1. IntroductionHair loss or hair thinning is a common complaint in clinical dermatology. Hair loss can be classified according to cause and presentation [1]. The four common hair loss disorders that occur in both men and women are androgenetic alopecia (AGA), telogen effluvium (TE), alopecia areata (AA), and scarring alopecia [1].Hair thinning has to be considered as an accumulation of multiple factors strictly linked to the complex biology of the hair follicle (HF) [2]. These factors include hormone unbalancing, genetics, diet, stress, oxidative damage, and ageing but also extrinsic factors such as ultraviolet radiation and pollution [2]. The combination of two or more of these factors leads to the dysregulation of physiological hair growth with the shortening of the anagen phase and the production of smaller, thinner, and less visible hairs.Despite its relatively benign behaviour, hair thinning can cause feelings of poor self-image, low self-esteem, anxiety, and depression [3,4], with a consequent negative impact on the quality of life of subjects [5]. In this view, therapies aiming to reduce the appearance of thinning by delaying, arresting, or reversing the course of hair thinning are highly desirable.A ground-breaking nutraceutical supplement (NS) has been developed to approach thinning in men and women (Bioscalin® NOVA•Genina). The supplement is a mixture of phyto-compounds (Ganoderma lucidum, Linum usitatissimum L., Cynara scolymus L., and Galeopsis segetum Neck.) with clinically proven dihydrotestosterone (DHT)-inhibiting and antioxidant properties [6,7,8,9,10,11,12]. It also contains polyunsaturated fatty acids (PUFAs), rutin, zinc, biotin, and ornithine with well-established efficacy on normal hair growth [13,14,15,16,17]. Indeed, nutritional deficiencies of minerals, essential fatty acids, amino acids, and vitamins are considered some of the major factors influencing hair loss [18,19]. 2. Materials and Methods 2.1. Subjects

A total of 60 subjects were enrolled in this monocentric randomized double-blinded placebo-controlled study with 2 arms of 2 parallel groups (1:1/1:1 each). The study was conducted under the approval of the Ethical Independent Committee for clinical, not pharmacological, investigation in Genoa (Italy) and following the ethical standards of the 1964 Declaration of Helsinki.

2.2. Inclusion and Exclusion Criteria

Before inclusion, subjects were asked to sign an informed consent form indicating that they are aware of the investigational nature of the study.

Participants in the study were healthy male and female subjects aged 18–65 with telogen effluvium-like hair loss and a percentage of anagen ratio at inclusion < 75%, accepting to not receive any drugs/cosmetic treatments able to interfere with the study results, and no participation in a similar study at the time or during the previous 6 months.

Exclusion criteria included: pregnancy and lactation (for female subjects); subjects not in menopause who did not use adequate contraceptive precautions to avoid pregnancies during the study; sensitivity to any compound of the investigational product; the presence of cutaneous disease in the tested area such as lesions scars, or malformations; a self-reported history of diseases such as diabetes, endocrine disease, hepatic, renal, cardiac, or pulmonary disorders, cancer, neurological or psychological diseases, inflammatory/immunosuppressive diseases, or drug allergy.

2.3. Test Product

Subjects were randomized to receive the study product (Bioscalin® NOVA•Genina, Giuliani Spa, Milan, Italy) or a placebo. The novel oral NS contains Ganoderma lucidum, Linum usitatissimum L., Cynara scolymus L., and Galeopsis segetum Neck., a patented composition comprising rutin and PUFAs, zinc, biotin, and ornithine. Placebo treatment consisted of inert tablets with the same excipients and appearance.

Subjects were asked to ingest their assigned treatment once daily immediately following breakfast.

2.4. Study Evaluations

Evaluations were performed at the baseline visit (T0), and after 15 days (T1) and one (T2), two (T3), and three (T4) months of treatment. Two follow-up visits one month (T5) and two months after the last assumption (T6) were also included in the protocol. The clinical efficacy of the products under study was evaluated by the TrichoScan® software (Trichology GmbH, Freiburg, Germany) to define the percentage of hair in the anagen phase (number of HFs in the anagen phase), the anagen hair density, and the percentage of vellus hair. The evaluation of the entity of hair loss in the telogen phase was also carried out through a pull test.

Each subject was asked to fill out a questionnaire regarding the perception of hair growth improvement (0–10 scale) at T1, T2, T3, T4, and T5, the efficacy of the study treatment on hair volume and hair shine (4-point scale for intense improvement, mild improvement, no effect, or worsening at T1, T2, T3, T4, and T5), and the product tolerance (score: bad, poor, medium, good, excellent) at T4. Moreover, during the treatment phase, volunteers’ compliance was verified, while the possible events which could have interfered with the test results were evaluated at each study visit.

2.5. Study Endpoints

The primary endpoints were the increase in the percentage of hair in the anagen phase (active hair growth phase), the density of hair in the anagen phase (n/cm2), and vellus hair (miniaturized, smaller, and thinner hair). The secondary endpoints were the evaluation of the entity of hair loss in the telogen phase (pull test) and responses to the subject self-assessment questionnaire. Safety endpoints were potential adverse events (AEs).

2.6. Statistical Analysis

The activity of the food supplementation was expressed in absolute values versus baseline (evaluation of tested product vs. T0) and in comparison to each other. Clinical data were compared as results at each visit versus basal conditions using the Friedman test followed, in case of a statistically significant result, by a Holm–Sidak adjusted test. Instrumental data were analyzed using a non-parametric test (Friedman test) when the normality hypothesis was rejected by the Shapiro–Wilk normality test (threshold at 5%) or a parametric test (ANOVA test for repeated measures) when the normality hypothesis was confirmed, followed in case of a statistically significant result by a Holm–Sidak adjusted test.

p-values less than 0.05 were considered clinically significant.

4. DiscussionThe results of the present study highlighted that the supplementation of a novel supplement (NS) containing Ganoderma lucidum, Linum usitatissimum L., Cynara scolymus L., and Galeopsis segetum Neck., a patented composition comprising rutin and PUFAs [7], zinc, biotin, and ornithine, to men and women with telogen effluvium-like phenomena was safe and effective, compared to placebo.

The results showed a significant increase in hair in the anagen phase, hair density, and vellus hair counts from fifteen days of treatment to two months of follow-up, compared to placebo. These improvements were accompanied by significant and progressive improvement in hair tensile strength and hair growth, and volume and brightness increase, as determined by the self-assessment questionnaire. Taking these results together demonstrated a steady improvement from baseline to the end of treatment (90 days) and two months of follow-up (150 days).

TE-like phenomena are very common among men and women. These phenomena are mainly linked to diffuse hair thinning.

The definition of “hair loss” in English-speaking culture now tends to define all the modifications of the bulb cycle, regardless of the specific cause, as a fundamental marker of hair loss. All the phenomena of “hair loss”, in the most modern sense, can be generally defined as “telogen effluvium-like” phenomena.

Several causes have been reported to be responsible for these phenomena, including heredity, comorbidities (e.g., thyroid disease), pregnancy, malnutrition or rapid weight loss, iron deficiency or vitamin D deficiency, or stress [2,3].Current research is highlighting the nutritional impact on hair growth [18,19]. The cell of the HF is characterized by a fast division and this makes it very sensitive to any nutritional reduction [19]. Therefore, the role of inflammation, oxidative stress, ageing, dihydrotestosterone, and stress has been well documented in hair loss [20,21,22].

The results of the present study demonstrate that nutritional supplementation may be beneficial in TE-like phenomena when there are no associated hormones. Treatment with NS showed positive significant results in all the parameters evaluated in the study, compared to placebo.

The ingredients in the NS supplement provide a unique therapeutic value to its multi-active clinical biological activity versus multiple causative factors of hair thinning in TE-like phenomena.

Triterpenoids isolated from G. lucidum (Fr.) (Reishi) showed a potent inhibitory activity of 5-alpha reductase [9]. This complements the documented effects of another botanical extract, C. scolymus L. leaf extract, which is a rich source of polyphenolic compounds with a strong antioxidant potential [12]. G. segetum Neck. is a herbaceous species belonging to the Lamiaceae family. The plant is present in the BELFRIT list and the lists of the Italian Ministry of Health with the claim: “Well-being of nails and hair” [23]. The botanical extract of this plant has been studied and patented for its antioxidant and inhibitory effect on 5α reductase (isoform 2), which is the main target of the drug finasteride [6,7]. G. segetum extract also stimulates the synthesis of cytokeratin 6A by outer-root sheet (ORS) cells in the HF [6,7].The NS supplement also contains a patented combination of rutin and PUFAs from L. usitatissimum L., having an inhibitory activity on 5α reductase [24]. L. usitatissimum L. is rich in the PUFAs omega-3 (alpha-linolenic acid) and omega-6 (linoleic acid) that can be useful in counteracting the production of DHT through inhibition of 5α reductase [8,9]. In vivo tests with omega-3, 6 PUFAs were effective in reducing telogen and hair miniaturization [10]. Rutin is a bioflavonoid found in plants (Ruta graveolens L. (Rutaceae), Sophora japonica L. (Fabaceae), etc.) with numerous beneficial activities for human health [13] as an iron chelator that prevents radical reactions and it also possesses antiandrogenic activity [13].Hair thinning may be improved with vitamin and mineral supplementation which are important for normal cell growth [10]. Zinc, in particular, plays a very important role in HF biology since it is a structural element of hair proteins [14,15,25]. It is also a regulatory factor of numerous enzymatic reactions (cofactor of superoxide dismutase, an antioxidant enzyme that counteracts the action of free radicals and is an inhibitor of 5-alpha reductase) [15].Biotin, also known as vitamin H, is a coenzyme important for the functioning of various enzymes involved in the metabolism of nutrients, and also for the hair follicle [14].Considering that the HF has also been shown to be dependent on polyamine synthesis for its growth, function, and cycling [16,26,27,28], the NS formulation also contains, as an essential amino acid, ornithine which is involved in the urea cycle and also in the biosynthesis of polyamines [28]The NS has also been formulated in patented dosage form, comprising a polymeric matrix containing hyaluronic acid and a non-ionic polymer, to obtain a gradual release in the intestine for optimal absorption of active ingredients [29].