Bacocalmine - clinical information - soothing, anti-irritant and fighting skin redness

This page contains the clinical data that Sederma provides on their patented ingredient Bacocalmine™. To view a more "user friendly" page that deals with Bacocalmine - the extract of Bacopa monnieri - please click here.

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Bacocalmine is used in the following products Problem Skin Day Gel Problem Skin Night Care Lotion Problem Skin Spot Treatment Problem Skin Mask

Taking as our starting point the studies of Dar and Vohora, we decided to demonstrate in vitro and ex vivo the following properties :

• Anti-free radical properties
  • DPPH°
  • ¹O₂ singlet oxygen
  • Anti-oxidant properties ex vivo
  • Anti-oxidant effect in cells
• Membrane anti-lipoperoxidation properties (cell tests)
  • Protection of cell viability
• Anti-inflammatory effects (anti-PGE2 tests in human biopsies after UV exposition)
  • Anti-irritant and anti-inflammatory properties in vivo
    • Volunteer inclusion criteria
  • Irritation by a-hydroxy-acids
  • Inflammatory reaction induced by 5% SDS on occlusive patch

Anti-free radical and anti-oxidant properties in vitro

DPPH°

The anti-free radical properties of BACOCALMINE™ are illustrated by its antagonistic action on DPPH°.

DPPH° is a highly reactive free radical with a non-paired electron which stabilizes itself by capturing a hydrogen atom : this stabilization reaction is accompanied by a change in absorbance which is measured at 517 nm.

Any substrate capable of conceding a hydrogen atom to DPPH° is positive in this in vitro test and the intensity of coloration decreases in proportion with the anti-free radical power of the substance enabling conversion of DPPH° to DPPH2.

Table 1 illustrates results obtained with increasing concentrations of BACOCALMINE™.

Results show a marked anti-free radical effect proportional to the concentration of BACOCALMINE™. Anti-free radical effect reached 80% when the active substance was incorporated at a concentration of 1%.

¹O₂ singlet oxygen

A second series of tests evaluated capacity to detoxify singlet oxygen, known for its powerful oxidant effect.

Certain dyes, including Rose Bengal, have the ability to produce singlet oxygen if exposed to UV-A or visible light. The singlet oxygen produced is capable of attacking an added target substrate such as uric acid, converting it to allantoin. Any substance capable of inactivating 1O₂ is positive in this test, by protecting uric acid.

Incremental concentrations of BACOCALMINE™ were added to the reactive mixture containing uric acid. After irradiation for 30 minutes in the visible spectrum, unchanged uric acid was assayed by measurement of absorbance at 292 nm. Results are shown in Table 2.

BACOCALMINE™ had a very effective protective dose-related action, reaching 69% at 3%. BACOCALMINE™ offers effective protection against oxidative effect by singlet oxygen.

Anti-oxidant properties ex vivo

In order to validate ex vivo the above results obtained with singlet oxygen, BACOCALMINE™ was formulated at 1 and 3% in a carbomer gel then applied to the skin of volunteers. After complete penetration of the product, the first and second layers of keratinocytes were collected using an adhesive then left in contact with uric acid for 1 hour. After removal of the adhesive, rose bengal was added, followed by irradiation. Non-converted uric acid was measured as before (Table 3).

While the dummy product was inactive, the anti-free oxidative activity of BACOCALMINE™ was confirmed ex vivo with a dose-related effect. About 20% protection was obtained after an application of 3% BACOCALMINE™ to the skin.

Anti-oxidant effect in cells

It is well established that lipid peroxidation is a major cause of damage to cell membrane lipids. This self-catalytic process starts by plucking of a methylene hydrogen atom (e.g. : in the presence of heavy metals, UV) from a polyunsaturated fatty acid, creating a fatty radical. After reaction with oxygen to form a radiculated lipoperoxide, secondary breakdown products are generated (self-catalytic process), in particular aldehydes such as 4-hydroxynonenal and malonaldehyde.

4-hydroxynonenal is one of the chief breakdown products of w6 polyunsaturated acids. Malonaldehyde is the breakdown product most heavily generated from lipoperoxides.

A cell model was used in our test : fibroblasts. BACOCALMINE™ was tested at different concentrations and its effect was compared with irradiated control cells in the absence of the product.

Two aspects were considered : damage done by UV-A to cell membrane lipids (12 Joules/cm²) and its consequences regarding cell viability (mortality induced at 16 J/cm²).

Prevention of membrane damage

The formation of 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) was monitored by spectophotometric assay at 586 nm of the appearance of a color reaction substance obtained in presence of the substrate N-methyl-2-phenylindole. Results were as follows :

BACOCALMINE™ decreases the peroxidation of cell lipids. This lipid protective effect is obtained immediately at low concentrations of the substance. Mean protection is 50% with 0.5% BACOCALMINE™.

Protection of cell viability

In order to illustrate this protective effect, a further experiment was done exposing cells to a larger dose of UV-A (16 J/cm²). In these conditions, membrane peroxidation results in a loss of viability of about 20%, measured by a Hoechst test.

It was of interest to see whether BACOCALMINE™ would improve cell survival, in accordance with its anti-oxidant action.

It can be seen here that the survival of cells irradiated in the presence of BACOCALMINE™ was good while cells irradiated in dummy medium showed about 20% mortality. The highly probable explanation of these results is decreased membrane peroxidation, in agreement with results already obtained previously (45% and 50% protection at 0,3 and 0,5% respectively).

This effect is illustrated by photo 1 showing an unprotected cell layer damaged after UV-A irradiation, in comparison with unaffected cell density in the presence of 0,5% BACOCALMINE™, photo 2

Untreated cells	Cells treated with Bacocalmine

Anti-inflammatory properties in human biopsies

The anti-inflammatory process induced by various types of cell stress, in particular cell membrane lipid peroxidation, is related to a so-called "cascade" of events started by membrane release of oxidated fatty acids producing arachidonic acid, which is then converted to prostaglandins (including PGE2).

At any event, a cell membrane protector must slow or even suppress the appearance of these inflammatory mediators of lipid origin : a reduced level of PGE2 production is a good index for evaluating the anti-inflammatory activity of a substance.

The following test used human biopsies (abdominal skin) maintained in survival in an appropriate culture medium (DMEM). Skin fragments were incubated for 6 hours in the presence of culture medium, with or without (control) the addition of various concentrations of BACOCALMINE™ :

The medium was discarded and skin samples rinsed, 2 batches were exposed to moderate and high dose UV-B irradiation : 200 and 1000 mJ/cm². They were then returned to culture medium incorporating BACOCALMINE™ for 24 hours. PGE2 release was assayed by ELISA (PGE2 antibodies). (Tests were done in quintuplicate, Table 5).

Prostaglandin release was seen to a similar degree in skin samples exposed to moderate and high UV irradiation. The presence of BACOCALMINE™ in media resulted in a dose-dependent decrease in production of the inflammation mediator. The highly significant (p<0.05) 43% and 58% decreases obtained with 5% BACOCALMINE™ clearly demonstrate an anti-inflammatory action on UV irradiated human skin. These results confirm in human skin earlier results concerning decreased membrane peroxidation and the persistence of cell viability after UV-B irradiation.

Anti-irritant and anti-inflammatory properties in vivo

Ten volunteers participated in a dual placebo-controlled clinical trial of 3% BACOCALMINE™ :

• A study of soothing effect on irritation induced by 7.5% a-hydroxy-acids (lactic acid, glycolic acid, citric acid)
• A study of anti-inflammatory effect after induction by 5% SDS.

Volunteer inclusion criteria

Volunteers were enrolled on the basis of the following specific inclusion criteria : individuals without allergic predisposition, and individuals not taking any systemic or topical anti-inflammatory, analgesic nor tranquillizer type treatment.

Irritation by a-hydroxy-acids

Protocol

The 10 female volunteers (age between 21 and 48, mean age 30) applied to half of the face at random either a carbomer gel containing 3% BACOCALMINE™ or a placebo gel.

Both substances were applied twice a day for 2 weeks. On day 16, at T0, 1 ml of a solution of a mixture of 7.5% a-hydroxy-acid in water-alcohol solution was applied to each half of the face by dabbing with cotton wool (Appendix 1). Irritation induced by a-hydroxy-acids takes the form of stinging. Each volunteer evaluated for 10 minutes the effect felt, using a scale from 0 (no stinging) to 4 (marked stinging).

This test is a variant of the so-called "stinging" test widely used in cosmetology. Here it measured an effect due to daily use of the product and provided a better evaluation of benefits to be expected by usage closer to standard conditions.

Results

Scores obtained (Table 6) were analyzed by a t test to compare variations in means between the site treated with 3% BACOCALMINE™ and the placebo site.

The figure below shows a clear difference between scores on the placebo and BACOCALMINE™ sites. The half-face treated with 3% BACOCALMINE™ is less irritated by a-hydroxy-acids comparatively to placebo, with an average difference of 60%, highly significant up to 10 minutes.

Volunteers were classified according to the difference in stinging felt between the two sites using the following criteria :

• Slight decrease if difference of 1 score unit
• Clear decrease if difference of 2 score units
• Marked decrease if difference of 3 score units

100% of panellists experienced a decrease in irritation, including 55.6% clear to marked.

These results justify the conclusion of an anti-irritant effect of BACOCALMINE™ used at 3% against a-hydroxy-acids.

Inflammatory reaction induced by 5% SDS on occlusive patch

Protocol

The same volunteers applied twice daily for 2 weeks 3% BACOCALMINE™ or placebo at random to the left or right forearm. On D16, two sites previously identified on each forearm were selected and an occlusive patch of 5% SDS (sodium dodecyl sulfate) applied for 4 hours. Patches were removed at t = 4 hours. Measurements were made t = 1 hour after removal of the patch and compared with those obtained before the patch was fitted at T0.

SDS-induced inflammation takes the form of redness of the skin and the possible appearance of micro-spots. Objective quantification being impossible just by visual reading, variations in micro-capillary blood flow were measured by Doppler-Laser.

Results

Among the 10 subjects exposed to 5% SDS patches, measurements in 3 volunteers led to no result in the absence of SDS-induced inflammation. Results below concern the 7 responders.

Both forearm sites were sensitive to irritation caused by 5% SDS, with an inflammatory type reaction leading to increased blood flow in skin capillaries and with visual evidence of typical redness.

Measurements revealed a 126% increase in micro-cutaneous flow on placebo while it was only 102% on BACOCALMINE™.

These results show a marked anti-inflammatory action of -24% in comparison with placebo.

In certain individuals, the effect persisted sufficiently long for a marked visual difference between the placebo site and BACOCALMINE™ site to be seen 24 hours after the final application, as illustrated by the photos below.

Conclusion

These 2 clinical trials involving irritation induced by a-hydroxy-acids and inflammation caused by 5% SDS offer proof of the efficacy of BACOCALMINE™ used daily at 3% as a soothing and anti-inflammatory agent.