The microflora is an important part of the skin. Sebum and corneocytes as well as sweat create the ideal feeding ground for the tolerated microbes. The present article describes a new active containing bioflavonoids from Maclura cochinchinensis, which was tested for its capability to rebalance the disturbed microflora of oily skin and, in turn, reduce acne blemishes as well as axillary odor.
The Microbial Settlement on the Skin
The skin is our largest organ, with a surface area of roughly 1.7 square meters. It protects us from the environment, is important for thermoregulation and is definitely key for our appearance. However, we are not alone in our skin—millions of small companions are settling on our skin's surface: microbes. In total, 100 billion bacteria and fungi use our skin as their habitat.1 This incredibly large number corresponds to the entirety of our skin cells.2
Recent advances in microbiome analysis techniques revealed a much higher bacterial diversity on skin than previously detected by culture-based methods. For example, studies by the authors' company on acne prone skin have revealed bacteria from six phyla, more than 80 families and no less than 500 different species. One can imagine this is only a fraction of what can be found in normal skin. Furthermore, skin of different ethnicities and of people who live on different continents may harbor even greater microbe diversity.
The skin's microflora is an important guard against potentially harmful intruders. By occupying all skin niches (see Figure 1), the settlement of germs is strongly reduced. Sweat also plays an important role to provide the best conditions for this beneficial microflora in that it creates a slightly acidic pH on the skin surface.3
Microflora produce a human being’s typical scent. In moist areas, in particular, apocrine sweat glands secrete a special sebum composition in which Staphylococci, Corynebacteria and Bacilli degrade sebum, dead cell material and components of the skin surface. These produce body odor by releasing their metabolites.
Skin Problems Due to Imbalanced Microflora
An imbalanced microflora condition—referred to as dysbiosis—can cause skin problems such as acne, atopic eczema and fungal infections. Despite this, the microflora is not the root cause of skin problems, but rather a reaction to dysregulated skin functions.
Microorganisms typically respond to the "food supply" of the skin. In the case of greasy skin, a lot of sebum is available, which supports the growth of Staphylococci and Propionibacteria. The latter, being trapped in anaerobic zones of clogged skin pores due to hyperkeratinization, are the main drivers of acne vulgaris and the formation of inflammatory spots.
Due to sebum overproduction, the sebaceous duct is filled with excess sebum and dead keratinocytes. Oxidation turns the greasy texture of sebum into a wax-like consistency, while melanin of dead keratinocytes oxidizes to become black—this is recognized as blackhead comedones. The following sebum will be trapped in an anaerobic tube. This is the best condition for the colonization of Propionibacterium acnes (P. acnes)—an anaerobic Gram-positive bacterium—settling on the sebum-rich parts of the body.
With an estimated density of 100–1,000,000 per square centimeter on the skin, P. acnes accounts for approximately half of the total skin microflora5 and may represent more than 90% of the microflora in acne conditions. It feeds on lipids and secretes metabolites such as porphyrins and lipopolysaccharides; all while attracting immune cells, which aim to fight the bacterial infection.6 In the late stage of an acne pustule, the epidermis around the hair shaft ruptures to create scarring in the skin (see Figure 2).
Therapies Against Acne and Oily Skin
Pharmacologically, there are two major strategies to fight oily skin and the development of acne. The first is to reprogram sebocytes to produce smaller amounts of sebum—i.e., the feeding ground for P. acnes. Second is to use strong cleansing products containing benzoyl peroxide to control the excess settlement of bacteria. This therapy targets P. acnes in particular; however, the entirety of the skin’s microflora is affected due to the radical nature of benzoyl peroxide. Due to this, benzoyl peroxide therapies should only be utilized under medical supervision. Furthermore, the regular use of benzoyl peroxide may provoke dry skin.
Antibiotics also are commonly used to fight a severely imbalanced microflora. However, the use of non-specific broad-spectrum antibiotics increases the risk of generating resistant Staphylococcus aureus strains, which according to latest investigations, also settle in pores and pimples.7
Cosmetic solutions for acne-prone skin can only mimic one’s dermatological armory to prevent the aggravation of a mild condition. And since a healthy microflora is important to reduce the growth of deleterious species, it is crucial to choose intelligent molecules that target a specific sub-population of bacteria; in the cases of oily skin or moist areas, these targets are predominantly P. acnes or Corynebacterium spec. (which creates malodor), respectively.
As such, a blend of propanediol and bioflavonoids extracted from Maclura cochinchinensis, commonly known as cockspur thorn, was developeda and tested for its anti-acne effects. Isoflavones, a subclass of bioflavonoids, are secondary plant metabolites that defend plants against pathogens. These were hypothesized to function as balancing ingredients in cosmetic formulations for the skin's microflora.
The M. cochinchinensis leaf extract was first characterized. As its main constituents, the prenylated isoflavones isolupalbigenin, 6,8-diprenylorobol and 6,8-diprenylgenistein were identified. These have the isoflavone core in common, with two prenyl residues attached at different positions on the core (see Figure 3). Combined with propanediol, the ingredient was tested in vitro and in vivo as described next to determine its efficacy on the skin's microflora and microbiome.
Material and Methods
MIC of skin microflora: The M. cochinchinensis ingredient was diluted in different concentrations in Müller-Hinton broth and the growth of selected bacterial strains was analyzed under aerobic or anaerobic conditions for P. acnes for 24–48 hr. The minimal concentration of the active at which no bacterial growth was observed corresponded to the minimal inhibitory concentration (MIC).
The microflora is not the root cause of skin problems, but rather a reaction to dysregulated skin functions.
In vivo guidelines: In vivo studies were performed in accordance with the principles of good laboratory practice (GLP), good clinical practice (GCP), and in compliance with the quality assurance system requirements. The studies also were in accordance with the World Medical Association’s Declaration of Helsinki. All study participants signed a written informed consent at the beginning of the studies.
Acneic skin microbiome, before and after treatment: A small application study was carried out using a formulation containing 3% of the M. cochinchinensis ingredient for 28 days on three female subjects, 18–45 years old (average 28.7 years old), with acneic and oily skin. The test formulation was applied twice daily to the face. At Day 0 and Day 28, swab samples of the skin surface in the nasolabial area were taken. Sequence analysis of the 16S rRNA revealed the microbial diversity of the skin.
Corynebacterium spec. density and axillary malodor: A a placebo-controlled study was carried out to substantiate a deodorant claim on 20 subjects, 11 male and 9 female, between 35–64 years old (average 52.6 years old) with pronounced axillary odor. After 10 days of applying a standardized cleansing product, subjects washed their armpits in a defined way at a test institute (pre-conditioning). Three evaluators judged malodor by sniffing subjects' armpits both 6 hr and 24 hr later. After 24 hr, the armpits were washed again and a simple spray, containing either 1% or 0% of the M. cochinchinensis ingredient, was applied. The development of malodor was then assessed in the same way as before. Swab samples were taken after 6 hr to investigate the colony-forming units (CFU) of bacteria and of Corynebacteria on Columbia blood agar.
Effect of active ingredient on acne-prone skin: In a double-blind, placebo-controlled, randomized study, 21 female subjects 16–24 years old (average 19.4) with healthy, oily Caucasian skin, applied an emulsion containing either 3% of the M. cochinchinensis ingredient or a placebo twice daily to half of their face. Inflammatory spots were assessed with the red channel of a VISIA photographic system while porphyrins were detected in the UV channel.
Results
Selective suppression of actinobacteria: The M. cochinchinensis ingredient differentially regulated the growth of the skin's microflora. This effect was attributed to the extract's prenylated isoflavones. Depending on the bacterial strain, they suppressed the growth of bacteria and were most active on both P. acnes, the main aggravating bacterium of acne vulgaris, and Corynebacterium spec., responsible for malodor (see Figure 4). At 0.5–1.0%, the M. cochinchinensis ingredient only affected these unfavorable Actinobacteria. At higher concentrations, the growth of Staphylococci and Bacillus subtilis were regulated as well.
Microbiome diversity in vivo: Analysis of the skin microflora in subjects having acne lesions before application of the M. cochinchinensis ingredient proved dysbiosis in each study participant. The majority of skin bacteria was represented by Propionibacteria (87–98%), of which 99% were P. acnes. In all study participants, the abundance of Actinobacteria, to which Propionibacteria and Corynebacteria belong, was reduced by application of the active (see Figure 6). After 28 days, the amount of P. acnes decreased by 10% or 12% in two-thirds of study participants. In contrast, biodiversity in the phyla Firmicutes (e.g. Staphylococcus sp. and B. subtilis) and Proteobacteria increased.
In relation, the median Shannon index was increased for all study participants, moving toward the more diversified microbiome of normal and balanced skin (see Figure 5). This result was in accordance with the antimicrobial specificity profile determined in Figure 4 but due to high inter-individual diversity and the small number of study participants, significance was not established.
Corynebacteria and axillary malodor: Analysis of swab samples after 6 hr of placebo or serum application in the armpits revealed a reduction of Coryneform bacteria by 44.4%, presumably due to the activity of the prenylated isoflavones in M. cochinchinensis (see Figure 7). With 1% M. cochinchinensis, the fraction of Coryneform bacteria among all colony-forming units was 5% while for the placebo, it was 9% after 6 hr. This aligns with the sniff test results of the deodorant study, since Coryneform bacteria are responsible for the creation of malodor.
Untreated armpits developed a pronounced malodor after 24 hr with a sniffing value of roughly 3.5. After application of a deodorant spray, the sniffing value was reduced by 1.2 units for the test active and 0.83 units for placebo (see Figure 8). Thus, M. cochinchinensis significantly outperformed the placebo after 6 hr. In addition, after 24 hr, the malodor in the armpits treated with active was still significantly decreased by 0.25 units while the placebo returned back to the initial value.
P. acnes and signs of acne: While application of the test formulation on the hemiface significantly increased the porphyrin count, the same formulation with 3% M. cochinchinensis continuously decreased the porphyrins in sebaceous ducts, which are a measure for P. acnes colonization, significantly over the placebo (see Figure 9). This effect was easily visible by VISIA imagine in the UV channel (see Figure 10).
As noted, red inflammatory spots also were measured by VISIA photography. After 28 days of application, M. cochinchinensis decreased the number of red spots by 8% in acne-prone skin. After 84 days, spots were reduced significantly over the baseline and placebo by 22%, while the placebo did not significantly change (see Figure 11).
Cosmetic solutions for acne-prone skin can only mimic one’s dermatological armory to prevent the aggravation of a mild condition.
Discussion
Normal skin harbors numerous different bacterial and fungal microorganisms on its surface. These strains cohabitate to create the larger beneficial microflora. Under certain conditions, though, the balance of the microflora can shift unfavorably.
An excess of P. acnes, for example, can lead to acne vulgaris; and the overgrowth of Staphylococci may lead to skin inflammation and atopic dermatitis.3 Corynebacteria, which grows primarily in moist areas, also can cause pitted keratolysis on the soles of the feet, manifested clinically as hyperhidrosis, unpleasant odor and pain.9
Even Staphylococci and Bacilli are desirable for healthy skin flora, under normal conditions, and since M. cochinchinensis was shown to act selectively on Propionibactrium spec. and Corynebacterium spec. (see Figure 4), the potential to develop antibiotic resistance to S. aureus can be excluded. Additionally, the suppressive activity was the smallest for this strain.
The prenylated isoflavones in M. cochinchinensis also appear capable of regulating the microbiome of oily skin. High biodiversity reflects skin health but this diversity is reduced in oily, acne-prone skin due to an abundance of Propionibacteria. Said biodiversity is measured using the Shannon index, where normal skin has a value of about 6.8 and the lower the Shannon index, the lower the diversity.10
In the described study, the Shannon index of one subject was extremely low at 0.15. This shows that predominantly, P. acnes strains were collected in the swab samples due to the severe acneic condition. However, treatment with 3% M. cochinchinensis for 84 days led to a 73% increase of the Shannon index and a large diversification of bacterial phyla except the Actinobacteria, which were reduced. Although after treatment, the Shannon index showed the overall bacterial diversity was still very low, the trend toward a normalized skin microflora was obvious. This result underlines the limitations of cosmetic active ingredients compared with drugs, which are only allowed under medical supervision.
To investigate the potential of M. cochinchinensis to inhibit the growth of Corynebacteria, a common sniff test was performed. Axillary malodor is a result of the decomposition of sweat and sebum by moist-loving Corynebacteria. Interestingly, the development of axillary malodor goes along with hormonal changes during puberty. As experience has shown, babies and children do not typically develop malodorous sweat until they enter adolescence.
In the present tests, Corynebacteria clearly were reduced after the use of the active ingredient, compared with the placebo. This reduction was manifested by a significant reduction in axillary malodor after 6 hr.
The suppression of P. acnes growth also was investigated in an 84-day study with a placebo vs. 3% active applied to the face. As a result, the porphyrins, indicative for growth of P. acnes, were reduced by 12% over the baseline of 38%. This translated to a visible reduction in red spots, most likely because P. acnes is known to aggravate inflammatory processes in acne-prone skin and lead to spots and pimples.
Taken together, the reduction of Corynebacteria in the axillary study, the reduction in porphyrin count, as well as the reduction of Actinobacteria in the 28-day facial study demonstrate the growth suppression profile of M. cochinchinensis, supporting the results observed in vitro in the broth culture experiments.
Conclusions
It is important to reduce the number of unwanted bacteria in favor of a balanced microbiota to maintain a healthy skin. The overgrowth of P. acnes or Corynebacteria can aggravate acneic conditions and malodor. However, the described M. cochinchinensis ingredient acts as a microbiota-regulating agent by selectively suppressing the growth of these unwanted species.
The number of porphyrins, a measure of P. acnes colonization, was reduced as well as the number Coryneform bacteria, in vitro and in vivo. This leads to a reduction in inflammatory spots and a normalized skin appearance.
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