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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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 3
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.
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 4
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 5
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
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
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 6
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%
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 7
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.
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 8
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 Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 9
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 10
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 11
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 12
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 13
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 14
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 %
Campo Ceramide 6 (VI) 03.00
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.
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 15
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
Campo Ceramide 6 (VI) 01.00
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.
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 16
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 19
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
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 20
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 24
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
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 25
CAMPO CERAMIDE 3a (IIIa) -
TRUE HUMAN SKIN IDENTICAL SPHINGOSINE
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.
The use of skin’s Bio-Aqueous liquid as an identical carrier/menstrum in the extraction of true
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
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, , 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 )
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 ( i.e.: 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 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
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 3 triggers the natural CoA fatty lipids
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 26
sensitivity, photo caused irritation as experienced in synthetic Retinol based ( Tretinoin )
creams application for acne, and fine wrinkles.
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 ( 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
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.
Campo THSI Ceramide 3a ( similarly identical to natural skin ceramide 3 ) 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 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
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.
TOXICOLOGICAL 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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 27
BOTANICAL INFORMATION – LATIN Aloe Vera flora callus
- ENGLISH Aloe leaf cells
INCI/CTFA NAME CERAMIDE III ( CERAMIDE 3 )
PLANT MATERIAL Aloe Leaf Cells - biotechnological
ACTIVE COMPONENTS OF PLANT Sphingosine and Transferases Enzymes, and
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
TOTAL MICRO COUNT < NIL cfu/ml
TOTAL YEAST/MOLD COUNT < NIL cfu/ml
PATHOGENIC BACTERIA 0
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.
CAMPO RESEARCH, SINGAPORE
Level 30, 6 Battery Road
SINGAPORE 049909
Tel: 63833202 / Fax: 63834034
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 28
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 Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 29
CAMPO CERAMIDE 3 (III) -
TRUE HUMAN SKIN IDENTICAL SPHINGOSINE
Art No. 96/10/00550-100
uman Skin Identical CERAMIDE III ( 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.
The use of skin’s Bio-Aqueous liquid as an identical carrier/menstrum in the extraction of true
human skin identical Ceramide IIIa from biotechnological olive fruit cells; the end-product
Ceramide III ( 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, , 3b, 4, 5, 6, 6I and 6 II to exhibit a high
HLB, Not less than a minimum HLB 15 .
Ceramide III is an exceptional to the HLB value.
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 ( water repelling ) , 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 ( i.e.: 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 III 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 3 triggers the natural CoA fatty lipids
present in the skin to metabolize the Retinol content ( increase in natural content ) and an
H
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 30
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.
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 ( 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
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.
Campo THSI Ceramide 3 ( similarly identical to natural skin ceramide 3 ) 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 3 ( III ) 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
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.
TOXICOLOGICAL 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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 31
BOTANICAL INFORMATION - LATIN Olea europea fruit callus
- ENGLISH Olive Fruit cells
INCI/CTFA NAME CERAMIDE III ( CERAMIDE 3 )
PLANT MATERIAL Olive Fruit Cells - biotechnological
ACTIVE COMPONENTS OF PLANT Sphingosine and Transferases Enzymes, and
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 III ) 36 - 45 %
pH-VALUE ( 1% in water ) 4.0 - 7.5
Water Content 6 - 9 %
EXTRACTION VEHICLE Liquid Nitrogen at -1,900 Deg C ( minusZero)
PRESERVATION None
TOTAL MICRO COUNT < NIL cfu/ml
TOTAL YEAST/MOLD COUNT < NIL cfu/ml
PATHOGENIC BACTERIA 0
Appearance Liquid
COLOUR Amber - golden brown
ODOUR Characteristic minimal
APPLICATION Anti-Acne products : 0.5 - 5 %
After Shave preparation : 0.2 - 03 %
Skin-Care preparations : 0.5 - 2.0%
Hair-Care preparations : 0.5 - 1.5%
Anti-Perspirant & Deodorant : 0.25 -0.5%
Store in a closed container in a dry &dark
place.
CAMPO RESEARCH, SINGAPORE
Level 30, 6 Battery Road
SINGAPORE 049909
Tel: 63833202 / Fax: 63834034
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 32
CAMPO CERAMIDE 3 (III)-Wax Granules (w/s & o/s -amphilic)
TRUE HUMAN SKIN IDENTICAL SPHINGOSINE
Art No. 96/10/00550-100wax-amphilic
uman Skin Identical CERAMIDE III ( 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.
The use of skin’s Bio-Aqueous liquid as an identical carrier/menstrum in the extraction of true
human skin identical Ceramide IIIa from biotechnological olive fruit cells; the end-product
Ceramide III ( 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, , 3b, 4, 5, 6, 6I and 6 II to exhibit a high
HLB, Not less than a minimum HLB 15 .
Ceramide III is an exceptional to the HLB value.
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 ( water repelling ) , 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 ( i.e.: 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 III 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 3 triggers the natural CoA fatty lipids
present in the skin to metabolize the Retinol content ( increase in natural content ) and an
H
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 33
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.
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 ( 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
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.
Campo THSI Ceramide 3 ( similarly identical to natural skin ceramide 3 ) 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 3 ( III ) 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
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.
TOXICOLOGICAL PROPERTIES
Dermal Evaluation ( 100% in 10ml of Water )
48 Hour 100/100 completely non-irritating
Human Patch Test ( non-erythema causing THSI ceramide )
100 Test Subjects
IN-VITRO OCULAR EVALUATION ( 10 % soluble In 10ml Water )
Ropak, Eyetex Eyetex Classification
Rapid Membrane Assay Minimal/ Mild
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 34
BOTANICAL INFORMATION - LATIN Olea europea fruit callus
- ENGLISH Olive Fruit cells
INCI/CTFA NAME CERAMIDE III ( CERAMIDE 3 )
PLANT MATERIAL Olive Fruit Cells - biotechnological
ACTIVE COMPONENTS OF PLANT Sphingosine and Transferases Enzymes, and
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 III ) 36 - 45 %
Water Content 6 - 9 %
EXTRACTION VEHICLE Liquid Nitrogen at -1,900 Deg C ( minusZero)
PRESERVATION None
TOTAL MICRO COUNT < - cfu/ml
TOTAL YEAST/MOLD COUNT < - cfu/ml
PATHOGENIC BACTERIA -
Appearance Waxy Hardened Granules
COLOUR Amber - golden brown
ODOUR Characteristic minimal
APPLICATION Anti-Acne products : 0.5 - 5 %
After Shave preparation : 0.2 - 03 %
Skin-Care preparations : 0.5 - 2.0%
Hair-Care preparations : 0.5 - 1.5%
Anti-Perspirant & Deodorant : 0.25 -0.5%
Store in a closed container in a dry &dark
place.
CAMPO RESEARCH, SINGAPORE
Level 30, 6 Battery Road
SINGAPORE 049909
Tel: 63833202 / Fax: 63834034
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 35
TOXICOLOGY PROFILE
Ceramide 6 (VI)
Ceramide 3a (IIIa) (powder)
Ceramide 3 (III) (liquid)
Clear Colourless Ceramide Oil Complex
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 36
CERAMIDE 6 (VI) (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)
Biotechnological HUMAN SKIN CERAMIDE
THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION
TOXICOLOGICAL & ECOTOXICOLOGICAL DATA
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.
When tested on the chorio-allantoic membrane of a chicken egg, according to the technique
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.
When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to
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.
* Test on healthy 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
protocols has proven to be free of comedogenic effect.
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
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 37
CERAMIDE 3a (IIIa) (Powder)
(A BIOTECHNOLOGICALHUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)
BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE
THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION
TOXICOLOGICAL & ECOTOXICOLOGICAL DATA
TOLERANCE
As to ensure a good level of innocuity THS Ceramide powder was tested in in-vitro 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 20% active hydrosoluble solution, THS Ceramide powder 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.
When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to
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
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%, 1.0%, 2.0%, 4.5% & 15%
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
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 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.
Campo Research Singapore
December 21st 1996
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 38
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
TOXICOLOGICAL & ECOTOXICOLOGICAL DATA
TOLERANCE
As to ensure a good level of innocuity THS Ceramide 3 (liquid) was tested in in-vitro 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 15% active liposoluble solution, THS Ceramide 3 (liquid) 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.
When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to
both hydrosoluble as well as liposoluble products). THS Ceramide 3 (liquid) in a 15% active
aqueous phase and/or 15% active oily phase, does not show any signs of toxicity towards fibroblasts
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
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%, 5.0%, 10.0%, 50.0% & 100%
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,
anti-acne, baby cream, face cream, etc. ).
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.
Campo Research Singapore
December 21st 1995
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 39
CERAMIDE 3 (III) WAX GRANULES (W/S & O/S - amphilic) (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)
BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE
THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION
TOXICOLOGICAL & ECOTOXICOLOGICAL DATA
TOLERANCE
As to ensure a good level of innocuity THS Ceramide wax granule was tested in in-vitro 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 20% active hydrosoluble solution, THS Ceramide wax granule 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.
When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to
both hydrosoluble as well as liposoluble products). THS Ceramide 3 wax granules 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 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
When patch tests were carried out at increasing concentrations (0.5%, 1.1%, 2.2%, 4.7% & 20%
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.
Campo Research Singapore
December 21st 1996
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 40
CLEAR COLORLESS CERAMIDE OIL COMPLEX (A BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE DERIVATIVE - EX-OLIVE FRUITS)
BIOTECHNOLOGICAL HUMAN SKIN CERAMIDE OIL-
THE ACTIVE NOVEL DRUG FOR THE COSMETIC FORMULATION
TOXICOLOGICAL & ECOTOXICOLOGICAL DATA
TOLERANCE
As to ensure a good level of innocuity THS Ceramide oil was tested in in-vitro 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, THS Ceramide oil 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.
When tested on human fibroblasts using a method patented by BIOGIR (which can be applied to
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
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% & 10%
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
protocols has proven to be free of comedogenic effect.
Due to its excellently good level of innocuity. THS 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 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.
Campo Research Singapore
December 21st 1996
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CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 41
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
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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 :-
Campo Novel Liquid & Soft-Paste Ceramides
CAMPO CD VERSION 3.7.2 dated 23 November 2013 © 2013 Library of Congress Wash.DC 43
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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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
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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
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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”.
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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:
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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
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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
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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)
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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.)
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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.
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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.
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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.
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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.