activity-guided purification identifies lupeol, a

Activity-Guided Purification Identifies Lupeol, aPentacyclic Triterpene, As a Therapeutic AgentTargeting Multiple Pathogenic Factors of AcneHyuck Hoon Kwon1,2,5, Ji Young Yoon2,5, Seon Yong Park1,2, Seonguk Min1,2, Yong-il Kim3, Ji Yong Park3,Yun-Sang Lee3, Diane M. Thiboutot4 and Dae Hun Suh1,2

Acne vulgaris is a nearly universal cutaneous disease characterized by multifactorial pathogenic processes.Because current acne medications have various side effects, investigating new pharmacologically activemolecules is important for treating acne. As natural products generally provide various classes of relatively safecompounds with medicinal potentials, we performed activity-guided purification after a series of screenings fromthe extracts of five medicinal plants to explore alternative acne medications. Lupeol, a pentacyclic triterpene,from the hexane extract of Solanum melongena L. (SM) was identified after instrumental analysis. Lupeol targetedmost of the major pathogenic features of acne with desired physicochemical traits. It strongly suppressedlipogenesis by modulating the IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt/sterol response element–bindingprotein-1 (SREBP-1) signaling pathway in SEB-1 sebocytes, and reduced inflammation by suppressing the NF-kBpathway in SEB-1 sebocytes and HaCaT keratinocytes. Lupeol exhibited a marginal effect on cell viability and mayhave modulated dyskeratosis of the epidermis. Subsequently, histopathological analysis of human patients’ acnetissues after applying lupeol for 4 weeks demonstrated that lupeol markedly attenuated the levels of both thenumber of infiltrated cells and major pathogenic proteins examined in vitro around comedones or sebaceousglands, providing solid evidence for suggested therapeutic mechanisms. These results demonstrate the clinicalfeasibility of applying lupeol for the treatment of acne.

Journal of Investigative Dermatology (2015) 135, 1491–1500; doi:10.1038/jid.2015.29; published online 5 March 2015

INTRODUCTIONAcne vulgaris affects nearly 90% of adolescents worldwideand can leave permanent scarring if it is not properly treated(James, 2005; Williams et al., 2012). Although currentmedications are moderately effective in treating acne, theymay be associated with various side effects (Strauss et al.,2007; Thiboutot et al., 2009). For example, oral isotretinoin,one of the most effective treatments, has potentially seriousside effects including teratogenicity and dyslipidemia. Topicalretinoids, as well as topical and systemic antibiotics, may

cause a burning sensation and antibiotic resistance. Therefore,the need to investigate new antiacne ingredients has beengrowing, and natural products provide important clues foridentifying novel drugs with relative safety that, so far, researchhas neglected (Ji et al., 2009). They are a matchless source ofpharmacologically active drugs even compared with high-throughput combinatorial screening, although only limitednumbers have been tested for the acne model. Because ofthe innate complexity of acne pathogenesis, a variety ofcandidate molecules should be screened simultaneously formultiple pathogenic processes involving seborrhea, inflam-mation, Propionibacterium acnes (P. acnes), and folliculardyskeratosis (Zouboulis et al., 2005). Therefore, stepwisefractionation and screening of natural products would be amore efficient and systematic approach compared with thehypothesis-driven study for a single molecule.

Lupeol (Lup-20(29)-en-3b-ol), a pentacyclic lupane-typetriterpene, is present in several species of the plant kingdomand is abundant in medicinal plants (Saleem, 2009; Siddiqueand Saleem, 2011). The compound possesses wide-spectrumpharmacological activities, including lipid-lowering, anti-inflammatory, and anticancerous effects (Lee et al., 2007;Liu et al., 2013). In addition, a few preclinical studies havesuggested its potential as an anti-inflammatory orchemopreventive agent (Saleem et al., 2001; Tarapore et al.,

ORIGINAL ARTICLE

1Department of Dermatology, Seoul National University College of Medicine,Seoul, South Korea; 2Acne and Rosacea Research Laboratory, Seoul NationalUniversity Hospital, Seoul, South Korea; 3Department of Nuclear Medicine,Seoul National University College of Medicine, Seoul, South Korea and4Department of Dermatology, Pennsylvania State University College ofMedicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania, USA

Correspondence: Dae Hun Suh, Department of Dermatology, Seoul NationalUniversity College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, South Korea. E-mail: [email protected]

5The first two authors contributed equally to this work as the first authors.

Received 6 July 2014; revised 12 January 2015; accepted 21 January 2015;accepted article preview online 3 February 2015; published online 5 March2015

Abbreviations: FFA, free fatty acid; PI3K, phosphatidylinositide 3 kinase; SM,Solanum Melongena L.; SREBP-1, sterol response element–binding protein-1;TLR-2, Toll-like receptor-2

& 2015 The Society for Investigative Dermatology www.jidonline.org 1491

http://dx.doi.org/10.1038/jid.2015.29

mailto:[email protected]

http://www.jidonline.org

2013). These observations, along with the fact that acne ismainly related to lipogenesis and inflammation, suggest that itmay improve acne.

In this research, we conducted activity-guided puri-fication after a series of screenings from five medicinal plants.Lupeol purified from the Solanum melongena L. (SM) extracthas potent antiacne effects. These include sebosuppressiveand anti-inflammatory effects on human SEB-1 sebocytes andHaCaT keratinocytes, as well as beneficial effects on folliculardyskeratosis. Further biochemical and cellular studies indicatethat the modulation of IGF-1R/phosphatidylinositide 3 kinase(PI3K)/Akt and NF-kB signaling pathways mediate lupeol’ssebosuppressive and anti-inflammatory effects, respectively.We subsequently confirmed these experimental results in vivoafter analyzing histopathological changes of human patients’acne tissues after applying lupeol for 4 weeks.

RESULTSAcidic hexane fraction of Solanum melongena L. demonstratedantiacne characteristics in the screening

To explore antiacne ingredients from natural compounds, wescreened five candidate medicinal plants (Agrimonia pilosa,Aleriana fauriei, Lycopodium clavatum, Solanum melongenaL., and Curcuma longa) that are known to be effective for acnefrom the literature or complementary medicine. Methanolextracts of each plant were separated on the basis of polarityand acidity (Figure 1a). Each fraction of these plant extractswas tested for biologic activities such as toxicity, antilipogen-esis, anti-inflammation, and antimicrobial activities. Amongthem, the acidic hexane fraction of SM showed most desiredresults, including antiproliferative effects on SEB-1 sebocyteswith no direct cytotoxicity on the HaCaT keratinocytes and3T3-L1 adipocytes, and suppression of intracellular lipidcontents per equal cell counts (Figure 1b and c). The fractionalso demonstrated anti-inflammatory effects by attenuatingcytokine gene expressions induced by P. acnes–stimulatedSEB-1 sebocytes (Figure 1d). Antimicrobial activity against P.acnes was also observed at a relatively high concentration(400mg ml�1) of the hexane fraction.

Lupeol was isolated and identified after activity-guidedpurification procedures from acidic hexane fraction of SM

On the basis of these initial screening tests, we isolated andidentified specific antiacne chemical components from the SMextract based on activity-guided purification procedures. Afterseparating SM extracts by open-column chromatography andfollowing preparatory HPLC steps, each subfraction wasevaluated by serial antiacne screening steps including toxicity,antilipogenic effects, anti-inflammatory effects, and antimicro-bial activities (Figure 2a). Subfractions showing the mostdesired effects were further purified. The results are summar-ized in Supplementary Table S1 online. With these proce-dures, a molecular structure of a single final fraction thatshowed the best antiacne traits was characterized afteranalyzing data from gas chromatography–mass spectrometry,Fourier transform infrared spectroscopy, and nuclear magneticresonance spectroscopy (Figure 2b–d). Lup-20(29)-en-3b(lupeol), a pentacyclic triterpene, was identified (Figure 2e).

Lupeol decreased lipogenesis in human SEB-1 sebocytesAlthough hyperseborrhea is critical in the pathogenesis ofacne, few topical drugs that are currently used can reducesebum secretion effectively. To examine whether lupeolmodulates lipid synthesis in human SEB-1 sebocytes, intracel-lular neutral lipid content was analyzed by Nile Red stainingafter treating the cells with different concentrations of lupeol.Lupeol significantly decreased lipid by 58% in SEB-1 sebo-cytes treated with 20mM lupeol (Figure 3a and b). Changes inspecific free fatty acid (FFA) components, which have impor-tant roles in the innate immunity of acne inflammation, werefurther analyzed using quantitative fatty acid methyl esteranalysis with gas chromatography–mass spectrometry(Figure 3c). Overall, fatty acid content was reduced as lupeolconcentration increased, with significant decreases in majorFFA components (palmitic acid [C16:0], stearic acid [C18:0],and oleic acid [C18:1]). Addition of lupeol robustly decreased14C acetate incorporation into fatty acids, cholesterol, andsqualene, which are major components of human sebum,dose dependently after cell count normalization, furtherconfirming the lupeol-induced reduction of intracellular lipidsynthesis (Figure 3d).

Downregulation of the IGF-1R/PI3K/Akt/SREBP pathway wasresponsible for the suppression of lipogenesis by lupeol in humanSEB-1 sebocytes

We tested the hypothesis of whether sterol regulatory element–binding proteins (SREBPs), major transcriptional factors respon-sible for the regulation of cholesterol/fatty acid metabolism,were involved in the antilipogenic effects of lupeol. Lupeolsignificantly decreased both precursor and mature forms ofSREBP-1 proteins after 24 hours (Figure 3e). Quantitative real-time PCR showed that lupeol also decreased mRNA levels ofSREBP-1a, SREBP-1c, and SREBP-2, as well as key downstreamtargets of all SREBPs such as fatty acid synthase, acetyl-CoAcarboxylase, HMG-CoA reductase, and HMG-CoA synthase.These suggest that lupeol inhibited the expression of lipogenicmolecules at the transcriptional level (Figure 3f).

To identify upstream regulators that are responsible for thelupeol-mediated suppression of the SREBP pathway, wefocused on the IGF-1R/PI3K/Akt pathway for the followingreasons: (1) previous studies show that lupeol mitigates thePI3K/Akt pathway in a CD-1 mouse model and in AsPC-1 celllines (Saleem et al., 2004, 2005), and (2) IGF-1 treatmentinduces lipogenesis via activation of the IGF-1R/PI3K/Aktpathway in SEB-1 sebocytes (Smith et al., 2008). Lupeoldecreased the IGF-1R/IRS-1/PI3K/Akt pathway in a dose-dependent manner after 3 hours and subsequentlydownregulated SREBP-1 protein expressions (Figure 3e). Co-treatment with lupeol and the PI3K/Akt inhibitor LY 294002potently blocked the expression of phosphorylated Akt.Importantly, this inhibition did not synergistically decreaseprotein content of precursor and mature SREBP-1 or intracel-lular lipid content (Figure 3g and h), suggesting that lupeolmainly suppressed lipogenesis through the PI3K/Akt pathway.Together, these data suggest that lupeol suppressed sebummainly through inhibition of the IGF-1R/PI3K/Akt/SREBP-1signaling pathway in human SEB-1 sebocytes.

HH Kwon et al.Lupeol Modulates Multiple Targets of Acne

1492 Journal of Investigative Dermatology (2015), Volume 135

Lupeol decreased inflammation induced by heat-inactivated P.acnes through the inhibition of the NF-jB pathway in both SEB-1sebocytes and HaCaT keratinocytes

In acne, P. acnes–induced inflammatory response aroundpilosebaceous gland, mainly through the secretion of variousproinflammatory cytokines, represents a key pathogenicfactor leading to disease initiation and aggravation(Kang et al., 2005; Nagy et al., 2006; Agak et al., 2014).We investigated anti-inflammatory effects of lupeol inSEB-1 sebocytes (Figure 4a–d) and HaCaT keratinocytes(Figure 4e–h), two major cutaneous parenchymal cells asso-ciated with acne. Heat-inactivated P. acnes induces severalproinflammatory cytokines including IL-8 and IL-6 in SEB-1sebocytes and tumor necrosis factor-a and IL-6 in HaCaTkeratinocytes, respectively (Figure 4a, b, d, e, and f).

To further examine underlying molecular mechanisms, wemeasured the activation of associated proteins related with theNF-kB pathway. We found that protein expressions of NF-kBp65 and phospho-IkB were significantly increased after P.acnes treatment, strongly suggesting that the activation of NF-kB may induce proinflammatory cytokine expressions(Figure 4c and g). Then, we tested the anti-inflammatory effectof lupeol in this P. acnes-induced inflammatory model. Lupeolsignificantly decreased protein expression of cytokines in adose-dependent manner for both SEB-1 sebocytes and HaCaTkeratinocytes, strongly supporting its anti-inflammatory effect.

Messenger RNA expressions of proinflammatory cytokines inHaCaT keratinocytes showed consistent patterns (Figure 4h).Furthermore, protein expressions of phospho-IkB and NF-kBwere reduced with lupeol, confirming that lupeol inhibitedinnate immunity of two major cutaneous cells associated withinflammatory acne by mitigating the NF-kB pathway inducedby heat-inactivated P. acnes (Figure 4a–h).

Lupeol exhibited only a marginal effect on cell viability, possiblymodulated dyskeratosis of epidermal keratinocytes, andinhibited growth of P. acnes

To test the possible toxicity of lupeol, we investigated theeffect of lupeol treatment on the viability of SEB-1 sebocytesand HaCaT keratinocytes. Using both MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) andcell counting kit-8 assays, we demonstrated that lupeoltreatment was not cytotoxic in conditions identical to othercellular experiments (concentration range 0–20mM for 24 hourof incubation) in either cell type. Only a marginal effect oncell viability was observed at 20mM after 48 hours in SEB-1sebocytes, and no toxicity was observed in HaCaT keratino-cytes (Figure 5a and b). Because follicular epidermal dysker-atosis is another major triggering factor of acne pathogenesis,we investigated the possible beneficial effects of lupeol on theabnormal differentiation of epidermal keratinocytes. Lupeolsignificantly suppressed protein and mRNA expressions of IL-

0

20

40

60

80

100

%

120

140

SEB-1 HaCaT24 Hours

3T3-L1 SEB-1 HaCaT48 Hours

3T3-L1 SEB-1 HaCaT72 Hours

3T3-L1

Control

Control

1 μg ml–1 5 μg ml–1 25 μg ml–1 50 μg ml–1 100 μg ml–1

5 μg ml–1

0

0.2

0.4

0.6

0.8

1

1.2

3h

Partitioned with n-BuOH

or base

Suspended in H2OPartitioned with n-Hexane

acid

Plants screened

MeOH extraction

Crude extracts

n-Hexane layer

• Agrimonia pilosa• Aleriana fauriei Briq• Lycopodium clavatum L.• Solanum melongena L.• Curcuma longa L.

or base

Hexanelayer

Aqueouslayer

Chloroform layeracid

Chloroformlayer

Aqueouslayer

Partitioned with CHCl3

n-BuOH H2O

0

20

40

60

80

100

120

Control 12.5 25(μg ml–1)

50 100

%

Rel

ativ

e ab

unda

nce

of m

RN

A

***

**

***

***

** *

*

**

*

GAPDH

TNF-α TNF-αIL-63h

IL-8 IL-66h

IL-8

Figure 1. Acidic hexane fraction of Solanum melongena L. (SM) demonstrated antiacne characteristics. (a) Extracts of five candidate medicinal plants were

separated on the basis of polarity and acidity. The acidic hexane fraction of SM demonstrated antiacne characteristics by displaying (b) antiproliferative effects in

SEB-1 sebocytes with no general cytotoxicity on the HaCaT keratinocytes and 3T3-L1 adipocytes measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay, (c) antilipogenic effects for SEB-1 sebocytes based on relative percentile quantification of Nile red assay after normalization by

cell numbers, and (d) anti-inflammatory effects in SEB-1 sebocytes stimulated by heat-inactivated P. acnes after 3 and 6 hours based on reverse transcription PCR

(RT-PCR) test for tumor necrosis factor-a (TNF-a), IL-6, and IL-8. All experiments were repeated a minimum of three times (mean±SEM). *Po0.05 between control

and each concentration in acidic hexane fraction of the SM-treated group (Student’s t-test).


www.jidonline.org 1493


1a, a strong inducer of hypercornification of the infundibulm,and Toll-like receptor-2 (TLR-2), a key molecule in IL-1arelease during comedogenesis, in HaCaT keratinocytes stimu-lated by heat-inactivated P. acnes (Figure 5c and d). In accordwith these findings, lupeol also downregulated keratin 16expression, a marker of abnormal differentiation and epider-mal proliferation, in inflamed HaCaT keratinocytes (Figure 5cand e). Then, we investigated the antibacterial effects of lupeolagainst P. acnes, playing critical roles in innate immunity ofacne. To examine this hypothesis, the minimal concentrationof lupeol needed to inhibit P. acnes growth in culture mediumwas compared with vehicle control used for dissolution. Only

lupeol-containing solution effectively inhibited the growth ofP. acnes at rather higher concentrations, suggesting that it mayalso partly contribute to suppress abnormal colonization of P.acnes for acne development in vivo (Figure 5f and g). Aschematic diagram of lupeol’s therapeutic mechanisms basedon whole experimental results is illustrated in Figure 5h.

Histopathological analysis of the patient’s skin tissues afterapplication of lupeol confirmed relevant therapeuticmechanisms in vivo

To determine the consistency with our in vitro findings, wefurther examined changing patterns of inflammatory skin

PrepHPLC #2

Fr 5–1, 2, 3, 4EtOAch/aqueous layer

3,345.9

2,942.3

2,356.11,638.7

1,455.81,043.8

882.7

2,872.1

Tra

nsm

ittan

ce (

% T

)

64

59

54

49

44

Wavcnumber cm–1

4,000 3,100 2,200 1,300 400

Prep HPLC #1

Screening #1 Screening #2

Crude extract(20.1%)

Residue

Solaneummelongena L.

Activity-guided purification procedures

1H NMR peaks: δ 4.69 and δ 4.57 (each 1H,

m, H-29), δ 3.18 (1H, tdd, H-3), δ 2.39 and δ

δ 1.39 (1H, q, H-6), δ 1.36 (1H, t, H-18),

Fr 5–5–2, 3

HOH

H

H

H

n-hexaneextract(1.3%)

Fr 5-5–1

Extractedwith MeOH

1.92 (each 1H, m, H-19), δ 1.68 (1H,t, H-15),

δ 1.66 (3H, s, H-30), δ 1.60 (1H, d, H-2),

δ 1.59 (1H, q, H-2), δ 1.42 (1H, d, H-16),

δ 1.33 (1H, m, H-21), δ 1.20 (1H, m, H-22),

δ 1.03 (1H, q, H-12), δ 0.99 (3H, s, H-23),

δ 0.97 (3H, s, H-27), and δ 0.83,

δ 0.79 (3H, s, H-25, H-28, H-24)

Fr 5

Fr 1–7

55.2

95.2A

bund

ance

Abu

ndan

ce

Minutes

1,000,000

800,000

600,000

400,000

200,000

25 27 29 31 33 350

135.2189.4

218.4

426.8

Molecular weight

35,000

25,000

15,000

10,000

5,000

0

29 41 53 6570

.279

.2 8593

.2 9710

7.2

111.

211

9.2

123.

212

913

5.2

140

147.

215

1.2

157

163.

217

0.2

177.

218

2.8

191.

220

1.2

207.

421

6.2

220.

223

1.2

243.

225

7.2

269

275.

228

6.2

300.

431

6.4

339.

234

4.2

383.

441

2.4

30,000

20,000

Fr 5–5

SiO2 C.CPetrolem ether

/CH2 Cl2-acetone

n-hexane/H2O

in acidiccondition

Figure 2. Lupeol was isolated and identified on the basis of activity-guided purification procedures. (a) Activity-guided purification procedures were performed

based on repetitive chromatographic separation steps with open column chromatography and preparatory HPLC methods for acidic hexane Solanum melongena L.

(SM) extract. Instrumental analyses including (b) gas chromatography–mass spectrometry (GC–MS), (c) Fourier transform infrared spectroscopy (FT-IR), and (d) 1H

nuclear magnetic resonance (NMR) were performed for molecular characterization of the final isolated product that had the most desired biologic activities (Fr 5-5-1)

in terms of toxicity, antilipogenesis, anti-inflammation, and antimicrobial activities. (e) Lup-20 (29)-en-3b (lupeol), a pentacyclic triterpene, was finally identified.



Peak c.p.m: 55

Peak c.p.m: 226

200

150

100

50

0

0 20 40 60

50.0

40.0

30.0

20.0

10.0

0.0

0 20 40 60

Control

Lupeol20 μM

62

120

0

20

40

60

80

100

0 5 μM 10 μM 20 μM

%

*

*

1 0.9 0.7 0.5**

PrecursorSREBP-1

MatureSREBP-1

0 5 10 20Lupeol (μM)

pIGF-1R 95

60

62

62

68

42

180

125

pAkt

tAkt

pPI3K

β-Actin

PrecursorSREBP-1

MatureSREBP-1

pAkt

tAkt

β-Actin

Control 5 μM

10 μM 20 μM

0

5

10

15

20

C14 0 C14 1 C16 0 C16 1 C18 0 C 181n9 cand t

C 182n6 cand t

C20 1 C203n3

Control 5 μM

10 μM 20 μM

pIRS-1

0 5 10 20Lupeol (μM)

LY 294002 – + + + +

0

20

40

60

80

100

120

ControlLupeol

LY 294002 + + + +42

62

125

68

0 Sizemarker

Rel

ativ

e ab

unda

nce

of m

RN

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

SREBP-1a

SREBP-1c

SREBP-2

FAS ACC HMGCR HMGCS

Control 5 μM 10 μM 20 μM

5 μM 10 μM 20 μM

**

*

*

**

*

*

*

*

*

*

**

* *

**

**

*

20

0

40

60

80

100

120

140

160

FA C SQ

Control

Lupeol 5 μM

Lupeol 10 μM

Lupeol 20 μM

*

*

**

**

*

**

**

MW(kDa)

MW(kDa)

1 0.9 0.7 0.5**

1 0.9 0.7 0.6**

1 0.9 0.8 0.6**

1 0.8 0.7 0.5**

1 0.8 0.7 0.5*

*

**

%

1 11.1 1.1

1 10.9 1.1

d.p.

m. p

er 1

04 cel

l cou

nts

μg p

er s

ampl

e of

104 c

ell c

ount

s

Figure 3. Lupeol decreased lipogenesis in SEB-1 sebocytes by suppressing the IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt/sterol response element–binding

protein (SREBP) signaling pathway. (a) Nile red assay was performed for SEB-1 sebocytes treated with lupeol for 24 hours (bar¼ 10mm). (b) Relative percentile

quantification after cell count normalization. (c) Fatty acid methyl ester–gas chromatography (FAME-GC) analysis was performed to detect quantitative changes in

fatty acid profiles. (d) Changes of 14C acetate incorporation levels in specific lipid components of SEB-1 cells after lupeol treatments were analyzed using thin-layer

chromatography (C, cholesterol; FA, fatty acid; SQ, squalene) (left). Representative radio-TLC data for fatty acid (red line) of control and 20mM lupeol samples

(right). After 24 hours of lupeol treatment, (e) western blotting of phospho-IGF-1R, phospho IRS-1, phospho-PI3K, phospho-Akt, precursor, and mature SREBP-1

was performed, and (f) RNA was subjected to quantitative real-time PCR to determine the abundance of SREBP-1a, SREBP-1c, SREBP-2, fatty acid synthase (FAS),

acetyl CoA carboxylase (ACC), HMG-CoA reductase (HMGCR), and HMG-CoA synthase (HMGCS). After pretreatment with LY294002 at 30 minutes before lupeol

treatment, (g) western blotting of phospho-Akt, precursor, and mature SREBP-1 was performed, and (h) quantification of Nile red assay was done. All experiments

were repeated a minimum of three times (mean±SEM). *Po0.05 between each lupeol-treated group and control (Student’s t-test).




pathologies and several target protein expressions associatedwith acne pathogenesis in the human tissue after lupeolapplication. Skin specimens including typical acne lesionswere acquired from patients before and after applying 2%lupeol twice daily for 4 weeks. The major findings ofhematoxylin and eosin staining demonstrated that lupeolattenuated the manifestation of infiltrated inflammatory cellsaround comedones or sebaceous glands (Figure 6a). This smallmolecule also significantly decreased expressions of IGF-1R,SREBP-1, NF-kB p65, and IL-8 in the human acne lesion,consistent with results from our cellular assays (Figures 6b–e).As a real control of the baseline state of skin, the expression ofthese four major pathogenic proteins in nonacne skin wassignificantly lower than in the skin with acne lesions at bothtime points (IGF-1R: 0.8, SREBP-1: 0.5, NF-kB p65: 0.8, IL-8:0.8). These hematoxylin and eosin staining and immunohis-tochemical analyses confirmed that lupeol decreased lipogen-esis and inflammation, two critical steps in acne pathogenesis,from human acne lesions in vivo.

We also found that expression levels of IL-1a and TLR-2were significantly decreased after lupeol application for 4weeks (Figure 6f and g). In addition, the expression of keratin16 was significantly downregulated around the whole

epidermis (Figure 6h). The expression of these three proteinswas also significantly lower in nonacne skin than in skincontaining acne lesions at both time points (IL-1a: 0.5, TLR-2:0.5, keratin 16: 0.3). These results suggest that lupeol mayhave preventive roles in the progression of pathogeniccornification in the early phase of acne pathogenesis.

DISCUSSIONDespite the near-universal prevalence of acne and correspond-ing demands for treatment, the development of effectiveantiacne medications has been hampered by the complexityof acne pathogenesis. In our previous study, we discovered thatepigallocatechin-3-gallate improves acne by modulating multi-ple pathogenic factors, including hyperseborrhea, inflamma-tion, and P. acnes overgrowth (Yoon et al., 2013). In spite ofgreat efficacy in properly controlled condition, relativemolecular instability of this antioxidant could be affected byvarious external factors including solvent, pH, temperature,ionic strength, and oxidative stress (Proniuk et al., 2002).Possible molecular degradation during drug formulation ordelivery processes may lead to significant decreases oftherapeutic potentials. To resolve this issue by exploring newclass of small molecules, we performed activity-guided

Control

p.acnes + lupeol 5 μM

p.acnes

p.acnes + lupeol 10 μM p.acnes + lupeol 20 μM

phospho-IκBα

NF-κBp65

β-Actin1 1.4* 1.3 0.8†1.0†

1 1.9* 1.4† 1.2† 1.0†

1 1.2* 0.9 0.7† 0.6†

SEB-1 sebocytes

IκBα(total)

phospho-IκBα

NF-κBp65

β-actin

IκBα(total)

1 1.3* 1.2 0.8† 0.7†

0

2468

10121416

IL-6 IL-8 TNF-α

*†

†

*

†

†

*

† †

HaCaT keratinocytes

P. acnes

Lupeol(μM)

+

0

+

20

IL-8

0

20

40

60

80

100

120IL-8

*

†

P. acnes + + +–

0 0

+

5 10 20

†65

42

40

39

39

*

0

5

10

15

20TNF-α

†

†

+ + +–

0 0

+

5 10 20

0

5

10

15

20

25IL-6

*

††

+ + +–

0 0

+

5 10 20

0 0 5 10 20Lupeol (μM)

Lupeol (μM)

pg m

l–1

pg m

l–1

pg m

l–1

pg m

l–1

Rel

ativ

e ab

unda

nce

of m

RN

A

P. acnes – + + + +

0 0 5 10 20

– + + + +MW

(kDa)

MW(kDa)

65

42

40

0

5

10

15

20

25

30IL-6

†

*

†

Lupeol(μM)

P. acnes

Lupeol(μM)

P. acnes

Lupeol(μM)

P. acnes

Lupeol(μM)

+ + +–

0 0

+

5 10 20

P. acnes

Figure 4. Lupeol decreased inflammation induced by heat-inactivated P. acnes through the inhibition of the NF-jB pathway in both SEB-1 sebocytes and HaCaT

keratinocytes. ELISA was performed for (a) IL-8 and (b) IL-6 in the supernatant of SEB-1 sebocytes stimulated with heat-inactivated P. acnes before and after

treatments with lupeol for 24hours. (c) Western blotting of NF-kB p65 and phospho-IkBa under the same condition. (d) Immunocytofluorescence staining was done

for IL-8 (green) with cell nuclei counterstained with 4,6-diamidino-2-phenylindole (blue) (bar¼ 50mm). Parallel experiments were performed with HaCaT

keratinocytes. ELISA for (e) tumor necrosis factor-a (TNF-a), (f) IL-6, (g) western blotting of NF-kB p65, and phospho-IkBa, and (h) quantitative real-time PCR for IL-6,

IL-8, and TNF-a in HaCaT keratinocytes. All experiments were repeated a minimum of three times (mean±SEM). *Po0.05 between control and each P. acnes–

stimulated group, wPo0.05 between P. acnes–stimulated, lupeol nontreated group, and each lupeol-treated group (Student’s t-test).



purification after a series of screening tests from extracts ofvarious medicinal plants, and finally isolated lupeol showingdesirable therapeutic effects against all major targets of acne.This systematic methodology would have implications forfuture research. Lupeol is quite lipophilic, has no ionizablemoiety in the physiologic pH, and it is chemicallystable (Laszczyk, 2009). It is also useful in maintaining skintexture and integrity in animal studies (Harish et al., 2008).These physicochemical and biological properties wouldenhance the absorption rate through the solid skin barrierand accumulation around the pilosebaceous unit during a longperiod of time without notable side effects (Mitragotri, 2003).

Excessive sebum secretion is crucial in acne pathogenesis,and only oral isotretinoin and hormonal therapy have beenshown to reduce seborrhea (Nelson et al., 2008). In thisstudy, lupeol had sebosuppressive effects mainly through thedownregulation of IGF-1R/PI3K/Akt/SREBP-1 signalingpathway in SEB-1 sebocytes with a marginal effect on cellviability. Significant decreases in typical FFAs, including

palmitic acid and oleic acid, were remarkable because theseFFAs enhance innate immune system by inducingantimicrobial peptides (Nakatsuji et al., 2010; Makrantonakiet al., 2011). However, the concentration of linoleic acid, apolyunsaturated FFA that attenuates related inflammation bydecreasing NF-kB-mediated host immune response (Zhaoet al., 2005), did not change with lupeol treatment.Therefore, changing patterns of these fatty acid componentsmay subsequently alleviate inflammation. We confirmed thisantilipogenic effect from other lipid-laden cell lines byshowing that lupeol also decreased intracellular lipidaccumulation in adipocytes without notable cytotoxicity(Supplementary Figure S1 online). Although lupeol normalizesthe serum lipid profiles in animals fed a high-fat andcholesterol diet, associated cellular mechanisms have neverbeen investigated extensively (Sudhahar et al., 2006;Ardiansyah et al., 2012). Taken together, lupeol may provideanother therapeutic strategy to target lipogenesis in acne orseborrhea itself.

P. acnes +lupeol 20 μM

0

1

2

3

4

5

Rel

ativ

e ab

unda

nce

of m

RN

A

IL-1α TLR-2

β-Actin

IL-1α1 1.5* 1.3 1.0† 0.8†

TLR-21 1.7* 1.3 1.1† 0.8†

**

*†

† †

† †Control Lupeol 5 μM Lupeol 10 μM Lupeol 20 μM

0

20

40

60

80

100

120

SEB-1 HaCaT SEB-1 HaCaTSEB-1 HaCaT SEB-1 HaCaT24 Hours 48 Hours

0

20

40

60

80

100

120

24 Hours 48 Hours

% %

MTT assay CCK-8 assay

**

33

42

90

Lupeol Control

1 2 3

4 5

1 2 3

4 5

Keratin 16

Keratin 16 481 1.3* 1.2 0.9† 0.8†

0 0 5 10 20Lupeol (μM)P. acnes – + + + +

MW(kDa)

Control

P.acnes + lupeol 5 μM

P.acnes



No 1 2 3 4 5

Conc.(μM) 2,300 1,150 575 287.5 143.8

Lupeol – – + + +

Control + + + + +

P. acnes

P.acnes

Activation

pIκB

IκB

NFκB IκB

IκB

P. acnes

IL-1α

NFκB

TNF-α

IL-1α

HaCaTkeratinocytes

TLR-2

IL-6IL-8

NFκB

NFκB

IL-6IL-8

1. Downregulation ofinflammation

2. Sebosuppression

Fatty acids ↓Cholesterol ↓Squalene ↓

Lupeol (Lup-20(29)-en-3β) targets all four major pathological processes of acne

3. Antibiotic effect forP. acnes

4. Improvement forfollicular dyskerationization

SREBP

FAS, ACC,HMGCR,HMGCS

Akt

PI3K

IRS-1

IGF-1R

Lupeol

Lupeol

TLR-2

p

Activation

Lupeol

K-16

P. acnes

PIP2

PIP3

SEB-1sebocytes

Figure 5. Lupeol exhibited only a marginal effect on cell viability, possibly modulated dyskeratosis of epidermal keratinocytes, and inhibited the growth of P.

acnes. Cell viability of both SEB-1 sebocytes and HaCaT keratinocytes was measured by both (a) MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) and (b) CCK-8 (cell counting kit-8) assays after treatment with lupeol for 24 and 48 hours. For HaCaT keratinocytes stimulated with heat-inactivated P.

acnes before and after treatments with lupeol for 24 hours, (c) western blotting of IL-1a, Toll-like receptor-2 (TLR-2), and keratin 16, and (d) quantitative real-time

PCR were performed. (e) Immunocytofluorescence staining was done for keratin 16 (green) with cell nuclei counterstained with 4,6-diamidino-2-phenylindole

(blue) (bar¼ 50mm). (f) P. acnes was incubated with increasing concentrations of lupeol and vehicle control. (g) Their antibacterial effects were measured and

minimal inhibition concentration of lupeol was determined. (h) Possible therapeutic mechanisms based on overall experimental results. *Po0.05 between control

and each P. acnes–stimulated group, wPo0.05 between P. acnes–stimulated, lupeol nontreated group, and each lupeol-treated group (Student’s t-test).




Many cutaneous diseases are associated with chronic inflam-mation that also aggravates acne and frequently leaves remnantscars (Harper and Thiboutot, 2003; Yamasaki et al., 2007). Wefound that lupeol alleviated the inflammatory response in well-established in vitro models of inflammatory acne by inhibitingthe NF-kB pathway (Lee et al., 2014). Although the exactmechanism by which lupeol mitigates NF-kB remains elusive,the decrease of phospho-IkB that we observed strongly suggeststhat lupeol acts upstream of IkB degradation (Pikarsky et al.,2004). The uniqueness of acne inflammation is not in thenature of the signaling cascade but in the localization of theprocess (specialized sebaceous follicles) (Kang et al., 2005).Downregulation of the NF-kB pathway, a representativeindicator of inflammation, may provide insight into themolecular mechanism underlying lupeol’s effects on thesignificant improvement of inflammatory skin pathologies.Consistent with our results, downregulation of NF-kB is the

major mechanism underlying the anticancer properties oflupeol against various cancer cell lines (Saleem et al., 2004;Lee et al., 2007). As NF-kB and Akt pathways are functionallyinterconnected (Guo et al., 2009), lupeol may target a proteincommonly shared by these two pathways.

We found elevated levels of the proinflammatory cytokineIL-1a in acne lesions in vivo. Previous works have shown thatexposing isolated infundibula to IL-1a in vitro induces epi-dermal dyskeratosis (Guy et al, 1996; Graham et al., 2004;Kim, 2005), and a recent report suggests that activation ofTLR-2 in basal and infundibular keratinocytes provokes therelease of IL-1a, thereby initiating comedogenesis (Selwayet al., 2013). Remarkably, lupeol decreased both IL-1a andTLR-2 from inflamed HaCaT keratinocytes in our study.Differentiation-inducing activities of lupeol have beenreported in other cell lines (Hata et al., 2005). Therefore,these results suggest that lupeol modulates a comedogenic

Keratin 16

IL-8

TLR-2

BL

0 1 2 3 4 5

*

* * *

*BL

4 Weeks

0 1 2 3 4 5

BL

4 Weeks

0 1 2 3 4 5

BL

4 Weeks

0 1 2 3 4 5

BL

4 Weeks

0 1 2 3 4 5

BL

4 Weeks

*

0 1 2 3 4 5

BL

4 Weeks

*

0

1

2

3

4

5

BL 4 Weeks0

1

2

3

4

5

BL 4 Weeks

*Baseline 4-Week lupeol 4-Week lupeolBaseline

4-Week lupeolBaseline4-Week lupeolBaseline4-Week lupeolBaseline

4-Week lupeolBaseline 4-Week lupeolBaseline 4-Week lupeolBaseline

SREBP-1 NF-κB p65

IL-1α

H & E IGF-1R

BL

Figure 6. Histopathological changes from human acne tissues after applying lupeol for 4 weeks confirmed proposed therapeutic mechanisms in vivo. To detect

changing patterns of both acne inflammation levels around comedones or sebaceous glands and expression of several target proteins associated with acne

pathogenesis in vivo, typical acne lesions were acquired from patients before and after applying 2% lupeol twice daily for 4 weeks. (a) Histopathological

inflammation severity assessment in hematoxylin and eosin staining (H&E), and immunohistochemical staining intensities of target proteins from patient skin

tissues for (b) IGF-1R, (c) sterol response element–binding protein-1 (SREBP-1), (d) NF-kB p65, (e) IL-8, (f) IL-1a, (g) Toll-like receptor-2 (TLR-2), and (h) keratin

16 were analyzed at baseline and final visit (bar¼ 100mM, arrows: inflammation around comedones or sebaceous glands). *Po0.05 compared with baseline

(Student’s t-test). BL, baseline.



process in the early phase of acne development. We alsodiscovered that P. acnes was vulnerable to lupeol. Althoughminimal inhibitory concentration was rather higher comparedwith the concentration range shown in other experiments, theantibiotic effect of lupeol, which has been reported in severalstudies (Tanaka et al., 2004; Ahmed et al., 2010), should befurther examined in vivo around acne lesions for the followingreasons: First, sebocytes may demonstrate less cytotoxicityin vivo. Second, P. acnes related to acne inflammation locatedabove the sebaceous glands (e.g., the gland duct) may beexposed to a much higher concentration of lupeol thansebocytes within the gland.

On the basis of these in vitro results, we further investigatedhistopathological changes of typical acne lesions of humanpatients after applying 2% lupeol for 4 weeks. The resultsconfirmed our proposed therapeutic mechanisms in humanacne tissue. Levels of both infiltrated inflammatory cellnumbers around comedones or sebaceous glands and sixmajor pathogenic proteins involved with lipogenesis, inflam-mation, and follicular dyskeratosis were significantlydecreased. Keratin 16 is the type I keratin partner of keratin6 in the intermediate filament heterodimer formation and isupregulated in all abnormally differentiating and hyperproli-ferative suprabasal keratinocytes (Aldana et al., 1998; Ramotet al., 2013). Therefore, decreases of typical inducers and amarker of abnormal differentiation in vivo further support thatlupeol not only has potential therapeutic effects against activeacne as described, but also exerts a prophylactic activity in theprevention of comedone formation. Follow-up studies arerequired.

In summary, lupeol modulated the key pathological factorscontributing to acne, including hyperseborrhea, inflammation,follicular dyskeratosis, and P. acnes overgrowth. These resultsstrongly suggest the potential clinical feasibility of lupeol inacne treatment.

MATERIALS AND METHODSCell culture

The SEB-1-immortalized human sebocyte cell line was generated by

transfection of secondary sebocytes with SV40 large T antigen, as

previously described (Thiboutot et al., 2003). SEB-1 cells were

cultured and maintained in standard culture medium containing

DMEM (Invitrogen, Carlsbad, CA), 5.5 mM glucose/Ham’s F-12 3:1

(Invitrogen), fetal bovine serum 2.5% (HyClone, Logan, UT), adenine

1.8� 10� 4M (Sigma, St Louis, MO), hydrocortisone 0.4 mg ml� 1

(Sigma), insulin 10 ng ml� 1 (Sigma), epidermal growth factor

3 ng ml� 1 (Austral Biologicals, San Ramon, CA), and cholera toxin

1.2� 10� 10M (Sigma) at 37 1C in a 5% CO2 incubator. The human

keratinocyte cell line HaCaT was maintained in DMEM (Invitrogen)

supplemented with 5% fetal bovine serum, 20 mM L-glutamine,

1 mM sodium pyruvate, and antibiotic/antimycotic solution

(10 U ml� 1 penicillin, 10mg ml� 1 streptomycin, and 0.25mg ml� 1

amphotericin; Invitrogen) at 37 1C in a 5% CO2 incubator.

Western blot analysis

Protein was extracted using cell lysis buffer (Cell Signaling Technol-

ogy, Beverly, MA). Protein contents in lysates were determined using

the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of

protein were run on 10% SDS-PAGE gels and then transferred to a

polyvinylidene difluoride membrane. The blots were primarily

probed with Akt rabbit antibody, Phospho-Akt (Thr308) rabbit anti-

body, Phospho-IRS-1 (Ser 307) rabbit antibody, IkBa rabbit antibody,

Phospho-IkBa (Ser32/36) mouse antibody, Phospho-PI3K p85(Tyr

458)/p55 (Tyr199) rabbit antibody (Cell Signaling Technology),

b-actin mouse antibody, NF-kB p65 mouse antibody, SREBP-1 rabbit

antibody, keratin 16 mouse antibody (Santa Cruz Biotechnology,

Santa Cruz, CA), Phospho-IGF1 receptor rabbit antibody, TLR-2 rabbit

antibody (Abcam, Cambridge, UK), and IL-1a mouse antibody (R&D

Systems, Minneapolis, MN). Secondary anti-rabbit IgG and anti-

mouse IgG antibody (Cell Signaling Technology) were used to detect

primary antibodies. Blots were developed with WESTSAVE Up

(LabFrontier, Seoul, South Korea) and exposed to the film. Films of

blots were analyzed and quantified using a densitometric program

(TINA; Raytest Isotopenmebgerate, Straubenhardt, Germany).

Thin-layer chromatography

SEB-1 cells were grown to 80% confluence in 60 mm dishes and

treated with control or lupeol (5, 10, and 20mM) for 24 hours. In all

experiments, cells were counted to normalize the data. The remaining

cells were suspended in a DMEM solution containing 2mCi 14C-acetate

and incubated for 2 hours at 37 1C with agitation, and extracted twice

with ethyl ether and nonradioactive carrier lipids. Samples were

dissolved in a small volume of ethyl acetate and spotted on 20-cm

silica gel thin-layer chromatography plates (Merck, Darmstadt, Ger-

many), which were run until the solvent front reached 19.5 cm in

hexane, followed by 19.5 cm in benzene, and finally to 11 cm in

hexane/ethyl ether/glacial acetic acid (69.5:30:1.5). Lipid spots were

visualized, excised, and radioactivity in each spot was quantified in a

liquid scintillation counter. The radio-TLC was also checked using TLC

aluminum sheets 20� 20 cm silica gel (Merck) as a stationary phase.

Fatty acid methyl ester analysis with gas chromatographyTo analyze changes of specific fatty acid components after lupeol

treatments, SEB-1 sebocytes treated with control or different concen-

trations of lupeol were grounded after freeze drying. Then, they were

placed in tubes with Teflon caps. Pentadecanoic acid (15:0) is used as

an internal standard. Methylation and extraction steps were per-

formed as previously described (Garces and Mancha, 1993). Gas

chromatography (7890 A, Agilent, Santa Clara, CA) with flame

ionization detector was used for detection.

CONFLICT OF INTERESTThe authors state no conflict of interest.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES

Agak GW, Qin M, Nobe J et al. (2014) Propionibacterium acnes induces an IL-17 response in acne vulgaris that is regulated by Vitamin A and VitaminD. J Invest Dermatol 134:366–73

Ahmed Y, Sohrab MH, Al-Reza SM et al. (2010) Antimicrobial and cytotoxicconstituents from leaves of Sapium baccatum. Food Chem Toxicol48:549–52

Aldana OL, Holland DB, Cunliffe WJ (1998) Variation in pilosebaceous ductkeratinocyte proliferation in acne patients. Dermatology 196:98–9



http://www.nature.com/jid

http://www.nature.com/jid


Ardiansyah Yamaguchi E, Shirakawa H et al. (2012) Lupeol supplementationimproves blood pressure and lipid metabolism parameters in stroke-pronespontaneously hypertensive rats. Biosci Biotechnol Biotech 76:183–5

Garces R, Mancha M (1993) One-step lipid extraction and fatty acid methylesters preparation from fresh plant tissues. Anal Biochem 211:139–43

Graham GM, Farrar MD, Cruse-Sawyer JE et al. (2004) Proinflammatorycytokine production by human keratinocytes stimulated with Propioni-bacterium acnes and P. acnes GroEL. Br J Dermatol 150:421–8

Guo C, Gasparian AV, Zhuang Z et al. (2009) 9-Aminoacridine-based antic-ancer drugs target the PI3K/AKT/mTOR, NF-kappaB and p53 pathways.Oncogene 28:1151–61

Guy R, Green MR, Kealey T (1996) Modeling acne in vitro. J Invest Dermatol106:176–82

Harish BG, Krishna V, Santosh Kumar HS et al. (2008) Wound healing activityand docking of glycogen-synthase-kinase-3-beta-protein with isolatedtriterpenoid lupeol in rats. Phytomedicine 15:763–7

Harper JC, Thiboutot DM (2003) Pathogenesis of acne: recent researchadvances. Adv Dermatol 19:1–10

Hata K, Hori K, Murata J et al. (2005) Remodeling of actin cytoskeleton inlupeol-induced B16 2F2 cell differentiation. J Biochem 138:467–72

James WD (2005) Clinical practice. Acne. N Eng J Med 352:1463–72

Ji HF, Li XJ, Zhang HY (2009) Natural products and drug discovery. Canthousands of years of ancient medical knowledge lead us to new andpowerful drug combinations in the fight against cancer and dementia?EMBO Rep 10:194–200

Kang S, Cho S, Chung JH et al. (2005) Inflammation and extracellular matrixdegradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo. Am JPathol 166:1691–9

Kim J (2005) Review of the innate immune response in acne vulgaris: activationof Toll-like receptor 2 in acne triggers inflammatory cytokine responses.Dermatology 211:193–8

Laszczyk MN (2009) Pentacyclic triterpenes of the lupane, oleanane andursane group as tools in cancer therapy. Planta Med 75:1549–60

Lee TK, Poon RT, Wo JY et al. (2007) Lupeol suppresses cisplatin-inducednuclear factor-kappaB activation in head and neck squamous cellcarcinoma and inhibits local invasion and nodal metastasis in anorthotopic nude mouse model. Cancer Res 67:8800–9

Lee WR, Kim KH, An HJ et al. (2014) The protective effects of Melittin onPropionibacterium acnes-induced inflammatory responses in vitro andin vivo. J Invest Dermatol 134:1922–30

Liu F, He Y, Liang Y et al. (2013) PI3-kinase inhibition synergistically promotedthe anti-tumor effect of lupeol in hepatocellular carcinoma. Cancer CellInt 13:108

Makrantonaki E, Ganceviciene R, Zouboulis C (2011) An update on the role ofthe sebaceous gland in the pathogenesis of acne. Dermatoendocrinol3:41–9

Mitragotri S (2003) Modeling skin permeability to hydrophilic and hydrophobicsolutes based on four permeation pathways. J Control Release 86:69–92

Nagy I, Pivarcsi A, Kis K et al. (2006) Propionibacterium acnes andlipopolysaccharide induce the expression of antimicrobial peptides andproinflammatory cytokines/chemokines in human sebocytes. MicrobesInfect 8:2195–205

Nakatsuji T, Kao MC, Zhang L et al. (2010) Sebum free fatty acids enhance theinnate immune defense of human sebocytes by upregulating beta-defensin-2 expression. J Invest Dermatol 130:985–94

Nelson AM, Zhao W, Gilliland KL et al. (2008) Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis ofhuman sebaceous gland cells. J Clin Invest 118:1468–78

Pikarsky E, Porat RM, Stein I et al. (2004) NF-kappaB functions as a tumourpromoter in inflammation-associated cancer. Nature 431:461–6

Proniuk S, Liederer BM, Blanchard J (2002) Preformulation study of epigallo-catechin gallate, a promising antioxidant for topical skin cancer preven-tion. J Pharm Sci 91:111–6

Ramot Y, Sugawara K, Zakany N et al. (2013) A novel control of human keratinexpression: cannabinoid receptor 1-mediated signaling down-regulatesthe expression of keratins K6 and K16 in human keratinocytes in vitro andin situ. Peer J 1:e40

Saleem M (2009) Lupeol, a novel anti-inflammatory and anti-cancer dietarytriterpene. Cancer Lett 285:109–15

Saleem M, Alam A, Arifin S et al. (2001) Lupeol, a triterpene, inhibits earlyresponses of tumor promotion induced by benzoyl peroxide in murineskin. Pharmacol Res 43:127–34

Saleem M, Afaq F, Adhami VM et al. (2004) Lupeol modulates NF-kappaB andPI3K/Akt pathways and inhibits skin cancer in CD-1 mice. Oncogene23:5203–14

Saleem M, Kaur S, Kweon MH et al. (2005) Lupeol, a fruit and vegetablebased triterpene, induces apoptotic death of human pancreatic adeno-carcinoma cells via inhibition of Ras signaling pathway. Carcinogenesis26:1956–64

Selway JL, Kurczab T, Kealey T et al. (2013) Toll-like receptor 2 activation andcomedogenesis: implications for the pathogenesis of acne. BMC Dermatol13:10

Siddique HR, Saleem M (2011) Beneficial health effects of lupeol triterpene: areview of preclinical studies. Life Sci 88:285–93

Smith TM, Gilliland K, Clawson GA et al. (2008) IGF-1 induces SREBP-1expression and lipogenesis in SEB-1 sebocytes via activation of thephosphoinositide 3-kinase/Akt pathway. J Invest Dermatol 128:1286–93

Strauss JS, Krowchuk DP, Leyden JJ et al. (2007) Guidelines of care for acnevulgaris management. J Am Acad Dermatol 56:651–63

Sudhahar V, Kumar SA, Varalakshmi P (2006) Role of lupeol and lupeollinoleate on lipemic-oxidative stress in experimental hypercholesterole-mia. Life Sci 78:1329–35

Tanaka T, Ikeda T, Kaku M et al. (2004) A new lignan glycoside andphenylethanoid glycosides from Strobilanthes cusia BREMEK. ChemPharm Bull 52:1242–5

Tarapore RS, Siddiqui IA, Adhami VM et al. (2013) The dietary terpene lupeoltargets colorectal cancer cells with constitutively active Wnt/beta-cateninsignaling. Mol Nutr Food Res 57:1950–8

Thiboutot D, Jabara S, McAllister JM et al. (2003) Human skin is asteroidogenic tissue: steroidogenic enzymes and cofactors are expressedin epidermis, normal sebocytes, and an immortalized sebocyte cell line(SEB-1). J Invest Dermatol 120:905–14

Thiboutot D, Gollnick H, Bettoli V et al. (2009) New insights into themanagement of acne: an update from the Global Alliance to ImproveOutcomes in Acne group. J Am Acad Dermatol 60:S1–50

Williams HC, Dellavalle RP, Garner S (2012) Acne vulgaris. Lancet 379:361–72

Yamasaki K, Di Nardo A, Bardan A et al. (2007) Increased serine proteaseactivity and cathelicidin promotes skin inflammation in rosacea. Nat Med13:975–80

Yoon JY, Kwon HH, Min SU et al. (2013) Epigallocatechin-3-gallate improvesacne in humans by modulating intracellular molecular targets andinhibiting P. acnes. J Invest Dermatol 133:429–40

Zhao G, Etherton TD, Martin KR et al. (2005) Anti-inflammatory effects ofpolyunsaturated fatty acids in THP-1 cells. Biochem Biophys Res Com-mun 336:909–17

Zouboulis CC, Eady A, Philpott M et al. (2005) What is the pathogenesis ofacne? Exp Dermatol 14:143–52



activity-guided purification identifies lupeol, a

Documents