEST (OCDE 301 B, guideline EEC 84/449, Annex V. Method C5).

Under these conditions, a level of biodegradability of THS Ceramide powder is 100% in 28 days at

50 mg/ml.

The level of biodegradability of THS Ceramide 3 powder is considered to be excellent.


December 21st 1996



CERAMIDE 3 (III) (LIQUID) (A BIOTECHNOLOGICAL HUMAN SKIN IDENTICAL CERAMIDE DERIVATIVE-

EX-OLIVE FRUITS)

BIOTECHNOLOGICAL HUMAN SKIN IDENTICAL CERAMIDE LIQUID THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION


TOLERANCE

As to ensure a good level of innocuity THS Ceramide 3 (liquid) was tested in in-vitro as follows:



developed by LUEPKE** in a 15% active liposoluble solution, THS Ceramide 3 (liquid) is



1986, 24, 6-7, 495-496.



both hydrosoluble as well as liposoluble products). THS Ceramide 3 (liquid) in a 15% active


in culture.

* Eyetex According to this technique, in a 25% active solution, THS Ceramide 3( liquid ) is totally non-

irritant.

* Skintex

According to this technique, in a 50% active solution, THS Ceramide3 (liquid )is non-irritant. This




respectively) on 20 healthy subjects, THS Ceramide 3 (liquid) did not show any significant irritant

reaction at all. Its tolerance is total satisfactory.

COMEDOGENESIS

THS Ceramide 3 (liquid) was tested in a 50% active solution on human volunteers, according to the

usual protocols has proven to be free of comedogenic effect.

Due to its excellently good level of innocuity. THS Ceramide 3 (liquid) has proved to be first class

moisturizer for a large number of formulae where tolerance is imperative ( dermatological cream,


BIODEGRADABILITY

The ultimate aerobic biodegradability of THS Ceramide 3 liquid is measured according to STRUM

TEST (OCDE 301 B, guideline EEC 84/449, Annex V. Method C5).

Under these conditions, a level of biodegradability of THS Ceramide 3 (liquid) is 100% in 28 days at

50 mg/ml.

The level of biodegradability of THS Ceramide 3 (liquid) is considered to be excellent.


December 21st 1995



CERAMIDE 3 (III) WAX GRANULES (W/S & O/S - amphilic) (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE



TOLERANCE

As to ensure a good level of innocuity THS Ceramide wax granule was tested in in-vitro as follows:



developed by LUEPKE** in a 20% active hydrosoluble solution, THS Ceramide wax granule is



1986, 24, 6-7, 495-496.

*Cytotoxicity on human fibroblasts.


both hydrosoluble as well as liposoluble products). THS Ceramide 3 wax granules in a 20% active


in culture.

*Eyetex

According to this technique, in a 20% active solution, THS Ceramide 3 wax granule is totally non-

irritant.

*Skintex

According to this technique, in a 20% active solution, THS Ceramide 3 wax granule is non-irritant.

This tolerance data is confirmed by the tests carried out in vivo on health humans.

*Test on healthy humans


respectively) on 20 healthy subjects, THS Ceramide wax granules did not show any significant

irritant reaction at all. Its tolerance is total satisfactory.

COMEDOGENESIS

THS Ceramide 3 wax granule was tested in a 20% active solution on human volunteers, according to

the usual protocols has proven to be free of comedogenic effect.

Due to its excellently good level of innocuity. THS Ceramide wax granule has proved to be first

class emulsifier for a large number of formulae where tolerance is imperative ( dermatological

cream, anti-acne, baby cream, face cream, etc. ).

BIODEGRADABILITY

The ultimate aerobic biodegradability of THS Ceramide 3 wax granule is measured according to

STRUM TEST (OCDE 301 B, guideline EEC 84/449, Annex V. Method C5).

Under these conditions, a level of biodegradability of THS Ceramide wax granule is 100% in 28

days at 50 mg/ml.

The level of biodegradability of THS Ceramide 3 wax granule is considered to be excellent.


December 21st 1996



CLEAR COLORLESS CERAMIDE OIL COMPLEX (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)

BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE OIL-



TOLERANCE

As to ensure a good level of innocuity THS Ceramide oil was tested in in-vitro as follows:



developed by LUEPKE** in a 10% active liposoluble solution, THS Ceramide oil is classified as

non-irritant.


1986, 24, 6-7, 495-496.



both hydrosoluble as well as liposoluble products). THS Ceramide oil in a 10% active aqueous phase

and/or 10% active oily phase, does not show any signs of toxicity towards fibroblasts in culture.

* Eyetex According to this technique, in a 10% active solution, THS Ceramide oil is totally non-irritant.

* Skintex

According to this technique, in a 10% active solution, THS Ceramide oil is non-irritant. This




respectively) on 20 healthy subjects, THS Ceramide Oil did not show any significant irritant reaction

at all. Its tolerance is total satisfactory.

COMEDOGENESIS

THS Ceramide oil was tested in a 10% active solution on human volunteers, according to the usual


Due to its excellently good level of innocuity. THS Ceramide oil has proved to be first class



BIODEGRADABILITY

The ultimate aerobic biodegradability of THS Ceramide oil is measured according to STRUM TEST

(OCDE 301 B, guideline EEC 84/449, Annex V. Method C5).

Under these conditions, a level of biodegradability of THS Ceramide oil is 100% in 28 days at 50

mg/ml.

The level of biodegradability of THS Ceramide oil is considered to be excellent.


December 21st 1996



Ceramides are sphingolipids present in all cell structures. They are one of the constituents of cells cytoplasmatic membranes. Thus, they are found in skin, in the central nervous system and in spinal

marrow. Ceramides are the result of the formation of an amide bond between a fatty acid and a

sphingosine.

Ceramides are also very present in the plant, yeast, and fungi, while recently another living organism(s) have been known to afford Ceramides and Sphingolipids –

Coral Algae Seaweed –a marine oddity (that is still in the evolutionary process of between a coral

and seaweed). (See also our Marine Moisturizing Factor(s) I & II brochures)

The traditional commercial vegetal, yeast, synthetic and fungal ceramides and their derivatives are

being proposed or sold as human skin identical ceramides and sphingolipids, but these traditional ceramides are implicated as being involved in the controversy of “Cell-Apoptosis” when introduced

on the human skin and other human cells. These ceramides (other than human) and derivative(s)

which may be quite similar in structure to the human ceramides but the functionality’s can be adverse as the case of ‘cell apoptosis’ (1,2,3,4,5, and 6) (cell suicide) when different cell signals

are generated (as a novel second messenger) by these non-human ceramides.

CAMPO Biotechnological human skin ceramides (Campo THSC) range is totally different from most

of these commercially available traditional phyto, yeast, fungal or synthetic (mainly consist of squalene derivatives)– Campo THS Ceramide (as true human skin ceramides with coupled ceramide

related enzymes (appendix A) are novel end- products extracted and isolated via true novel extraction techniques (Modified Huddle Technique; high thermostable Polymerize Cell Reaction (high

heat stable PCR) for heat stable enzymes characterization, extraction & isolation) (PCR methodology-See Appendix B) and polar solvent extraction followed by liquid re-crystallization in

an organic solvent) from Biotechnological Olive Fruit Cells.(7)

Biotechnological Olive Fruit Cell are olive fruit cells which are incorporated with human DNA

structure(s) into the Olive Fruit Cellular DNA structure via use of the restriction enzymatic & slicing techniques (8) to induce bio-cellular signal transduction human skin ceramides and the related

ceramide enzymes formation.

GENERAL STRUCTURE OF THE COMPONENTS IN THE CERAMIDE RW COMPLEX

CAMPO Biotechnological HUMAN SKIN CERAMIDE RW-

(AN AQUEOUS & ETHANOL SOLUBLE- LIQUID –CRYSTALLIZED CERAMIDE)

.

Campo THS Ceramides as true identical human skin ceramides, similarly as those of all true human ceramides

exhibits antioxidant, free-radical scavenging and bacteriostatic properties, along with the other known

preventive action of Trans-Epidermal Water Loss (TEWL) etc. These combinations of properties are the

hallmark of true human skin ceramides and are responsible for the maintaining of young skin and

spontaneously exhibits non-cell-apoptosis.

CH2

OH O

CH2 O CH CH CH (CH2)13 CH3

OH OH NH

CO

R

SUGAR

PHYTOSPHINGOSINE

FATTY ACID

OH

OH



Campo THS Ceramide as a true identical human skin ceramides –contains Dihydroceramides and DAG –

(Diacylglycerol) ceramides fractions and the active enzymes, which are heat stable. These properties as

described above of True Human Skin Ceramides should be imperatively reflected as one of the property or as a

number of properties (other than common TEWL) in any of the commercially available ceramides and their

derivatives.

Otherwise such commercial ceramides are of dubious nature and should be mainly consist of the squalene

biosynthesis pathway derivatives – with ceramide like structure(s) and their only property of any viable

cosmetic use is Trans Epidermal Water Loss (TEWL)

The Campo Biotechnological Human Skin Ceramides are mainly of the six heterogeneous ceramide as found in Human skin and are the subjects involve in the functionality of maintaining Young Skin

or the lack of it (them) in Aging Skin conditions.

CAMPO BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE – CERAMIDE III

( A BACTERIOSTATIC ACTIVE CERAMIDE)

CAMPO Biotechnological CERAMIDE III (C3)

Common name: N-steroyl-sphingosine

Strurcture:

(A BACTERIOSTATIC ACTIVE CERAMIDE)CAMPO Biotechnological HUMAN SKIN

CERAMIDE –

CAMPO NOVEL CLEAR, COLORLESS CERAMIDE (LIQUID) OIL

(AN ANTIOXIDANT & FREE RADICAL SCAVENGING – EXHIBITING CERAMIDE) Due to the chemical properties, Campo Biotechnological Human Skin Ceramides have multiple usage’s in

Cosmetics, Cosmetceuticals, Pharmaceuticals and Nutriceuticals areas.

Campo Biotechnological Human Skin Ceramides were subjected to numerous studies; in analytical, in vitro &

in-vivo toxicological, and biochemical studies.

(see summary of contents of the full monograph or for information contact :-

[email protected])



II CYTOTOXICITY

a) Cell increase measurement:

Study method:

The influence of Biotechnological human skin ceramides RW ex-olive fruits on cell proliferation is evaluated

by means of two techniques:

- either cell multiplication is determined from a living cell count after an exclusion test with trypan blue

- or the cell proliferation is measured from a tritiated thymidine incorporation.

RESULTS: in-vitro analysis of fibroblast proliferation

Biotechnological human skin ceramide (ex-olive fruits) do not show any toxicity vis-à-vis the fibroblasts up to

500 mg/ml. Only a very high concentration of 2.5 mg/ml stopped cell multiplication.

In-vitro analysis of keratinocyte proliferation

Keratinocytes appeared to be a little less resistant than the fibroblast at high concentration of

Biotechnological human skin ceramides RW (ex-olive fruits, for 100 mg/ml concentrations did not inhibit cell growth.



b) Transmission electronic microscope study of Biotechnological human skin ceramides RW (ex-olive fruits) toxicity on fibroblasts:

Method:

- batch No 91200/09 (pure biotechnological human skin ceramides RW (ex-olive fruits)only at

100 mg/ml concentration,

- application: 48 hours in a D-Mem medium, containing 0.1% BSA,

- trypsinization, glutaraldehyde and epon fixation.

Results:

- absence of toxic effect, even at high biotechnological human skin ceramides RW (ex-olive fruits)

(100mg/ml): good development of ergastoplasma, presence of Golgi apparatus and of mitochondria’s, showing an active cell metabolism:

- multiple pinocytic vesicles;

- some lamellar inclusions of complex lipid type.



III Electronic microscopy of in-vitro regenerated epidermis+

a) Method:

Transmission electron microscopy is a technique frequently used for studying cell culture, as it gives

accurate information about molecular cell organization.

b) Results:

Photographs of a regenerated epidermis incubated for 48 hours with 100 mg/ml of Biotechnological human skin ceramides RW (ex-olive fruits) show a very high excess of complex lipids at the level of

the epidermis keratinocytes cytoplasm, and the presence of numerous lyposomial pseudo-myelinic figures, also at the level of the generated epidermis fibroblasts. The absence of the multilamellar

lipidic forms in the control sections (not have been incubated with the Biotechnological human skin

ceramides RW (ex-olive fruits)) (See also comments below on the comparison culture sections i.e.: Vegetal, Fungal & Yeast ceramides of competition+) seems to confirm that these structures are

Biotechnological human skin ceramides RW (ex-olive fruits) which penetrated into the cells.



Comments +Comparison cultures’ tests results of vegetal, fungal and yeast obtained ceramides incubated with well

cultures produced a detrimental results of a broad-spectrum cell apoptosis (programmed cell-death) of which

comparison results are omitted from this paper as further studies are required, as to find & understand the

cause and mechanism of the pathways involved in cell’s apoptosis.

4. Analysis of epithelial proliferation of purified Biotechnological human skin ceramides RW (ex-

olive fruits)

a) Method: A skin is regenerated in-vitro, by mixing fibroblasts with type I collagen, then implanting an

epidermis.

b) Results:

Control: Good epithelial differentiation in the presence of delipidated SVF

-One to two layers of basal cells, two three layers of large intermediary cells, and three to four

flattened superficial cells.

Absence of lipidic inclusions.

- 1% Biotechnological human skin ceramides RW (ex-olive fruits) batch

#1200/010:

Good differentiation, as with the control lattice. Multiple lamellar inclusion probably

corresponds to Biotechnological human skin ceramides RW (ex-olive fruits); equally found at the level of the intracellular spaces and in contact with desmosomes.



CLINICAL TESTS

1 EVALUATION OF CUTANEOUS SAFETY

STUDY CONDUCTED AT KOBE PHARACEUTICAL UNIVERSITY, DERMATOLOGAL POLUCLINIC.

Tested Compound: Basic cream containing 2% of Biotechnological human skin ceramides RW (ex-olive-fruits); clinical

study performed on a 20 males and 20 females-patient population of age varying between 18 to 56 years.

Results: After 8 days of daily 3X application, safety was shown to be very good, even excellent in all cases.

No patient reported or suffered any itching or stabbing pains; no desquamation was reported, nor any pityriasis, spots, papules nor pustules.

2 STUDY OF CONTACT ALLERGY:

This study was realized in the service Dermatogological Polyclinic.

Different products were tested:

- Biotechnological human skin ceramides RW (ex-olive fruits) 1% solution in propylene glycol, batch # 91/571

- Clobetasol vectorized by Biotechnological human skin ceramides RW, batch # 5134/1

- Hydrocortisone vectorized by Biotechnological human ceramides RW, batch #5136/02

- Dexamethasone vectorized by Biotechnological human skin ceramides RW, batch 4167

- Privalone diflucortolone vectorized by Biotechnological human skin ceramides RW, batch

#5123

- Betamethasone vectorized by Biotechnological human skin ceramides RW, batch#5145

- L-lactic acid 100% (ex-Campo Tomatoes ) 5% in solution of distilled water (ex-whey) and with 21% Biotechnological human skin ceramides RW in same solution, batch #9157/005

The material utilized as support is the fine chamber. The tested products were let on the patient’s back and removed 48hours later for the lecture. The test was realized on 31 patients.

RESULTS:

- 18 patients (56.06%) have negative effect balance sheet,

- 5 patients (16.13%) have a monosensibilization;

- 8 patients (25.81%) have a polysensibilization.

The products 2, 3, 4, 5, 6 and 7 do not provoke any reaction. The product 1 has provoked a

reaction on 2 patients: low soap effect, which corresponds to minimal irritation.

CONCLUSION:

NONE OF THE STUDIED PRODUCT HAVE NOTABLE ALLERGICAL OR IRRITICAL PROPERTIES.



MOISTURIZING PROPERTIES OF

Biotechnological HUMAN SKIN CERAMIDE RW [THSC] (ex-Olive fruits)

Tested compound: Cosmetic cream containing 0.5% Biotechnological Human Skin

Ceramide RW

Apparatus: Corneometer: moisturizing measurement

Study: Study performed on 7 patients for 8h15. Mean of the 7 values is expressed.

Results:

Time (hours)

0

0.5

1

2

4

6

7

% Moisturizing (mean of 7 subjects)

100

150.3

159.8

148.6

127.2

125.7

123.9

The moisturizing properties of THS Ceramide RW were studied (at Kobe Pharm. Univ. Dermatological Dept.) in a Cream, compared to its placebo.

The moisturizing kinetic was compared at different intervals and it has been shown that a cream containing 1% THS Ceramide RW was significantly different after 15 minutes at 1%, and

significantly different at 2%, after 8days of the cream application versus a placebo.

The measurement where taken with a corneometer.



Analytical Report on

1) Biotechnological human skin-identical vegetal

ceramide using Thin-layer chromatography

2) Biotechnological human skin-identical vegetal

Ceramide Analysis by infrared spectrometry



ANALYSIS REPORT ON Biotechnological HUMAN SKIN-IDENTICAL VEGETAL CERAMIDES USING THIN-LAYER CHROMATOGRAPHY

I Reagents and material:

- Standards (sigma Chemical - Co; St Louis MO): type III ceramides of bovine origin (CA),

glucocerebrosides of human origin (GLC),

mixed galactocerebrosides (GAC),

- Solvent system : chloroform - hexane - methanol - acetic acid - water (24:14:8:6:06; v/v/v/v/v),

- Derivation reagent: solution of copper sulphate (10g) in 8% phosphoric acid,

- Silicagel 60 HPTLC plates (without fluoresence indicator), 10 x 20 cm, (E.Merch, Darmstadt, Germany),

- Densitometer (CD 60 Desaga ) interfaced with a Donatec 385 x 16 computer.

II Procedures:

1 General procedure:

All separations are realized in a classic tank at room temperature; HPTLC plates are washed in the development system. After solvent evaporation using compressed air, samples and

standards are settled.

2 Standard and sample preparation:

The following solutions are prepared in chloroform - methanol mixture (2: 1,v/v):

A) Standard solutions:

* type III ceramides (CA) at 1,06 g/l, * glucocerebrosides (GLC) at 1.26 g/l,

mixed galactocerebrosides (Gac) at 1.04 g/l

B) Solutions to be analysed:

non-hydrolized Biotechnological human skin-identical vegetal

ceramides at 19.63 g/l, hydrolized Biotechnological human skin-identical vegetal ceramides

at 13.98 g/l,(appendix l).

3 Standard and sample application:

The solutions are settled according to the instructions listed in table I.

4 Chromatographic analysis

After the plates have been dried, they are developed in a tank with the solvent system up to 6 cm.

Again the plates are dried, then revealed in a copper II sulphate solution and

Heated at 1600 C for 20 minutes.

Densitometric reading is taken at 400 nm.



III Analysis report

The thin-layer chromatography of non-hydrolized Biotechnological human skin-identical vegetal

ceramides shows more than 10 spots. Two of them have an RFs of 0.81 and 0.83 (tableau II) similar

to standard. The other RF spots of 0.1; 0.2; 0.34; 0.47; are related to mono- and polyglycosyled

ceramide. The spot corresponding to type III ceramides with RF 0.83 has a poor intensity confirmed by gas chromatography coupled with mass spectrometry.

On the other hand, 0.1 and 0.47 RF spots have high intensities. This indicates a high concentration of mono- and polyglycosyled ceramides and other glycosylceramides, compared to that of

ceramides: this is why the compound was hydrolysed using Klenck's method.

The thin-layer chromatography of hydrolized compounds shows four RF spots going from 0.83 or

1.0, with a high intensity of the spots related to type III animal ceramides, a poor intensity for the sediment spots and near removal of other spots, especially those of RF 0.2; 0.34; 0.47. We may

therefore conclude that mono- and polyglycosyled ceramides were partially hydrolyzed into ceramides.

TABLE 1

TLC sediments of ceramides

TABLE II

RF Values of ceramides analyzed using TLC

COMPOUNDS RF Standards:

Type III Ceramides 0.83

Gludocerdbrosides 0.79 0.81

Gelactocerebrosides 0.75 0.77

Samples Non Hydrolized THIS-Vegetal Ceramides 0.10

0.20

0.34 0.47

0.60 0.81

0.83

0.90 1.00

Hydrolized THIS –Vegetal Ceramides 0.83 0.89

1.00

C O M P O U N D Q U A N T I T Y ( U l ) Q U A N T I T Y ( u g )

T y p e I I I c e r a m i d e s ( C A ) 5 . 0 5 . 3 0

G l u c o c e r e b r o s i d e s ( G I C ) 5 . 0 5 . 3 0

G a l a c t o c e r e b r o s i d e s ( G a C ) 5 . 0 5 . 2 0

H y d r o l y z e d T H S I - v e g e t a l 5 . 0 6 9 . 9 0

c e r a m i d e s 1 0 . 0 1 3 9 . 8 0

N o n - h y d r o l y z e d T H S I - v e g e t a l 1 0 . 0 1 9 6 . 3 0

c e r a m i d e s 2 0 . 0 3 9 2 . 6 0



ANALYSIS REPORT ON BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE RW (THSC)

1. Analysis by infrared spectometry:

1. Material

Infrared spectrometer NICOLET SX 730, Fourier trasform; infrared source: GLOBAR (silicon carbide)

2. Analysis report:

The infrared specturm reveal the presence of biatomic groups. They are essentially functional groups fractions.

The different absorption strips present in the infrared spectrum of the product correspond to the functions of the chemical structure. This observation is based on the correlation table.

CH2 lengthening is present at about 250 cm-1. The amide strip in both of its forms (symetrical

and asymetical), may easily be identified between 1550 and 1650cm-1

CH2 and ester deformation strips are found respectively at about 1420 and 1750cm-1. The secondary hydroxyl group is identified at about 3400 cm-1 and

1250 cm-1

Infrared spectrum of biotechnological human skin ceramide

References: 1) Dbaibo, G. S., Pushkareva, M. Y., Jayadev, S., Schwarz, J. K., Horowitz, J. M., Obeid, L. M. and Hannun, Y.A. (1995) Rb as a Downstream Target for a

Ceramide-Dependent Pathway of Growth Arrest. PNAS 92: 1347-1351.

2) Jayadev, S., Liu, B., Bielawska, A. E., Lee, J. Y., Nazaire, F., Pushkareva, M. Y.,

Obeid, L. M. and Hannun, Y.A. (1995) Role for Ceramide in Cell Cycle Arrest. J. Biol. Chem. 270: 2047-2052.

3) Hannun, Y. A. and Obeid, L. M. (1995) Ceramide: An Intracellular Signal for

Apoptosis. TIBS 20: 73-77.

4) Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman, J. A., Mckay, C.

and Hannun, Y. A. (1996)

5) (1S,2R)-D-Erythro-2-(N-Myristoylamino)-1-Phenyl-1-Propanol(D-e-MAPP) as an

Inhibitor of Ceramidase. J. Biol. Chem., in press.

Biotechnological Human Skin Ceramide



6) Zhang, J., Alter, N., Reed, J., Borner, C., Obeid, L. and Hannun, Y. (1996) Bcl-2

interrupts the ceramide-mediated pathway of cell death, in press.

7) European Initiative for Biotechnology Education 1995, University of

Reading,NCBE; United Kingdom

8) AaaI (restriction enzyme);ET R2; Source: Acetobacter aceti ss aceti, PT XmaIII , RS

CGGCCG, 1; RN [1], Tagami H., Tayama K., Tohyama T., Fukaya M., Okumura H., Kawamura Y., RA Horinouchi S.,

Beppu T.; FEMS Microbiol. Lett. 56:161-166(1988).

Appendix A Manual number # KAMPOYAKI Bio-molecular Drug Discovery; Training manual for enzymes

production 1989-45-05-ex.

Ceramide Enzymes: Classification, Structure, Mechanism

Introduction Enzyme Characteristics: Catalytic Power , Specificity Structure

Classification and

Mechanism of Action: Fit of Substrate and Active Site

Balancing Bond Breaking and Bond Making

Discussion

Ceramide related Enzymes (@) are the catalysts which make possible biochemical reactions in the ceramide

molecule. An important step of consideration is that biochemistry of ceramide takes place at about 37 degrees

C in -vivo and contrast that to typical reaction conditions in laboratory organic chemistry. Similar to

hydrolyzing (saponify) of fats (and similarly some commercial types of ceramide II and Ceramide III for

cosmetic formulations) which are boiled with concentrated sodium hydroxide solution for a few hours.

Ceramide transferase enzymes act similarly at body temperature in minutes.(@) Ceramides without the

coupling of related enzymes, would be occlusive, wax-like, with negligible HLB properties and extreme

hydrophobic (water-repelling) nature.

The ceramide biochemistry would not occur, and long lasting effect of smooth skin would not exist without

the ceramide related enzymes. This illustrates the impressive power of ceramide enzymes as catalysts.

These ceramide catalysts increase the rate of a reaction, but are not themselves consumed or produced by the

reaction. Also, they do not change the equilibrium constant of a reaction. This means that any catalyst which

catalyzes a reaction in one direction (e.g., esterification) also catalyzes the reverse (e.g., ester hydrolysis)

reaction.

That is; catalysts do not change the energy balance between reactants and products; catalysts do lower the

energy barrier between reactants and products.

These statements are true of ceramide enzymes as well as other types of catalysts. (see Fig A)

Ceramide Enzymes (as coupled in Campo THS Ceramides) differ from other enzymes in a very important way

– “thermal stability”.



As most Enzymes are low thermal-sensitive and degenerate in low heat(as low as 3 deg.Centigrade) as in most

cosmetic emulsion formulae – heating is used extensively).

As these enzymes are so specialized that they will catalyze a reaction of one molecule, but will leave

untouched a very similar structure-molecule. For example, any occlusive wax-like ceramide may not be

hydrolyze by a ceramide enzyme, but will hydrolyze any real ceramide of liquid or amphilic in nature.. Even

though both molecules may be characterized with identical ceramide-like structural properties.

In-house laboratory studies has shown that these structural-identical ceramides with these different activities is

due to difference in the orientation of one bond or multi-bonds at the junction of sugar (glucose) units.

These ceramide enzyme characteristics in the structure of enzymes are almost all proteins (globular proteins).

They are in terms of their primary, secondary, tertiary, and in many cases, quaternary structure are long chains

of amino acid units held together by peptide bonds, looped and folded into secondary and tertiary (and often

quaternary) structures by disulfide bonds, hydrophobic interactions, and salt bridges.

These ceramide enzymes, are active enzymes usually involve "cofactors." These co-factors are small

molecules (sometimes inorganic ions) which are needed complete the catalytically active structure of the

ceramide enzymes. Enzyme without the cofactor is called an apoenzyme, and the apoenzyme-cofactor

complex is called a holoenzyme. Protein chain of an apoenzyme can have functional groups on its side chains

(R groups) which are important to its catalytic function, but other important functional groups are introduced

by way of cofactors.

Ceramide related Enzymes are classified according to the reactions they catalyze. In some cases, the terms

used are fairly clear; in others, less so. Examples:



Transferases:

These enzymes catalyze the transfer of a group of atoms from one molecule to

another. Example of EC 2.3.1.24 involves transfer of an Acyl-CoA with + sphingosine molecule. While EC

2.3.1.76 involves transfer of an Acyl-CoA with + Retinol

ENTRY EC 2.3.1.24

NAME Sphingosine N-acyltransferase

CLASS Transeferases

Acyltransferases

Acyltransferases

SYS.NAME Acyl-CoA : sphingosine N-acyltransferase

REACTION Acyl-CoA + sphingosine = CoA + N-Acylsphingosine

SUBSTRATE Acyl-CoA

Sphingosine

PRODUCT CoA

N-Acylsphingosine

COMMENT Acts on sphingosine or its 2-epimer.

PATHWAY PATH: MAP00570 Sphingophospholipid biosynthesis

PATH: MAP00600 Sphingogylcolipid biosynthesis

DBLINKS (ref.) University of Geneva ENZYME DATABASE EC 2.3.1.24

ENTRY EC 2.3.1.76

NAME Retinol O-fatty-acyltransferase

CLASS Transeferases

Acyltransferases

Acyltransferases

SYS.NAME Acyl-CoA : Retinol O-acyltransferase

REACTION Acyl-CoA + Retinol = CoA + Retinyl ester

SUBSTRATE Acyl-CoA

Retinol

PRODUCT CoA

Retinyl ester

COMMENT Acts on palmitoyl-CoA and other logn-chain fatty-acyl derivatives of CoA.

PATHWAY PATH: MAP00830 Retinol metabolism

DBLINKS (Ref) University of Geneva ENZYME DATA BANK : 2.3.1.76



Hydrolases:

As the name suggests, these enzymes catalyze hydrolysis reactions (and their reverse reactions). The

hydrolysis of an ester would be an example of such a reaction.

ENTRY EC 3.5.1.23

NAME Ceramidase

Acylsphingosine deacylase

CLASS Hydrolases

SYSNAME N-Acylsphingosine amidohydrolase

REACTION N-Acylsphingosine + H2O = Carboxylate + Sphingosine

SUBSTRATE N-Acylsphingosine

H2O

PRODUCT Carboxylate

Sphingosine

COMMENT Acting on carbon-nitrogen bonds, other than peptide bonds in linear amides.

PATHWAY PATH: MAP00570 Sphingophospholipid biosynthesis

PATH: MAP00580 Phospholipid degradation

PATH: MAP00600 Sphingogylcolipid biosynthesis

PATH: MAP00610 Sphingogylcolipid degradation

DBLINKS University of Geneva ENZYME DATA BANK : 3.5.1.23

N-Acylsphingosine Related Enzymes (Total 9 listed.)

1.2.3.1.24 Sphingosine N-acyltransferase

2.2.4.1.47 N-Acylsphingosine galactosyltransferase

3.2.4.1.80 Ceramide glucosyltransferase

4.2.7.8.3 Ceramide cholinephosphotransferase

5.3.1.4.12 Sphingomyelin phosphodiesterase

6.3.2.1.45 Glucosylceramidase

7.3.2.1.46 Galactosyceramidase

8.3.2.1.62 Glycosylceramidase

9.3.5.1.23 Ceramidase



Conclusion

Each of these classes has more specific subclasses as well. The key to using this classification scheme

is to look at the reaction the enzyme catalyzes, decide which type of reaction it is, and apply the appropriate

name.

Specific enzyme names are systematically derived by specifying the substrate (the molecule being acted upon -

- the reactant), the type of reaction, and appending the suffix ase. Ceramidase thus is an enzyme which acts on

carbon-nitrogen bonds, other than peptide bonds in linear amides : it is therefore classified as a hydrolase

Catalytic power and specificity are the two characteristics of ceramide related enzymes which require

explanation of the activity. The structure of the enzyme's active site [the part of the enzyme's structure where

the substrate, the enzyme's functional groups and the cofactor (if any) come together] will provide us with the

beginnings of an explanation.

Since a catalyst must come in contact with the substrate to initiate any reaction, there must be a fit

between the substrate and the active site. Right away, some substrate molecules will fit and others will not, so

some substrates will react and others will not. This is specificity. The fit can come about either because the

molecule fits easily into the enzyme's active site (lock-and-key model) or because the enzyme's structure

adjusts to the substrate's entry (induced fit model).

How does catalysis occur, or, what reduces the energy barrier for reaction. Let's keep in mind that

making bonds lowers the energy of a molecule, and breaking bonds raises it. Since reactions involve both bond

breaking and bond making, a reaction's energy barrier is reduced if, in each step, the energy required to break

one bond is supplied by making another. Let's illustrate this idea by tracing through the mechanism of a

studied reaction, the hydrolysis of a peptide bond by the enzyme ceramidase. (Acting on carbon-nitrogen

bonds, other than peptide bonds in linear amides)



All You Ever Wanted To Know About

Enzymes

What is an enzyme?

Simple definition=organic catalyst

More complex definition= an organic substance(usually a globular protein) that lowers the activation

energy needed for a reaction to occur(be sure to check out the graph of activation energy that is in

your biology text book.)

Enzyme structure.

An enzyme's 3 dimensional structure is critical to its function. The "lock and key" analogy is often

used, a substrate fits an enzyme like a key fits a lock.

The site on the enzyme that "fits" the substrate is called the active site.

Basic Information.

An enzyme has only one function, it catalyzes only one reaction or one specific type of reaction.

There are thousands on enzymes in living things, each doing only one thing.

Enzymes are not used up in the reaction.

Enzymes work on a variety of reactions; syntheses, lysis, energy transfers, etc.

Enzymes do not change the equilibrium state of the reaction, they just speed up the time to

equilibrium.

Enzymes cannot cause a reaction to occur that does not otherwise occur.

The suffix -ase often is used in enzyme names.(e.g. maltase, catalase, reverse transcriptase, etc.)

Factors affecting the rate of enzyme catalyzed reactions.

Enzyme concentration

Substrate concentration

Product concentration

pH

Temperature

Concentration of salts

Presence of cofactors(inorganic) and coenzymes(organic)

Presence(or absence) of enzyme inhibitors

Enzyme Inhibition

Enzyme inhibitors are substances that inactivate the enzyme by changing the shape of the enzyme

molecule or blocking the active site.

Competitive inhibitors block the active site(they "compete" with the substrate).

These competitive inhibitors resemble the shape of the substrate and fit into the active site.

Noncompetitive inhibitors change the shape of the enzyme by binding to the molecule at a site other

than the active site.

Many poisons are enzyme inhibitors(e.g. pesticides, antibiotics, cyanide, etc.)



Enzyme Turnover

Enzyme activity is measured by turnover number, the number of molecules acted upon per unit of time. The

following table gives some turnover numbers in molecules per second.

(From McMurry and Castellion.1992. General, Organic, and Biological Chemistry. Prentice Hall.)

Enzyme Turnover number(per second)

Carbonic anhydrase 600,000

Acetycholinesterase 25,000

-Amylase 18,000

Penicillinase 2,000

DNA Polymerase 15

Prepared by Campo Professor Date: September 5. 1996

Appendix B

PCR Thermal Profiles for Biotechnological Human Skin Ceramides coupled with related

ceramide enzymes in a high heat (thermo-stable) extraction environment of cellular DNA material of

Biotechnological olive cells Soup in GeneAmp Reaction Tubes.

Manual number # KAMPOYAKI Bio-molecular Drug Discovery Extraction and characterization

methodology1990-23-05

Using the GeneAmp PCR System 9600 (or GeneAmp PCR System 2400) with the two-temperature

PCR protocol and GeneAmp PCR Reagents, amplification of the Lambda control target DNA is guaranteed

with a 15-second, 94 °C denaturation step and a 1-minute, 68 °C primer annealing/ extension step. This will

amplify a 500 bp product at least 105-fold in 25 cycles, taking about 2.3 minutes per cycle.

Using the DNA Thermal Cycler 480, with the two-temperature PCR protocol and GeneAmp PCR

Reagents, amplification of the Lambda control target DNA is guaranteed with a 1-minute, 94 °C denaturation

step and a 2-minute, 68 °C primer annealing/ extension step. This will amplify a 500 bp product at least 105-

fold in 25 cycles, taking about 4.25 minutes per cycle.

Using the DNA Thermal Cycler with the three-temperature PCR protocol and GeneAmp PCR

Reagents, amplification of the Lambda Control DNA is guaranteed with a 1-minute, 94 °C denaturation step, a

1-minute, 37 °C primer annealing step and a 2-minute, 72 °C primer extension step. This will amplify a 500 bp

product at least 105-fold in 25 cycles, taking about 6.5 minutes per cycle.

DNA denaturation is the critical step in the GeneAmp PCR process and is often the focus of attention

if PCR experiments fail. The practical range of effective denaturation temperatures for most samples is 94

°C96 °C.

On the GeneAmp PCR System 9600 (or GeneAmp PCR System 2400), the computed sample

temperature is used to time the incubation periods and 15-second denaturation times are routinely used.

Sufficient time must be allowed for thermal equilibration of the sample at this high-temperature

plateau. On the DNA Thermal Cycler 480 and the DNA Thermal Cycler, the average block temperature is used

to time the incubation periods. At least 1 minute must be specified for sample temperature equilibration for the

most reliable amplification in standard 0.5 mL GeneAmp PCR Reaction Tubes. Denaturation can often occur

in the final seconds of the 1-minute incubation segment. (Shorter hold times may be specified only when using

GeneAmp Thin-Walled Reaction Tubes on the DNA Thermal Cycler 480.)

Annealing temperature is based on the Tm (melting temperature) of the oligonucleotides chosen for

PCR amplification. If unwanted bands are observed, the annealing temperature is raised in 2 °C5 °C

increments in subsequent optimization runs. While the primer annealing temperature range is often 37 °C55

°C, it may be raised as high as the extension temperature in some cases. In fact, high-temperature annealing

should result in enhanced specificity. The merging of the primer annealing and primer extension steps results

in a two-step GeneAmp PCR process, which has been successful in many applications, including those using

the GeneAmp PCR Reagent Kit bacteriophage Lambda Control DNA.



Primer extension, in most applications, occurs effectively at a temperature of 72 °C and seldom needs

optimization. In the two-temperature GeneAmp PCR process, this temperature range may be 60 °C70 °C. All

GeneAmp PCR Instrument Systems are able to automatically increase the extension time linearly with cycle

number.

This technique may enhance yield, especially In situations where the enzyme concentration limits

amplification in late cycles. Typically, 2545 cycles are required for extensive amplification (i.e., 106-fold) of

a specific target.

Hot Start PCR

The major obstacle to routine, sensitive and specific PCR amplification appears to be competing side reactions

such as the amplification of non-target sequences in background DNA (mis-priming) and primer-

oligomerization. This mis-priming and primer-oligomerization occurs mainly during pre-PCR setup when all

reactants have been mixed, usually at room temperature, before thermal cycling is started.

In the Hot Start technique, reagent addition to the reaction tube is designed so that all reactants do not

mix until reaching a temperature high enough to suppress primer annealing to non-target sequences. Typically,

in manual Hot Start, all reactants except Taq DNA Polymerase are mixed at room temperature below the

mineral oil cap. Then, after all tubes have been loaded into a GeneAmp PCR Instrument System, and the

temperature has been raised to 70 °C80 °C, enzyme is added separately to each tube, changing pipet tips after

each sample. Although manual Hot Start can increase amplification specificity and yield, it is inconvenient and

can cause reproducibility and contamination problems.

AmpliWax PCR Gem 100 (P/N N808-0100) and AmpliWax PCR Gem 50 (P/N N808-0150) are

precisely aliquoted beads of specially cleaned and formulated wax. After a single AmpliWax PCR Gem is

added to a reaction tube containing a subset of amplification reagents, the tube is heated to 70 °C80 °C for 5

to 10 minutes, then cooled to create a wax barrier over the aqueous layer. At the time of use, the omitted

reagent(s) and the test sample are added above the solid wax layer. After all tubes for an experiment have been

assembled at room temperature, conventional thermal cycling is started. Rapid heating to the first denaturation

segment melts the wax layer, denatures the DNA template and creates enough thermal convection to assure

complete mixing of all PCR components under the melted wax (which also serves as a vapor barrier during

cycling). After cycling, the wax forms a solid shield preventing spillage and evaporation. It is simply

penetrated by a pipet tip to withdraw the PCR product for analysis and can be reheated to seal for long-term

storage. Chemical Hot Starts can be achieved using AmpErase Uracil N-Glycosylase or the novel AmpliTaq

Gold.

In the presence of AmpErase and dUTP, pre-PCR primed products will be cleaved at the dU-

containing sites by UNG with a pre-PCR incubation at room temperature and subsequent denaturation at 94

°C. Specific amplification can then be achieved following recommendations for amplification with the use of

AmpErase. AmpliTaq Gold is a modified version of AmpliTaq DNA Polymerase provided in an inactive form

which is then heat activated. Hence, PCR setup on many samples can be performed at room temperature

without concern for extension at misprimed sites. AmpliTaq Gold can be completely or partially activated in a

pre-PCR heat step (conventional Hot Start) or can be allowed to activate slowly during thermal cycling (Hot

Start and Time Release PCR). By increasing the amount of AmpliTaq in the reaction slowly with cycle

number, specific product yield is increased without buildup of misprimed products.



Suggestions for Successful PCR Product Analysis

PCR products are usually analyzed by ethidium bromide-stained agarose gel electrophoresis,

Southern blotting/probe hybridization, fluorescence assay or with the Perkin-Elmer HPLC System for PCR

Analysis.

If there is no yield of the desired PCR product, reproducible addition of the enzyme should be confirmed,

preferably in a master mix. Complete DNA denaturation should be ensured in each cycle by using optimized

GeneAmp Reaction Tubes and by allowing sufficient time at the denaturation plateau temperature. Slightly

higher denaturation temperatures should be checked and the chemical integrity of the

primers should be considered. Preincubation at 95 °C for 5 to 10 minutes in the absence of enzyme to

inactivate harmful proteases, or nucleases in the sample, is often helpful. This preincubation also ensures

complete denaturation of complex starting templates. Consider performing the Hot Start technique.

If a smear of PCR products is seen on the agarose gel, consider the following options: reduce the AmpliTaq

DNA Polymerase concentration and use master mixes; increase the annealing temperature and consider the

two-temperature PCR protocol; reduce the magnesium ion concentration; minimize the incubation times of the

annealing and extension steps (use at least 1 minute for best uniformity in GeneAmp Reaction Tubes);

decrease the number of cycles; do a second amplification with a set of nested primers; and run molecular

weight standards to check gel electrophoresis. (Avoid changing too many parameters simultaneously focus on

enzyme, primer and magnesium ion titrations and test higher annealing temperatures.)

If a primer-dimer artifact band is seen on the agarose gel, check primer sequences for 3' complementarity;

design longer primers; increase the amount of target DNA; reduce the primer concentration; reduce the number

of cycles; and raise the annealing temperature.



DISCLAIMER : The information contained herein is accurate to the best knowledge and belief of Campo Research Pte Ltd, and specification quoted may change without prior notice. Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, Campo Research Pte Ltd, however, cannot assume any liabilities or risks involved in the use of its natural products or their derivatives or raw materials or ingredients, since the conditions of use are beyond Campo Research Pte Ltd’ s control. Statements concerning the possible use are not intended as recommendations to use our materials in the infringement of any patents or infringements of mandatory regulatory requirements or without any safety evaluations conducted when used in combination with materials of other suppliers.. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specifications. Campo Research Pte Ltd accepts no liabilities of whatsoever either expressed or as otherwise arising out of the information supplied, the application, adaptation or processing of the products described herein, or the use of other materials in lieu of the Campo materials or the use of Campo raw materials or ingredients in conjunction with any other products and raw materials. The use of Campo Research Pte Ltd's raw materials or ingredients in any formulations are to be compulsory tested and to be assayed for safety and toxicology profiles evaluations and according the mandatory regulations as required by the laws and regulations of the countries where the evaluation and use of Campo Research Pte Ltd's raw materials or ingredients has been formulated as single components in any carrier systems or as in multi-components formularies. The end-users, marketers; manufacturers, formulation laboratories or importers of Campo Research Pte Ltd' raw materials and ingredients which are incorporated into any formularies as formulated or re-sold or re-exported or assayed in accordance with any mandatory regulatory requirements of any country or infringement of any patents assume all liabilities as that may arise out of the use of Campo Research Pte Ltd's raw materials and ingredients in any formularies in combination with raw materials and ingredients of other suppliers or as single components in any carriers. The definition of users as mentioned in these instances are manufacturers, marketers, formulation laboratories, consultants, and importers assumed all liabilities arising as either personal injuries suits, infringements of patents suits, infringements of or failures to meet regulatory requirements suits of a formulary either as single components in any carrier systems or in as multi-components formularies in which are may consist of a Campo Research Pte Ltd's raw material or ingredients.

IMPORTANT NOTICE Specifications may change without prior notice. Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk involved in the use of its natural products or their derivatives, since the conditions of use are beyond our control. Statements concerning the possible use are not intended as recommendations to use our products in the infringement of any patent. We make no warranty of any kind; expressed or implied, other than that the material conforms to the applicable standard specifications.

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