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Inhibition of Propionibacterium acnes-induced mediators of inflammation by Indian herbs.

Summary

Propionibacterium acnes, an anaerobic pathogen, plays an important role in the pathogenesis of acne by inducing certain inflammatory mediators. These mediators include reactive oxygen species (ROS) and pro-inflammatory cytokines. In the present study, ROS, interleukin-8 (IL-8) and tumor necrosis factor-[alpha] (TNF-[alpha]) were used as the major criteria for the evaluation of anti-inflammatory activity. To prove the anti-inflammatory effects of herbs, polymorphonuclear leukocytes (PMNL) and monocytes were treated with culture supernatant of P. acnes in the presence or absence of herbs. It was found that Rubia cordifolia, Curcurna longa, Hemidesmus indicus, and Azadirachta indica caused a statistically significant suppression of ROS from PMNL. Sphaeranthus indicus caused a smaller, still significant suppression of ROS. Aloe vera had no effect on ROS production. In the case of proinflammatory cytokine-induced monocytes, maximum suppression was shown by Azadirachta indica and Sphaeranthus indicus, followed by H emidesmus indicus, Rubia cordifolia, and Curcuma longa. Aloe vera showed insignificant inhibitory activity. Thus, these herbs shows anti-inflammatory activity by suppressing the capacity of P. acnes-induced ROS and pro-inflammatory cytokines, the two important inflammatory mediators in acne pathogenesis.

Key words: Propionibacteriurn acnes, reactive oxygen species, human, polymorphonuclear leukocytes, herbs

* Introduction

Acne is a chronic inflammatory follicular disorder of the skin, occurring in specialized pilosebaceous units on the face. Comedonal bacteria, "Propionibacterium acnes ", plays an important role in the pathogenesis of acne inflammation by inducing polymorphonuclear leukocytes (PMNL) and monocyte and/or macrophages to produce pro-inflammatory mediators. These organisms produce neutrophil chemotactic factors, which attract neutrophils to release inflammatory mediators such as reactive oxygen species (ROS) and lysosomal enzymes, resulting in disruption of the integrity of the follicular epithelium (Webster and Leyden, 1980). ROS are considered to be one of the most potent stimuli for inflammation. They also stimulate monocyte/macrophages for increased production of pro-inflammatory cytokines such as TNF-[alpha], IL-8 and IL-1[beta], which induce many of the hallmark mediators of infilamatory response. Interleukin-8 (IL-8) and tumor necrosis factor-[alpha] (TNF-[alpha]) have been detected in acne lesions and are c onsidered to have a role in pathogenesis of acne vulgaris (Ghezzi and Dinarello, 1998; Durum and Oppenheim, 1989). Inhibition of the mediators, such as production of ROS from PMNL and IL-8 and TNF-[alpha] from human PBMC, could be employed as a criterion for the evaluation of anti-inflammatory activity.

Plants used in folk medicine have been accepted as one of the main sources of drug discovery and development. In India, there is a rich treasury of ethnobotanical knowledge and over the past decades much research has been carried out on this subject. During our field studies, we have noticed the following herbal remedies being used in the treatment of skin diseases and related inflammatory diseases: Rubia cordifolia (Majistha, Rubiaceae) roots, Sphaeranthus indicus Linn. (Gorkha Mundi, Compositae) leaves and branches, Aloe vera (Aloeaceae) leaves, Azadirachta indica (Neem, Meliaceae) leaves and bark, Curcuma longa (Haldi, Zingiberaceae) roots, and Hemidesmus indicus Linn. (Anant mul, Asclepiadaceae) roots.

We undertook the present study to investigate the anti-inflammatory properties of aqueous extracts obtained from the above-mentioned herbs, using an in vitro screening method based on inhibition of the production of ROS from PMNL and pro-inflammatory cytokines (IL-8, TNF-[alpha]), by human peripheral blood mononuclear cells (PBMC).

* Materials and Methods

Materials

Plant materials/herbs were procured from the National Botanical Research Institute, Lucknow, India. RPMI-1640, Histopaque, TNF-[alpha] and IL-8 ELISA kits were obtained from R&D system (Minneapolis, USA). FCS, dithiothreitol (DTT) and antibiotic mix were obtained from Life Technologies (Rockville, USA).

Extraction of plant materials

Each material was first extracted using distilled water ([H.sub.2]O extract) and evaporated to dryness in vacuo. This aqueous fraction was used as such throughout the experiment (Yesilada et al. 1997).

Reactive oxygen species production assay

* Preparation of culture supernatant of Propionibacterium acnes (CS-P): P. acnes was cultured in 10% serum broth with and without herbs (0.1 mg/ml of broth) for 72 h at 37 [degrees]C in anaerobic conditions. The supernatant was harvested, filtered (through a 0.22-[micro]m-pore size filter) and stored at -20 [degrees]C until use.

* Nitroblue terazolium dye (NBT) reduction test: The NBT test was performed according to the method of Park et al. (1968). The test determines ROS activity. Briefly, to 500 [micro]l venous blood of healthy volunteers, 50 [micro]l of stimulants and positive/negative controls (CS-P with and without herbs/zymosan/culture media [CM]) were added and the solution incubated at 20 [degrees]C for 15 min. 100 [micro]l of NBT solution (1 mg/ml of PBS) were added to the above 100 [micro]l primed blood and incubated at 37 [degrees]C for 30 min and then again at 20 [degrees]C for 20 min. Finally, smears were prepared and stained by Leishman stain for differential counting of formazan deposits in PMNL.

Cytokine production assay

* Preparation of heat killed Propionibacterium acnes (P. acnes): The P. acnes (MTCC No. 1951) was procured from IMTECH, Chandigarh, India. It was grown in 10% serum broth for 72 h at 37 [degrees]C in an anaerobic atmosphere ([N.sub.2]:[CO.sub.2]:[H.sub.2] = 80:10:10). The log phase bacterial culture was harvested, washed thrice in phosphate-buffered saline PBS, pH: 7.2) and incubated at 80 [degrees]C for 30 min to heat-kill the bacteria. The heat-killed P. acnes suspension was stored at 4 [degrees]C until use.

* Isolation of peripheral blood mononuclear cells (PBMCs): Peripheral blood mononuclear cells (PBMCs) were separated from the venous blood of healthy volunteers (Boyum, 1967). Blood was diluted 1:2 with phosphate-buffered saline (PBS) (pH ~7.2), layered on Histopaque, washed thrice with PBS and resuspended in complete RPMI-1640 supplemented with 10% FCS. The cells were counted and resuspended at a concentration of 1 x [10.sup.6] cells/ml in RPMI supplemented with 10% FCS. Cell viability was determined using the trypan blue dye exclusion test. Viability of more than 95% was satisfactory.

* Cell culture: A one-ml culture of PBMC (1 x [10.sup.6] cells) was setup in 24-well tissue culture plates (Nunc, Denmark) and stimulated with heat-killed P. acnes ([10.sup.8] organisms/ml) in the presence or absence of different aqueous extract of herbs at a concentration of 50 and 5 [micro]g/ml. Cultures were incubated at 37 [degrees]C for 18 h in a humidified atmosphere containing 5% [CO.sub.2]. Cultures without stimulants were set up as controls. The following day, cultures were transferred to microfuge tubes and centrifuged to collect cell-free supernatant containing secreted cytokines.

* Quantification of cytokines: Cell-free supernatants were analyzed for IL-8 and TNF-[alpha] (R&D systems, Minneapolis, USA) by the use of commercially available sandwich ELISA according to the manufacturer's protocol. The sensitivities of the IL-8 and TNF-[alpha] assays were 32 pg/ml, and 15 pg/ml respectively. The ratio (%) of inhibition of the cytokine release was calculated using the following equation: Inhibition (%) = 100 x (1 - T/C); where T represents the concentration of the cytokine in the culture supernatant with the test compound, and C represents the concentration of the cytokine in the culture supernatant with the solvent (Yesilada et al. 1997).

* Results

Medical utilization of the selected plants in Indian folk medicine is summarized in Table 1. All the herbs are well known in folk medicine and used for the treatment of various ailments, including skin diseases.

Effect of herbs on the production of cytokines by PBMCs.

For interpretation of the results, percentage values are classified under four groups; an inhibition between 70 and 100% is accepted as high, between 40 and 69% as moderate, between 20 and 39% as low. An inhibition of less than 20% is considered to be insignificant.

To prove the inflammatory effects of these aqueous extracts, in Vitro screening test systems in various concentrations were applied. Inhibitory effects of the test sample on JL-8 and TNF-a production are shown in Table 2 as the inhibitory percentages.

As shown in Table 2, the highest inhibition of TNF-[alpha] and IL-8 production in P acnes stimulated cultures was observed with Azadirachta indica and Sphaeranthus indicus. Hemidesmus indicus showed moderate inhibitory activity against IL-8 and TNF-[alpha] production. Rubia cordifolia and Curcuma longa showed a moderate inhibitory activity on production of TNF-[alpha], while these extracts had low activity against IL-8 production. Aquous extract of Aloe vera had an insignificant inhibitory effect on IL-8 and TNF-[alpha].

Effect of herbs on generation of ROS by PMNL

The superoxide radical anion production was approximated by counting all the PMNL containing formazan deposit. When CS-P was used as a stimulant, 69.2[+ or -]`4% (Fig. 1) of PMNL contained the deposit of formazan, while stimulation by culture media showed that only 32 [+ or -]` 5% of cells had deposits. Stimulation in the presence of herbs was tested and the percent increase/decrease in PMNL containing formazan deposit was compared with CS-P as shown in Fig. 1 and Table 3).

On comparing the result using the one-way ANOVA test, Rubia cordifolia (26 [+ or -] 4.1%), Curcuma longa (28.9 [+ or -] 3.1%), Hemidesmus indicus (37.2 [+ or -] 1.8%) and Azadirachta indica (42 [+ or -] 5.4%) caused statistically significant suppression in the production of ROS induced by CS-P. Sphaeranthus indicus (52 [+ or -]3.9%) was observed to have little inhibitory activity against ROS production as compared to the above drugs. Aloe vera (68.2 [+ or -] 5.7%) had an insignificant inhibitory effect on ROS induced by CS-P.

* Discussion

Herbs have been used in clinical medicine for thousands of years. However, it is only in recent times that we have been able to employ scientific methods to prove the efficacy of these herbs and to give us a better understanding of their mechanisms of action. To prove the claimed effects of traditional medicine, the aqueous extracts were tested in two different in vitro experiments, as both ROS and cytokines play an important role in acne pathogenesis.

A potent stimulant for inflammation is reactive oxygen species (ROS) and it is seen that the ability of polymorphonuclear leukocyte (PMNL) to produce ROS varies according to the severity of acne (Akamatsu and Horio, 1998). It is considered that neutrophil chemotactic factors released from P. acnes may accumulate and induce the migration of PMNL at the site of infection, subsequently leading to release of ROS for the lysis of the invading microorganism. In the present study, P. aenes culture supernatant caused a significant increase in ROS production from PMNL as compared to control (CM), in the NBT dye reduction test. This signifies the role of ROS in the pathogenesis of acne rosacea. We also evaluated the effect of herbs on the P. acnes culture supernatant-mediated production of ROS from PMNL and it was found that Rubia cordifolia, Curcuma longa, Hemidesmus indicusi and Azadirachta indica caused significant suppression. The inhibitory effect of these herbs was more or less similar with cytokine and ROS produ ction, except that Sphaeranthus indicus caused significant decrease of cytokines and not ROS. Aloe vera caused no decrease of ROS or cytokines.

It is suggested that tissue macrophages are the main source of these cytokines. P. acnes has been shown to possess macrophage-activating activities (Cummins and Johnson, 1974), suggesting that it may be capable of eliciting the production of pro-inflammatory cytokines (Bomford and Christie, 1975). Vowels et al. (1995) suggested that PG-polysaccharide (PG-PS) complex and lipoteichoic acid are the compounds present in the bacterial cell wall which induce production of pro-inflammatory cytokines.

Thus our findings, together with earlier reports (Ohezzi and Dinarello, 1998; Durum and Oppenheim, 1989), suggest that there is an important role for pro-inflammatory cytokines in disease pathogenesis. In the present study we found that Azadirachta indica and Sphaeranthus indicus caused the maximum suppression of P. acnes-induced TNF-[alpha] and IL-8 production.

The anti-inflammatory activities of Azadirachta indica and Sphaeranthus indicus found in this study are in agreement with the traditional utilization of these plants. Sphaeranthus indica has been shown to possess antibacterial and anti-inflammatory activity, besides having anthelminthic, blood purifier, aphrodisiac and anti-stomachache properties. The aqueous extract obtained from root of Sphaeranthus indicus was found to be moderately active in down-regulating P. acnes-induced TNF-[alpha] and IL-8 production.

Hemidesmus indicus roots are used externally. A warm root decoction is used thrice a day for one month as a blood purifier and also in skin diseases.

Rubia cordifolia and Curcuma longa are both moderately effective againt TNF-[alpha] and show low activity against IL-8. Rubia cordifolia is regarded as astringent and useful in external inflammations like ulcers and skin diseases. The moderate activity of Curcuma longa against TNE-[alpha] and IL-8 is supported by a study of Gupta et al. (1982). They suggested that the anti-inflammatory activity of Curcuma longa might be due its ability to scavenge oxygen radicals, which has been implicated in the inflammation process (Kunchandy and Rao, 1990). Collectively these observations suggest that anti-inflammatory activity of Curcuma longa may be due to its effect on pro-inflammatory cytokine as well as its capacity to scavenge oxygen radicals.

Aloe vera shows no activity against JL-8 and TNF-[alpha] production in P. acnes-stimulated cultures. It is an established anti-inflammatory drug and has been shown to inhibit other inflammatory mediators like prostaglandin biosynthesis and platelet activating factor (PAF)-induced exocytosis (Tonun et al. 1995). It seems likely that its anti-inflammatory action involves neither suppression of pro-inflammatory cytokines nor scavenging of oxygen radicals.

The results presented in this study need to expanded through using different in vitro and in vivo test models to confirm and evaluate the anti-inflammatory activity of the plant materials. The in vitro screening system employed in this study seems to correlate well with traditional anti-inflammatory use of these plant extracts.

[FIGURE 1 OMITTED]
Table 1

Medical utilization of the herbs in Indian folk medicine.

Plant name Part used Medical utilization

Rubia cordifolia Root Anti-inflammatory activity
(Manjistha) (Ext), skin diseases and
 ulcers

Sphaeranthus Leaves Skin diseases, antihelminthic,
indicus Branches aphrodisiac and stomachache
(Gorkha Mundi)

Aloe vera Leaves Burns (minor), mouth ulcers,
(Aloe) diabetes, wound healing
 (topical), Crohn's disease,
 ulcerative colitis

Azadirachra Leaves Skin disease, boils,
indica (Neem) Bark antibacterial activity

Curcuma longa Root Root Antiseptic, stomachache,
(Haldi or blood purifier, vermicide,
Turmeric) carminative and tonic

Hemidesmus Root Skin disease and blood
indicus purifier
(Sarsaparilla or
Anant mul)

Table 2

Inhibitory effect of the aqueous plant extracts on production of IL-8
and TNF-[alpha] by human PBMCs.

Plant name Final con- Inhibitory ratio (%)
 centration
 ([mu]g/ml) IL-8 TNF-[alpha]

Rubia cordifolia 50 32.8 45.5
 5 22.4 24.8

Sphaeranthus indicus 50 71.6 75.3
 5 49.3 51.3

Aloe vera 50 4.8 9.9
 5 0.9 -22.2

Curcumalonga 50 29.8 57.6
 5 18.1 43.5

Azadirachta indica 50 85.9 73.8
 5 58.1 38.9

Hemidesmus indicus 50 49.7 50.4
 5 32.8 31.9

Table 3

Inhibitory effect of herbs on on P. acnes-induced oxidative burst.

Plant name % increase/decrease
 compared with CS-P

Rubia cordifolia 43.2 *
Sphaeranthus indicus 17.2 *
Aloe vera 1.0 **
Curcuma longa 40.3 *
Azadirachta indica 27.2 *
Heinidesmnus indicus 32.0 *

* significant

** non-significant


Acknowledgements

This work was supported in part by a grant from Uttar Pradesh Council of Science and Technology (UPCST). We thank the National Botanical Research Institute, Lucknow, India, for providing plant material.

* References

Akamatsu H, Horio T (1998) The possible Role of reactive oxygen species generated by neutrophils in mediating acne inflammation. Dermatology 196: 82-85

Bomford R, Christie GH (1975) Mechanisms of macrophage activation by Corynebacterium parvum. II. In vivo experiments. Cell Immunol 17: 150-155

Boyum A (1967) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97: 77-89

Cummins CS, Johnson JL (1974) Corynebacterium parvum: a synonym of Propionibacterium acnes? J Gen microbial 80: 433-442

Durum SK, Oppenheim JJ (1989) Macrophage-derived mediators: interleukin 1, tumor necrosis factor, interleukin 6, interferon and related cytokines. In: Fundamental Immunology, 2nd ed (ed Paul WE): 639-661. Raven Press, New York

Ghezzi P, Dinarello CA (1988) Specific inhibition of IL-[gamma] production by IFN-[gamma]. The journal of Immunology 140: 4238-4244

Gupta SS, Chandra D, Mishra N (1982) Anti-inflammatory and antihyaluronidase activity of volatile oil of Curcumin analogs. Indian journal of medical research 75: 574-578

Kunchandy E, Rao MN (1990) Oxygen radical scavenging activity of Curcumin. Internation J Pharmacognosy 58: 237-240

Park BH, Fikrig SM, Smithwick EM (1968) Infection and nitroblue tetrazolium reduction by neutrophils. Lancet 532

Tunon H, Olavsdotter C, Bohlin L (1995) Evaluation of anti-inflammatory activity of some Swedish medicinal plants. Inhibition of prostaglandin biosynthesis and PAF-induced exocytosis. Journal of Ethnopharmacoloty 48: 61-67

Vowels BR, Yang S, Leyden JJ (1995) Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne. Infect Immun 63: 3158-3165

Webster GF, Leyden JJ, Tsai CC, Baebni P, McArthur WP (1980) Polymorphonuclear leukocytes lysosomal release in response to Propionibacterium acnes in vitro and its enhancement by sera from patients with inflammatory acne. J Invest Dermatol 74: 398-401

Yesilada E, Ustun O, Sezik E, Takaishi Y, Ono Y, Honda G (1997) Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1[beta] and tumor necrosis factor [alpha]. Journal of Ethnopharmacology 58: 59-73
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Author:Jain, A.; Basal, E.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Jan 1, 2003
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