ผลของสมุนไพรไทยต่อการอักเสบจากเชื้อแบคทีเรียก่อสิว

Effects of Thai Medicinal Plants on Inflammation caused

by Acne-Inducing Bacteria

สุวิมลสุรัสโม Suvimol Surassmo

วีณานุกูลการ, Veena Nukoolkarn

วันดีกฤษณพันธ, Wandee Gritsanapan

มัลลิกาชมนาวัง, Mullika T. Chomnawang

1

1

Department of Microbiology, 2

Department of Pharmacognosy, Faculty of Pharmacy,

Mahidol University, Bangkok, Thailand.

บทคัดย่อ :

                เชื้อPropionibacterium acnes มีความสัมพันธกับการเกิดสิวอักเสบการศึกษานี้จึงมีจุดมุงหมาย เพื่อตรวจสอบฤทธิ์    ต้านการอักเสบของสมุนไพรไทยโดยพิจารณาจากฤทธิ์ต้านอนุมูลอิสระและผลลดระดับ  ของไซโตคายน์ ผลการทดลองพบว่าสุมนไพรทั้ง19 ชนิดมีฤทธิ์ต้านอนุมูลอิสระโดยมังคุดให้ผลดีที่สุดคือมีค่าIC  50  เท่ากับ6.17 มคก./มล. ส่วนพลูคาวสาบเสือและชุมเห็ดเทศให้ผลปานกลางมีค่าIC  50 เท่ากับ 32.52, 67.55, 112.46 มคก./มล. ตามลําดับในการทดลองโดยวิธีNBT reduction พบว่ามังคุดสามารถลด ระดับROS ได้ถึง77.80± 1.28% นอกจากนี้ยังสามารถยับยั้งการสร้างTNF-αได้ถึง94.59% จากการศึกษา นี้จะเห็นว่ามังคุดเป็นสมุนไพรไทยที่สามารถกําจัดอนุมูลอิสระและลดปริมาณสารสื่ออักเสบได้ดีมาก ดังนั้นมังคุดจึงเป็นสมุนไพรที่มีศักยภาพในการนํามาพัฒนาในการรักษาสิวอักเสบต่อไป

Abstract:

        Propionibacterium acnes has been associated with inflammatory acne. In the present study, Thai medicinal plants were examined for anti-inflammatory property in both free radical scavenging and cytokine reducing activities. The result showed that all 19 plants had some antioxidant effects. Among those, Garcinia mangostana possessed the most significant antioxidant activity at the IC 50 of 6.17 µg/ml. Houttuynia cordata, Eupatorium odoratum, and Senna alata had moderate antioxidant effect with the IC

50 of 32.52, 67.55, and 112.46 µg/ml, respectively. In NBT dye reduction assay, Garcinia mangostana significantly reduced ROS production with the highest inhibitory ratio at 77.80± 1.28%. In addition,

Garcinia mangostana extract could reduce the TNF-α production with the inhibition ratio of 94.59%. Taken together, the results suggested that Garcinia mangostana was effective in scavenging free radicals and was able to reduce the production of proinflammatory cytokines. Therefore, Garcinia mangostana has a potential to be developed as an anti-inflammatory agent used in acnetreatment. Introduction: Propionibacterium acnes has been recognized as an obligate anaerobic organism which is usually found as a normal skin commensal. It acts as an immunostimulator which can produce a variety of enzymes and biologically active molecules, which are implicated in the development of inflammatory acnes. The immunologic response in P.acnes infection involves both humoral and cell-mediated pathways (1). The main components of pilosebaceous unit, keratinocytes and sebocytes, can be activated by P.acnes leading to the production of inflammatory cytokines (2). Moreover, P.acnes significantly induce the interleukin-8 (IL-8) mRNA expression and selectively stimulate the expression of human beta-defensin-2 mRNA in keratinocytes (3). It has been reported that a secreted 31st Congress on Science and Technology of Thailand at Suranaree University of Technology, 18 - 20 October 2005 JC 317444 Botcode 0324 0666 0539 0209 0122 0121 0220 0447 0683 Maincode B peptidoglycan of P.acnes can stimulate the production of the proinflammatory cytokines such as IL-8, IL-1, and tumor necrosis factor-alpha (TNF-α) by human monocytic cell lines and freshly isolated peripheral blood mononuclear cells from acne patients (4). Previous findings suggest that P. acnes has a major role in the inflammation of acne by both antigenic and

mitogenic reactions (5). Additionally, P. acnes can evoke mild local inflammation by producing neutrophil chemotactic factors. Consequently, neutrophils, which are attracted to the acne lesion, release inflammatory mediators such as reactive oxygen species (ROS) (6). The ROS including superoxide radical anion, hydrogen peroxide and hydroxyl radical, generated by neutrophils play a critical role in irritation and disruption of the integrity of the follicular epithelium and are responsible for the inflammatory progression of acne (7). Removal of the ROS can significantly reduce cell damage that may occur during acne

inflammation. Current treatment for acne is mostly based on antibiotics such as clindamycin and

tetracycline derivatives. However, antibiotic-resistant microorganisms are widely spreaded and become a critical problem all over the world (8). Therefore, an alternative treatment of acne must be studied and developed. Medicinal plants have been extensively studies in their biological activity, especially during this decade. In the present study, the 19 Thai medicinal plants were examined for anti-inflammatory property against inflammatory acne induced by P.acnes.

    Methodology: The free radical scavenging ability of the plant extracts was examined by DPPH assay as described by Alessandral et al. (9). Briefly, aliquots of sample were added to DPPH solution and the reaction mixture was shaken vigorously. The reaction involved a color change from violet to yellow which could be measured by the decrease in absorbance at 515 nm and calculated for the radical scavenging activity. Cytokine production assay was performed by the method of Jain and Basal (10). The experiment used an in vitro screening method based on inhibition of pro-inflammatory cytokines (TNF-α), by human peripheral blood mononuclear cells (PBMC). The NBT test was performed according to the method of

Park et al. (11). The test was aimed to determine ROS activity. The stimulated venous blood of healthy volunteers with or without crude extracts was added into the NBT solution, incubated at 25 oC for 15 min and finally stained by Leishman for differential counting of formazan deposits in PMNL.

    Results, Discussion and Conclusion: In this study, the plant extracts were screened for antioxidant activity by DPPH free radical scavenging assay. The result showed that all 19 plants had some antioxidant effects with ranges in activity degree. Among those, Garcinia mangostana had very strong antioxidant activity observed by dark yellow spots that appeared immediately after spraying the DPPH solution. Andrographis paniculata, Eupatorium odoratum, Senna alata, Senna occidentalis, Senna siamea and Tagetes erecta had moderate antioxidant effect whereas the rest showed very little activity. Plant extracts that possessed good antioxidant effect were selected to determine the IC 50 . Garcinia mangostana had strong antioxidant activity and could inhibit 50% of free radicals at the concentration of 6.17 µg/ml.

Houttuynia cordata, Eupatorium odoratum, and Senna alata had moderate antioxidant effect

at the IC 50 concentrations of 32.525, 67.55 and 112.46 µg/ml, respectively. The mangostein

standard, Barleria lupulina, Senna alata and Andrographis paniculata showed no significant

activity in IC 50 values. 31st Congress on Science and Technology of Thailand at Suranaree University of Technology, 18 - 20 October 2005 B31159 ANTIOXIDANT ACTIVITY B31003 ANTIINFLAMMATORY ACTIVITY, ANTIARTHRITIS, ANTIIRRITANT

B14146 TUMOR NECROSING FACTOR INHIBITION

     The next experiment was performed to determine the effect of plant materials on reactive oxygen species production. The PMNLs were exposed to a stimulant alone or with plant extracts. The results showed that Garcinia mangostana could significantly reduce ROS production with the inhibitory ratio at 77.80± 1.28%. Houttuynia cordata, Senna siamea, Eupatorium odoratum, and Senna alata showed moderate inhibitory activity whereas Andrographis paniculata and Barleria lupulina had very little activity on the generation of ROS. In vitro screening test was conducted to determine proinflammatory cytokine activating activity of the plant materials. The result showed that the concentration of TNF-α

in culture supernatant without a stimulant was 8.54 pg/ml. The result demonstrated an increase in the production of TNF-α upto 112.87 pg/ml after stimulated with heat-killed P.acnes while the positive control, zymosan, showed an increase in the production of TNF-α upto 78.71 pg/ml. The result implied that P.acnes could stimulate the proinflammatory cytokines and also played an important role in the pathogenesis of inflammatory acne. We studied the inhibitory effect of the plant extracts on TNF-α synthesis. The highest inhibition of TNF-α (98.38%) was observed at the concentration of 50 µg/ml of the mangostin standard. Garcinia mangostana extract could lower the level of TNF-α at about 94.59%. The

other plant extracts at the same concentration also showed high inhibitory activity against TNF-α production (> 50%) while Senna alata, Houttuynia cordata and Andrographis paniculata at various concentrations had insignificant inhibitory effects on TNF-α. In conclusion, this study demonstrated the potential of Garcinia mangostana to be developed as an anti-inflammatory agent for acne treatment.

References:

(1) Masahiko T, Masaaki M. (2001) Medical Electron Microscopy 39: 29-40.

(2) Leeming JP, Holland KT and Cunliffe WJ (1985) Journal of Medical Microbiology 20: 11-16.

(3) Richard AB, Keith TH (2004) Clinics in Dermatology 22: 375-379.

(4) Gollnick H, Schramn M (1998) Journal of the European Academy of Dermatology and

Venereology (Suppl.1): s8-s12.

(5) Craig GB, Craig NB, Paul FL (1999) Postgraduate Medicine Journal 75: 328-331.

(6) Jame, J, Leyden, MD. (1997) New England Journal of Medicine 336: 1156-1162.

(7) Goulden V, Mcgeown CH, Cunliffe WJ. (1999) British Journal of Dermatology 141: 297-300.

(8) Stathakis V, Kilkenny M, Marks R.(1997) Australian Journal of Dermatology 38: 115- 123.

(9) Braca A, De Tommasi N, Di Bari L, Pizza C, Politi M, Morelli I. (2001) Journal of

Natural Products 64: 892-895.

(10) Jain A, Basal E. (2003) Phytomedicine 10: 34-38.

(11) Park J, Lee J, Jung E, Park Y, Kim K, Park B, Jung K, Park E, Kim J, Park D. (2004)

Journal of Ethnopharmacology 496: 189-195.

Keywords: Acne; Propionibacterium acnes; inflammation

31st Congress on Science and Technology of Thailand at Suranaree University of Technology, 18 - 20 October 2005

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