Efficacy and safety of persimmon leaf formulated with green tea and sophora fruit extracts (BLH308) on hair growth: A randomized, double‐blind, placebo‐controlled clinical trial

Abstract Background Recent research suggests that persimmon leaf extract (PLE) has an effect on inflammatory skin diseases. Previously, PLE is revealed to inhibit not only nitric oxide production but also inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression levels in mouse macrophages in vitro. Moreover, it significantly reduced IL‐6 production and 5α‐reductase expression in human follicle dermal papilla cells (HFDPCs). This study aimed to determine whether the PLE‐containing BLH308 complex improves hair growth in clinical trials. Materials and Methods A total of 88 participants were recruited, and were instructed to orally take BLH308 or the placebo twice a day for 24 weeks. The mean age of the test group was 38.52 ± 7.98 years and that of placebo group was 38.98 ± 8.80 years. The study was conducted for 24 weeks, and hair density, thickness, and gloss were evaluated. All participants completed a satisfaction survey questionnaire. Results The test group showed significantly increased hair density and hair diameter at week 24 compared with the placebo group (p = 0.0015 and p = 0.0001, respectively). Although not statistically significant, the degree of gloss also showed higher improvement in the test group compared to the placebo group. Conclusions Our data demonstrated that oral consumption of the BLH308 complex containing PLE significantly increased hair density and thickness compared to the placebo group, showing its possible role in promoting hair growth.


INTRODUCTION
Persimmon (Diospyros kaki) leaf extract (PLE) has been widely used in East Asian traditional medicine for its antioxidant properties. 1 It is mostly composed of anti-inflammatory flavonoids, such as quercetin, isoquercetin, hyperin, astragalin, and kaempferol. 2 Recent studies have revealed that the extract from the persimmon leaves possesses a range of biological properties, including radial scavenging, neuroprotection, thrombosis inhibition, anti-atherosclerosis, and anti-allergy. 3 The antioxidant and anti-inflammatory effects of PLE can also lead to a photoprotective and anti-aging effect, 4 which can affect the maintenance of healthy hair growth activity.
A few dermatologic studies have explored the effect of PLE on inflammatory dermatologic diseases. PLE was reported to have an inhibitory effect on T cell activation via reduction of interleukin (IL)-2 production in vitro; an additional in vivo animal model of atopic dermatitis resulted in reduced IgE levels, mast cell infiltration, and pro-inflammatory cytokine expressions, especially Th2 cytokines, after oral administration of PLE, suggesting its role in inflammatory skin diseases such as atopic dermatitis. 1 Another study on the effect of PLE in atopic dermatitis assessed its clinical therapeutic effect in NC/Nga mice, showing that oral administration of PLE for 4 weeks into the atopic dermatitis mouse model with overt dermatitis resulted in dosedependent decreases in not only the skin severity score but also in transepidermal water loss (TEWL), serum IgE, and skin scratching behavior. 5 Although the anti-inflammatory effect of PLE on inflammatory skin diseases such as atopic dermatitis seems obvious, the effect of PLE on other dermatologic conditions remains unknown. The effect of PLE in vitro was investigated, and it was found that PLE inhibited not only nitric oxide production but also inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels in mouse macrophages. Moreover, it significantly reduced IL-6 production as well as the expression level of 5α-reductase in human follicle dermal papilla cells (HFDPCs). 6 Recently, novel therapeutic interventions for promoting hair growth in alopecia have been garnering considerable scientific interest, such as platelet-rich plasma 7 and the use of oral supplements containing hydrolyzed collagen and aminoacids. 8 Therefore, in this study, we designed a randomized double-blind placebocontrolled study to evaluate the efficacy and safety of BLH308, the study product, which contained PLE as the major active ingredient formulated with green tea (Camellia sinensis) and sophora fruit (Sophora japonica) on hair growth and overall hair health when orally consumed. A total of 88 healthy Korean adults were recruited and were instructed to orally take BLH308 or a placebo containing PLE twice daily for 24 weeks. Consequently, the participants who consumed the test product showed significantly increased hair density and hair diameter compared with the control group. In addition, although not statistically significant, the degree of gloss assessed by the glossymeter also showed higher improvement in the test group compared to the control group.

Study participants
Participants in this study were recruited between the ages of 19 and 60 without any underlying medical conditions (men = 34, women = 54).

Study design and test product
The study was designed as a randomized, double-blinded, placebocontrolled clinical trial and was performed at the Global Medical Research Center, Seoul, Korea, from September 2021 to April 2021.
The study was conducted for 24 weeks and included a total of five visits for each participant (screening, week 0, week 8, week 16, and week 24).
The permitted window periods were ±7 days for week 8, 16, and 24.
The active ingredient of the test product, BLH308, was a PLE formulation of green tea and sophora fruit extract, both of which are known for their anti-inflammatory and anti-oxidant properties. These extracts were formulated in a 2:1:1 compounding ratio, respectively, considering the contents of tannic acid in PLE (ca. 12.5%), epigallocatechin-3gallate (EGCG, ca. 50%) in green tea extract, and sophoricoside (ca. 3%) in sophora fruit extract. The information regarding the test and placebo products is shown in Table S1.

Hair density and thickness
The efficacy of the test product for improvement in hair density and thickness was evaluated by phototrichogram using   (Table   S2), and the results were evaluated as average values.

Safety assessment
At each visit, subjects underwent physical examinations to assess adverse events caused by the test product. In the clinical trial, adverse events, including abnormalities observed in clinical pathology tests (hematological/blood chemical tests, urine tests), vital signs (blood pressure, pulse), and physical measurements (body weight), were evaluated. Any adverse event during the clinical trial was recorded and assessed in the Case Report Form (CRF). The adverse event occurrence ratio was calculated.

Statistical analysis
The subject data consisted of three types: Safety Set (SS) analysis, Full   Table 1.

Clinical efficacy on hair density and thickness
During the 24 weeks of oral BLH308 supplementation, we measured hair density at baseline, 8 weeks, 16 weeks, and 24 weeks after taking the test product. In the PPS analysis (Figure 2A) In the hair thickness analysis ( Figure 2B)

Clinical efficacy on hair gloss
In the analysis of the change in hair gloss, we did not find a significant difference in change from the baseline of the test groups compared to the placebo group after 8, 16, and 24 weeks of oral ingestion. However, as shown in Figure 3 we confirmed an increase in the test group F I G U R E 3 Comparison of change in hair gloss after BLH308 orally ingestion. The result of PPS analyzing the change from baseline of hair gloss at 8, 16, and 24 weeks after oral consumption of the test product. ‡p < 0.05, ‡ ‡p < 0.01, Paired t-test (A). Representative images of change in hair surface using SEM. All images were obtained from X500 magnification. PPS, per protocol set; SEM, scanning electron microscope.
when compared to the placebo group. In particular, after 8 weeks of ingestion, the hair gloss was highly improved in the test group compared to the placebo group. The test group significantly increased by 0.07 ± 0.14 scores (p = 0.0019, paired t-test), and the placebo group increased by 0.01 ± 0.13 scores (p = 0.4742, paired t-test), but no significant differences were observed between the test group and placebo group (p = 0.0651, Wilcoxon rank-sum test).

Adverse events
Safety assessment was conducted by analyzing subjects who had consumed the test product at least once after randomization in a clinical trial. During the clinical trial, there were 31 adverse events in a total of 20 subjects (39.22%) in the test group and 32 adverse events in a total of 22 subjects (44.f00%) in the placebo group during clinical trial, but there was no statistically significant difference between the groups (p = 0.6257). Clinical pathological tests were conducted at visits 1 and 5 and were evaluated by dividing them into hematological, blood chemistry, and urine tests (Table S3). No serious adverse reactions occurred, and there were no dropouts due to adverse events.

DISCUSSION
Persimmon is an East Asian fruit that is renowned for its nutritional value due to its high content of antioxidant compounds, especially flavonoids. Previous studies have confirmed that persimmon leaves contain a wide range of bioactive compounds such as flavonoids, terpenoids, polysaccharides, caffeine, carotenoids, amino acids, vitamin C, minerals, and chlorophylls. 4  In this study, we explored the efficacy and safety of PLE formulated with green tea and Sophora fruit extract (BLH308) on hair growth of healthy adult participants. After 24 weeks of oral intake of the test product, the participants showed significantly increased hair density and thickness compared to the control group. Notably, the hair density showed a significant increase from week 8 compared to the control group, indicating that oral consumption of BLH308 potentially contributed to a relatively early response in hair growth.
The antioxidant and anti-cancer properties of green tea (Camellia sinensis) are well known, and they are mediated by apigallocatechin-3-

gallate (EGCG), a key component of polyphenols. Previous studies have
shown that EGCG potentially contributes to an additive effect on hair growth activity by selectively inhibiting 5α-reductase activity. 10 Other investigations have also shown that EGCG promotes hair growth by upregulating phosphorylated Erk and AKT in vivo and in vitro using HFDPCs. 11 Meanwhile, Sophora fruit (Sophora japonica) possesses estrogenic, anti-oxidant, anti-inflammation, and immunomodulatory properties. Its extract contains isoflavone glycosides, which can inhibit the bioactivities of IL-6 and COX-2. 12 These anti-inflammatory activities might significantly affect the hair growth activity, as shown in previous studies that found increased levels of IL-6, along with MCP-3, IFNγ-inducible protein-10, and MIP-1α, in balding zones of androgenetic alopecia compared with non-balding zones. 13 Therefore, the formulation of PLE in combination with green tea and Sophora fruit extracts may have additive effects on PLE in term of increasing hair density and thickness.

CONCLUSION
The oral consumption of PLE formulated with green tea and Sophora fruit extracts showed significantly increased hair density and thickness compared to the control group, showing its possible role in promoting hair growth. Although not statistically significant, the degree of hair gloss in the test group after the oral consumption of BLH308 was also increased compared to that of the control group. As the mechanism involving skin tissue injury caused by extrinsic factors revolves primarily around the installation of oxidative stress, the antioxidative and anti-inflammatory activities of PLE along with green tea and Sophora fruit extracts could be the major factors resulting in the general improvement of hair health. Further clinical studies with the test product applied to participants with photo-aged scalps will be necessary to confirm its role in oxidative stress induced by extrinsic aggregations.

ACKNOWLEDGMENTS
This work was supported by the Ben's Lab Co., Ltd.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ETHICS STATEMENT
The Institutional Review Board from the study institution approved the study (IRB no. GIRB-21722-EA), and informed consents were obtained from each participant before enrolment in the study. This study was registered with the Clinical Research Information Service (CRIS) as PRE20221115-001.