Innovacos has developed a hair care ingredient that stimulates the expression of main skin components providing hydration and helping regenerate the hair structure. According to in vitro studies, the bamboo vacuolar extract promotes the viability of essential hair components (HHFK and HHFDPC) and prevents hair ageing increasing KAP5 expression

The hair makes us unique, it serves to frame and decorate our face, and defines our personal style. People invest a lot in the appearance of their own hair, so much so that hair significantly contributes to the first impression that we make on others.¹

Historically, hair has defined a precise membership to a social group. Just think about the coloured crests of punk, or the dreadlocks that symbolise Rastafarian culture, or the extravagant hairstyles of Marie Antoinette, or the cut of Cleopatra, still famous after millennia

When Delilah cut Samson’s hair, he lost his power and strength. This biblical situation shows the symbolism associated with the loss of our hair. Nowadays, career portals provide tips for correct hairstyling in application photos, introductory meetings and appointments with management, while magazines provide information about the unconscious messages we send with our hairstyle.

Yet hair is not just an aesthetic ornament, it also contributes to protecting parts of our body from trauma, UV rays, and the penetration of external agents, such as microbes and bacteria. The shield function implemented by the hair prevents some environmental pollutants from entering and coming into contact with the scalp.

Hair anatomy

Strands of hair originate in the hair follicle, an epidermal penetration of the dermis. The hair shaft is the visible portion that comes out of the scalp, while the hair root is anchored in the follicle and lies below the surface in the dermis. The hair bulb surrounds the hair papilla, which is made of connective tissue and contains blood capillaries and nerve endings from the dermis (Figure 1).

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New cells are constantly forming in the hair bulb. These cells stick together and harden. As the new hardened cells continue to stick to the old ones from below, the hair is gradually pushed out of the skin. A single hair on the head grows at a rate of about 1cm per month.

As long as new hair cells continue to grow in the hair bulb, the hair continues to grow longer. At any point in time, about 90% of a person’s total amount of hair is in this growth phase.

At the end of the growth phase, the hair root separates from the papilla, and the supply of blood is cut off. The hair is gradually pushed out of the skin and eventually falls out. The resting phase can last several months. New hair cells then start to multiply at the base of the ‘empty’ hair follicle to form new hair, and the growth phase of the hair growth cycle starts all over again.

Because hair continues to enter the resting phase and then fall out, we are constantly losing hair. A healthy adult may lose about 70 to 100 hair on their head per day. Yet because new hair is always growing and is replaced, this natural hair loss is not noticeable.

There are periods of the year when hair loss becomes more pronounced, in conjunction with spring (April-May) and autumn (OctoberNovember); it is the so-called seasonal hair loss. The causes can be sought in different areas, such as hormonal balance, stress situations or climatic factors.

The seasonal fall is, however, part of the physiological process of hair replacement and has a limited duration, equal to a few weeks. The situation becomes worrying when hair loss becomes more noticeable and does not stop autonomously

The condition of the scalp can influence the quality of the hair which is produced. Biochemical changes in proteins and lipid components have been observed in hair emerging from unhealthy skin.

A number of observations have found that premature hair loss can be caused by poor health of the scalp which would result in an impaired fibre anchoring force with the follicle.²

Innovacos has developed Plant C-Stem™ Bamboo (INCI: Glycerin, Water, Phyllostachys Pubescens Meristem Cell Lysate), hereafter known as the bamboo vacuolar extract. This active ingredient has been studied to offer a solution against hair loss.

It is a different concept of plant extraction because through technological know-how, the vacuole content of bamboo stem cells is selectively extracted at low temperatures and without solvents or enzymes to maintain the maximal biological potential of bamboo

The vacuole is an essential organelle for plants; it works as a site of storage for bioactive components required for plant growth and repair. The content of the vacuole is a cocktail of minerals, proteins, nutrients, and growth factors that the plant can use to be repaired and grow again in case of damage. Using the plant cell vacuole extract is like using the plant ‘serum’

The bamboo variety chosen for the bamboo vacuolar extract is Phyllostachys pubescens.

This plant is described as the giant bamboo par excellence; in optimal climatic conditions, the growing of this vegetable species is prodigious reaching 30 metres in height, 30cm in diameter, and a daily growth of 1 metre.

After cutting, bamboo spontaneously regrows from its roots up to 60 times per single life and it doesn’t need to be replanted. The rods are used as a construction material for roofs and floors for their strength and flexibility due to the high content of silicon (Si).

Thinking that these characteristics could be exploited in the hair, Innovacos has harnessed the power of Phyllostachys pubescens plant “serum” to develop an ingredient with an essential role in reducing hair loss and increasing hair density.

In vitro studies

Elastin and collagen fibres, the main components of the dermis, maintain the right elasticity of the skin, giving support, resistance, and firmness to the tissues. These molecules can deeply moisturize the scalp thus preventing its dehydration and helping regenerate the hair structure. A healthy scalp produces healthy hair.

The quantity of type I collagen in human dermal fibroblast was determined using a commercially available kit (Takara Bio, Japan). This kit is capable of detecting procollagen type I carboxy-terminal peptide (PIP) using polyclonal antibodies, rather than directly measuring collagen.

The quantity of type I collagen in human dermal fibroblast was determined using a commercially available kit (Takara Bio, Japan). This kit is capable of detecting procollagen type I carboxy-terminal peptide (PIP) using polyclonal antibodies, rather than directly measuring collagen.

Cells were incubated in the presence or the absence of the bamboo vacuolar extract for 48 hours, and then the culture supernatants were harvested and measured with a sandwich immunoassay kit, which was utilized in accordance with the manufacturer’s instructions (Takara Bio, Japan).

The measurement was performed with a microplate at 450 nm. The bamboo vacuolar extract increases collagen synthesis in a dosedependent manner (Figure 2A).

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Elastase is the proteinase enzyme that is capable of breaking down elastin. Low levels of elastin are linked with a decrease in the mechanical properties of connective tissue. The activity of elastase [PPE (Type IV); Sigma Chem. Co.] was examined using N-Suc-(Ala)3- nitroanilide as the substrate, and the release of –nitroaniline at 410 nm was measured.

The reaction was carried out in a 200 mM Tris-HCl buffer (pH 8.0) containing 5 mM N-Suc-(Ala)3-nitroanilide and 10 μg/mL elastase. The sample was added to the reaction mixture to reach a final concentration of 1%, and elastase inhibition was assessed at 25°C.

The reaction mixture was pre-incubated for ten minutes before adding the substrate. The change in absorbance was measured at 410 nm using a 96-well reader. The percentage inhibition of elastase was calculated as follows: Inhibition (%)= [(A-B)/A] × 100. The bamboo vacuolar extract showed an inhibitory effect on elastase activity in a dose-dependent manner (Figure 2B)

Glycation occurs in all living cells and it occurs when excess glucose molecules stick to collagen and elastin fibres, forming advanced glycation end-products (AGE), also known as glycotoxins. Young collagen and elastin are cross-linked in a particular way that promotes firmness and flexibility

Once these collagen and elastin fibres in the skin are glycated, they lose their normal functions. The body cannot break them down and replace them, so they become stiff and brittle

Albumin (1 mg/ml final concentration) was incubated with glucose (500 mM final concentration) in the presence of the bamboo vacuolar extract (5% final concentration), aminoguanidine (positive control), or PBS as the control buffer at the specified concentration. The reaction was allowed to proceed at 600°C for 48 hours.

The reaction mixture was allowed to cool at room temperature, and then 0.2 ml reaction mixture was transferred to a new tube and the reaction was stopped by adding 10 μl of 100% (w/v) trichloroacetic acid (TCA). The TCA-added mixture was kept at 40°C for ten minutes before centrifugation at 15,000 rpm.

The precipitate was redissolved with 0.8 ml alkaline PBS (pH 10), and the relative amount of glycated bovine serum albumin (BSA) was immediately determined on the basis of fluorescence intensity by use of a spectrofluorometer Infinite 200 Pro (Tecan, Austria).

The excitation and emission wavelengths used were 370 and 440 nm, respectively. Results are expressed as percentage inhibition of the formation of the glycated protein.

The bamboo vacuolar extract decreases the formation of the glycated protein in a dosedependent manner (Figure 2C) ensuring elasticity and flexibility to collagen and elastin fibres.

The evaluation of hair essential components has been carried out to validate the efficacy of the bamboo vacuolar extract in hair care. In particular, the viability of human hair follicular keratinocyte (HHFK) (Figure 3A), and human hair follicle dermal papilla cell (HHFDPC) (Figure 3B) was analyzed by exposing those cells (5X103 cells/well) to growing concentrations of the active ingredient for 72 hours.

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HHFK and HHFDPC are respectively responsible for the synthesis of keratin in the hair shaft and the supply of oxygen and nutrients to the hair follicle. The bamboo vacuolar extract increased the cell viability of both groups of cells which will result in increased keratin synthesis and nutrient intake for the new hair.Hair keratin-associated proteins (KAP) are the major components of the hair fibre. Like all fibrous proteins, they perform a structural function. Hair resistance comes from the process of keratinization, where the cells reaching the final stage of maturation, lose the nucleus and fill with keratin. This is why keratin is important for strong, shiny, and soft hair.^3,4

KAP5 is an age-related keratin protein that has been demonstrated to decrease with age. This diminution is correlated with a decrease in hair strength5 . The mRNA expression of KAP5 has been evaluated by exposing HHFK (1X105 cells/well) to the bamboo vacuolar extract for 24 hours. Some 100ppm of the active ingredient is enough to increase by three times the mRNA expression level of KAP5 (Figure 4).

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In vivo studies

In vivo tests have been carried out to evaluate hair loss (wash test) and density. The panel was composed of 20 subjects (males and females 30-60 years old) with seasonal hair loss. A lotion containing 2% of the bamboo vacuolar extract was applied three times a week for 12 consecutive weeks on the scalp (after washing) and gently rubbed. Subsequently, the hair has been dry.

Evaluations of the primary (T0) and secondary endpoints after 84 days (T84) have been done. The data on the quantitative discrete endpoints were described using the normal position and dispersion measurements: mean and standard deviation-median and interquartile range. A significance level of <0.05 was considered.

During the wash test, the hair of the subjects enrolled in the study is washed in a sink by covering its drain with gauze and then collecting and counting lost hair. The subject is taught to avoid shampooing for two days before the set test date

Compared to the baseline value (T0), a significant decrease of 18% in the number of hair lost during the wash test was observed after 84 days of treatment. The use of the bamboo vacuolar extract has a significant effect in reducing hair loss during washing (Figure 5A)

Hair density (number of hair/cm2 ) was measured by HIROX – RX 2000 3D, a 3D digital microscopy system. Hair density analysis is carried out by acquiring images of the scalp and counting the number of hair in a 1cm2 area

Every measurement collected is the average of hair density calculated in five different areas of the scalp. A statistically significant increase in hair density (+3%) between T0 and T84 has been observed. The treatment with the bamboo vacuolar extract has a significant effect on the growth of new hair (Figure 5B).

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Ex vivo studies

Ex vivo evaluation has been done to investigate the possibility of hair growth using the bamboo vacuolar extract.

The hair follicles isolated from the human body were separated and stored in Earle’s balanced salt solution (EBSS; Sigma Aldrich). Anagen phase follicles were separated using a stereomicroscope (Olympus, Tokyo, Japan) to avoid any damage.

A total of three tests were carried out. Six to 12 follicles were separated and used for the experiment

The isolated hair follicles were cultured in Williams medium E (Gibco, Grand Island, NY, USA) with 2 mM L-glutamine (Gibco, NY, USA), 10 μg/ml insulin (Sigma Aldrich), 10 ng/ ml hydrocortisone (Sigma Aldrich), 100 unit/ml penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO2.

On the first day of hair follicle culture, the photographs were taken and the shape and length were confirmed. The hair follicular tissues were treated with the bamboo vacuolar extract (250 ppm and 1000 ppm) for seven days. Minoxidil (10µM) was used as a positive control.

After seven days, the follicular tissues were fixed in 10% neutral buffered formalin (NBF). The hair follicle elongation was analyzed using Image J (version 1.52a, NIH, Bethesda, MD., USA). Hair treated with the bamboo vacuolar extract 1000 ppm showed the same results as the drug Minoxidil in promoting hair shaft elongation in hair follicles (Figure 6)

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Conclusion

Plant C-Stem™ Bamboo stimulates the expression of main skin components providing hydration and helping regenerate the hair structure. According to in vitro studies, the bamboo vacuolar extract promotes the viability of essential hair components (HHFK and HHFDPC) and prevents hair ageing increasing KAP5 expression

Clinical studies demonstrate that the active ingredient helps in reducing hair loss and increasing hair density, while ex vivo experiments comparing the activity of the bamboo vacuolar extract with the drug Minoxidil showed the same efficacy in promoting hair shaft elongation

References

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