Introduction

The unique and diverse flora of Sudan and its prominent traditional medicinal plants could probably be a rich source of bioactive compounds [1]. Information about Sudanese folk medicine was documented during comprehensive ethnobotanical investigations [2]. Acne vulgaris is a common skin disease of humans caused by bacteria inducing non-inflammatory and inflammatory skin lesions [3]. Propionibacterium acnes has been recognized as an obligate anaerobic organism which is usually found as a normal skin commensal. This organism has been implicated over other cutaneous microflora in contributing to the inflammatory response of acne. It acts as an immunostimulator which can produce a variety of enzymes and biologically active molecules involved in the development of inflammatory acnes. These products include lipases, proteases, hyaluronidases, and chemotactic factors [4]. Current treatment for acne is mostly based on antibiotics such as clindamycin and tetracycline derivatives. However, antibiotic resistance for P. acnes is widely spread and is a critical problem worldwide [5]. During this decade, they have been extensively studied for new compounds from natural substances possessing antibacterial activity against acne-inducing bacteria [6].

Reactive oxygen species (ROS) are subsequently generated from the hyper-colonization of P. acnes and from ultraviolet exposure [7]. Therefore, substances that inhibit the growth of skin microorganisms and have anti-oxidant activity are of great interest, since they may cure or prevent various diseases related to the effects of free radicals and may be useful in the treatment of adult pimples.

In the present study, the first anti-acne screening of Sudanese medicinal plants is shown by antibacterial activity against Propionibacterium acnes, lipase inhibition and antioxidant activity.

Materials and methods

Reagents

Dimethyl sulfoxide (DMSO), GAM Broth Modified “Nissui”, glucose, (+)-catechin, yeast extract (Difco, France), nutrient broth (Difco, France), Folin Ciocalteu, bovine serum albumin (BAS) and Tween-80 (MP Biomedicals LLC, France) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Tetracycline hydrochloride was obtained from Sigma Chemical Co. Ltd (China). 2-(N-Morpholino)ethanesulfonic acid (MES) and gallic acid were from Nakalai Tesque, Inc. (Kyoto, Japan). Isopropyl methylphenol (IPMP), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and vanillin were from TCI (Tokyo, Japan). Lipase kits were from Pharma Biomedical (Osaka, Japan).

Plant materials

The plants were collected in Khartoum and Gadarif states of Sudan in March 2011. Voucher specimens are deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum. A list of voucher numbers of the investigated species is shown in Table 1.

Table 1 Anti-acne properties of Sudanese medicinal plant extracts
Full size table

Preparation of plant extracts

Plant materials were shade dried at room temperature and powdered before being extracted with methanol and 50 % (v/v) ethanol in water (ratio of 1 g sample:10 ml solvent) for 12 h three times. The extracts were filtered and the solvent was removed under vacuum using rotary evaporator. The concentrated extracts were then dried under freeze drying. The crude extracts in yields are listed in Table 1.

Test microorganism

Propionibacterium acnes ATCC 6919 was obtained from Biological Resource Center (NBRC), National Institute of Technology and Evolution, Chiba, Japan. The bacterial colonies were maintained in a medium consisting of GAM broth modified “Nissui” 0.5 %, glucose 1.0 %, yeast extract 0.3 %, nutrient broth 0.5 %, and 0.2 % Tween-80.

Evaluation of antibacterial activity

The broth dilution method was used to determine the minimum inhibitory concentration (MIC) of each plant extract. This assay was determined as described by Chomnawang et al. [8]. Briefly, 100 μl of each extract was serially diluted twofold with 10 % DMSO; 95 μl of sterilized medium and inoculum 5 μl were added to each well of a 96-well plate. The inoculum was prepared at the density of 1 × 106 CFU/ml approximately. The broth culture was incubated for 72 h under anaerobic conditions. Extract concentrations at which there was no visually detectable bacterial growth were described as the MIC. Tetracycline hydrochloride and IPMP were used as a positive control. Each experiment was carried out in triplicate.

DPPH radical scavenging assay

This assay was performed as described previously by Batubara et al. [9]. The free radical scavenging activity of the extract was evaluated by decolorization of DPPH at 510 nm.

Preparation of crude lipase from Propionibacterium acnes

Lipases were produced under the same culture conditions. The cell suspension was centrifuged at 900×g for 10 min at 4 °C. The precipitate was diluted in phosphate buffer saline (PBS) at pH 6.98. The bacteria in this solution were destroyed by micro homogenizing system (TOMY Micro Smash MS-100) at 4000 rpm for 30 s and centrifuged at 5000×g for 60 s. The filtrate was collected and placed in a dialysis tube for 6 days. In a freeze drier, the crude enzyme was lyophilized to a powder form [9].

Lipase inhibitory activity assay

Lipase inhibitory activity assay determined by BABLB-DTNB method [10] was carried out using a commercially available kit (Lipase kits). Tetracycline hydrochloride and IPMP were used as the positive controls.

Analysis of phenolics

Determination of total phenolic

The total phenolic assay was performed as described previously by Ainsworth and Gillespie [11]. Data are expressed as milligram of gallic acid equivalents.

Vanillin assay for flavonoids

This assay was performed as described previously by Hagerman [12]. The flavonoids in the plant extracts were expressed as (+)-catechin equivalents.

BSA assay for Tannin

Tannin content of plant extracts was determined by a protein precipitation method using BSA. This method was described by Batubara et al. [13] by analyzing the BSA content of the supernatant liquid. Samples were diluted in 50 % (v/v) ethanol/water to prepare 2 mg/ml of concentration. A 200 μl volume of sample solution was added to 200 μl of BSA solution (10 mg/ml, dissolved with 0.1 M acetate buffer, pH 5). After reaction at room temperature for 1 h, the solution was centrifuged at 13000×g for 2 min. The remaining BSA in the supernatant was determined by HPLC with a reversed phase Develosil300C4-HG-5 column (4.6 i.d. ×150 mm, Nomura Chemical Co, Ltd, Japan) monitored at 280 nm. The solvent system used was as follows: a linear gradient elution for 20 min from 80 to 20 % solvent A (0.01 % TFA in water) in solvent B (90 % (v/v) CH3CN/water containing 0.01 % TFA) at flow rate 1 ml/min. The column temperature was 35 °C.

Results and discussion

In continuation of our research group studies to obtain anti-acne agents from plant origin, we deal in the present work with the screening of 80 extracts belonging to 29 plant species distributed among 20 families. Generally, polar organic solvents are most effective at producing extracts high in phenolic compounds and antioxidant activity [14]. In this study, we used methanol and 50 % ethanol as solvent for plant extraction. The anti-acne activity of some Sudanese medicinal plant extracts were determined using an antibacterial assay, lipase inhibitory activity and a DPPH radical scavenging assay. Table 1 summarizes the scientific name, family, part used and voucher specimen of the medicinal plants. Within these selected Sudanese medicinal plants, Lawsonia inermis L., Combretum hartmannianum, Acacia seyal var fistula and Solanum dubium are used for preventing the dryness and bacterial infection of the skin; other plants are used for anti-inflammation, anti-malaria, anti-diarrhea, antimicrobial infection and other diseases [15].

The antibacterial MIC values of the extracts of 29 medicinal plants used are presented in Table 1. Gibbson [16] suggested that isolated phytochemicals should have MIC <1 mg/ml, so that in this study MIC value 0.13 mg/ml was considered to be an indication of excellent antibacterial activity. Among the extracts used in this study, the species belong to Combretaceae family comparatively showed a good antibacterial activity, specifically all extracts from T. laxiflora showed the best antibacterial activity against P. acne (MIC 0.13 mg/ml), followed by methanol extract of T. brownii (bark) and all of C. hartmannianum (wood) extracts exhibited a medium (MIC 0.5 mg/ml) antibacterial activity. According to Elegami et al. [17], water and methanol extracts of leaves, fruit and stem bark from C. hartmannianum exhibited an activity against gram-positive bacteria which was due to flavonoids, saponins, terpenes and tannins. Antimicrobial investigation of some species of Terminalia and Combretum that belong to Combretaceae indicated that the most effective extracts were the methanol extracts of roots of T. sambesiaca, T. kaiserana, T. sericea, C. fragrans and C. padoides. All these extracts showed a remarkable growth inhibition against gram-positive bacteria [18]. Although the activity of T. arjuna (bark) against acne bacteria is known [19], potency of T. laxiflora (wood) against P. acnes has not yet been investigated.

DPPH is a stable radical that is used as a popular method of screening for free radical scavenging ability, antioxidant activity in particular of plant extracts [20]. This assay was used to evaluate the free radical scavenging activity of our plant extracts. The result in Table 1 showed that ten plant extracts have partially good potent activity with <5 μg/ml of IC50, comparing with positive control of (+)-catechin (IC50 2.39 μg/ml). In addition, methanol extract of Acacia nilotica (pod) demonstrated the lowest IC50 for scavenging activity of the DPPH radical (IC50 1.32 μg/ml) and this could be due to some isolated compounds from this plant, such as galloylated catechins and gallocatechin derivatives [21]. Furthermore, Singh et al. [22] have mentioned that the strong antioxidant activity of A. nilotica (bark and leaves) was due to umbelliferone.

Results of the lipase inhibitory activity shown in Table 1 clarified that the methanol extract of A. nilotica (pod), 50 % ethanol extracts of Abrus precatorius (seed) and T. laxiflora exhibited the strongest inhibitory effect on lipase with more than 70 % inhibition. These values were higher than those of the positive controls such as tetracycline hydrochloride and IPMP at the same concentration. Methanolic extract of T. laxiflora and A. nilotica bark showed the second strongest activity with an inhibition of 69.5 and 66.0 %, respectively. Seven plant extracts expressed moderately strong activity with more than 50 % inhibition. Batubara et al. [9] reported that some extract from Indonesian medicinal plants had better activities than the positive controls (chloramphenicol, tetracycline, and IPMP).

A study done by Falcocchio [23] indicated that (+)-catechin and kaempferol are promising candidates for treatment of acne due to their strong inhibitory activity on P. acnes lipase GehA (glycerol-ester hydrolase A), as well as to their wide anti-acne properties and their low toxicity. Previously, phytochemical studies of A. nilotica resulted in the identification of a variety of phenolic constituents, among which kaempferol was identified [24]. The best extract based on comprehensive activities was T. laxiflora 50 % ethanol extract with better MIC value 0.13 mg/ml, antioxidant activity (IC50 3.45 μg/ml) and lipase inhibitory activity (74.1 %).

The chemical analysis was performed to find the perspective chemical group which was responsible for biological activity. Total phenolic and flavonoid contents of active extracts were determined spectrophotometerically, while total tannin content was determined by the ability of BSA precipitation, which refer to condensed and hydrolysable tannins presence in the extracts. Vanillin HCl method is widely used to estimate the proanthocyanidin in the extracts. The result for total phenolic, flavonoid and tannin contents of selected extracts are presented in Table 2. Almost all selected extracts contained phenolics ranging from 34.9 to 47.9 μg/mg. Total phenolics content previously reported had relationship with antioxidant activity [25]. Many useful properties of flavonoids have been reported including anti-inflammatory activity, enzyme inhibition and antimicrobial activity [26]. The highest amounts of flavonoids (38.87 μg/mg) were detected in methanol extract of T. brownii bark and the lowest amount (0.14 μg/mg) was detected in methanol extract of T. laxiflora wood.

Table 2 Total phenolic, flavonoid and total tannin contents of selected plant extracts as anti-acne activities
Full size table

Tannins exhibit many biological activities. In previous studies, tannins have been evaluated as antibacterial, antiviral, radical scavenging, antitumor activities and inhibitory activities for some enzymes [27, 28]. No tannin content was detected in A. precatorius fruit. Methanolic extracts of A. nilotica (L.) bark, T. brownii bark and wood extracts (methanol, 50 % ethanol) of T. laxiflora have partially high percentage of total tannin (88.01, 82.83, 81.91 and 73.3 %) respectively. Among them, the extracts of T. laxiflora wood have less or no flavonoids; these results indicate that hydrolysable tannins may included in these extracts, while condensed tannin would be included in A. nilotica (L.) and T. brownii barks.

In conclusion, these findings demonstrated that the study of medicinal plants from Sudan confirmed a promising inhibitory effect in anti-acne. Fractionation and bioassay-guided isolation of the anti-acne active pure compounds would be made on the crude extract of T. laxiflora. We also aim in our future studies to perform the chemical characterization (structure elucidation) of some of the anti-acne potent compounds. Toxicity and pharmacological studies are also needed to support the safety of these plants for cosmetic uses.