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. 2017 Oct 27;22(11):1829.
doi: 10.3390/molecules22111829.

Inhibition of Human Kallikrein 5 Protease by Triterpenoids from Natural Sources

Affiliations

Inhibition of Human Kallikrein 5 Protease by Triterpenoids from Natural Sources

Yosuke Matsubara et al. Molecules. .

Abstract

Stratum corneum tryptic enzyme kallikrein 5 (KLK5) is a serine protease that is involved in the cell renewal and maintenance of the skin barrier function. The excessive activation of KLK5 causes an exacerbation of dermatoses, such as rosacea and atopic dermatitis. Some triterpenoids are reported to suppress the serine proteases. We aimed to investigate whether bioactive triterpenoids modulate the KLK5 protease. Nineteen triterpenoids occurring in medicinal crude drugs were evaluated using an enzymatic assay to measure the anti-KLK5 activity. The KLK5-dependent cathelicidin peptide LL-37 production in human keratinocytes was examined using immunoprecipitation and Western blotting. Screening assays for evaluating the anti-KLK5 activity revealed that ursolic acid, oleanolic acid, saikosaponin b₁, tumulosic acid and pachymic acid suppressed the KLK5 protease activity, although critical molecular moieties contributing to anti-KLK5 activity were unclarified. Ursolic acid and tumulosic acid suppressed the proteolytic processing of LL-37 in keratinocytes at ≤10 μM; no cytotoxicity was observed. Both triterpenoids were detected in the plasma of rats administered orally with triterpenoid-rich crude drug Jumihaidokuto. Our study reveals that triterpenoids, such as ursolic acid and tumulosic acid, modulate the KLK5 protease activity and cathelicidin peptide production. Triterpenoids may affect the skin barrier function via the regulation of proteases.

Keywords: Jumihaidokuto; LL-37; cathelicidin; kallikrein 5; kallikrein 7; skin barrier; triterpenoid; tumulosic acid; ursolic acid.

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Conflict of interest statement

S.A. and K.Y. received a research grant from Tsumura & Co. Y.M., T.M., J.K., and A.K. are employees of Tsumura & Co.

Figures

Figure 1
Figure 1
Chemical structure of 19 triterpenoids. 1: Ursolic acid; 2: Oleanolic acid; 3: Polygalacic acid; 4: Platycodigenin; 5: Platycodin D; 6: Betulinic acid; 7: 18β-Glycyrrhetinic acid; 8: Saikosaponin a; 9: Saikosaponin c; 10: Saikosaponin d; 11: Saikosaponin b1; 12: Saikosaponin b2; 13: Saikogenin A; 14: Saikogenin D; 15: Dehydrotumulosic acid; 16: Dehydropachymic acid; 17: Eburicoic acid; 18: Tumulosic acid; 19: Pachymic acid.
Figure 2
Figure 2
Dose-dependent anti-KLK5 activity of triterpenoids. All samples were evaluated at the described concentrations. The percentage of inhibition was calculated based on the formula: (1 − (A − B)/(C − B)) × 100, where A, test sample RFU; B, basal RFU without KLK5; and C, vehicle RFU. Data are presented as mean ± SEM of triplicate tests.
Figure 3
Figure 3
Decrease in LL-37 production in human keratinocytes treated with triterpenoids. Normal human epidermal keratinocytes (NHEKs) were treated with ursolic acid (0.5 μM and 5 μM) or tumulosic acid (1 μM and 10 μM) or vehicle for 23 h in the presence of 10 μM cycloheximide. LL-37 peptide in the lysate of cultured cells was detected using immunoprecipitation and Western blotting. Representative results are shown. Recombinant human LL-37 (0.2 ng) was loaded in the leftmost lane as a standard.
Figure 4
Figure 4
No cytotoxic effect of triterpenoids on cell proliferation. NHEKs were cultured with ursolic acid (0.5 μM and 5 μM) or tumulosic acid (1 μM and 10 μM) or vehicle in the presence or absence of 10 μM cycloheximide. After 24 h culture, cell proliferation activities were measured using an XTT-based cell viability assay kit. Optical density at 465 nm was measured by subtracting the reference absorbance at 630 nm. Data are presented as mean ± SEM of triplicate tests.
Figure 5
Figure 5
Plasma pharmacokinetic analysis of active triterpenoids of Jumihaidokuto. Plasma samples of rats were obtained at 0.25, 0.5, 1, 2, 4, 6, 10 and 24 h after single oral administration of Jumihaidokuto (JHT) (2 g/10 mL/kg, p.o.). Ursolic acid (and/or oleanolic acid) and tumulosic acid in the plasma were measured by LC-MS/MS. Each data point represented the mean ± SEM of triplicate results.

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