International Journal of Drug Development and Research

Keywords

Clinacanthus nutans, Phytochemical Screening, Total Phenolic Content, Total Flavonoid Content, Antibacterial Activity, Microbroth Dilution

Introduction

Clinacanthus nutans (Burm.) Lindau (Family: Acanthaceae) is a shrub and found in deciduous forests in Malaysia, Indonesia, Thailand, Vietnam and parts of China. It has been used traditionally to treat envenomation, dysentery, burns, scalds, fever, herpes skin infections and diabetes [1,2]. Recent studies have shown that C. nutans has significant antiviral activities against herpes simplex virus type-2, varicella-zoster virus, antioxidant activity and anti-inflammatory activity [3-6]. The chemical constituents of C. nutans are flavonoids, stigmasterol, β-sitosterol, lupeol, betulin, C-glycosyl flavones, vitexin, isovitexin, shaftoside, isomollupentin, 7-O-β-glucopyranoside, orientin, isoorientin, cerebrosides, monoacylmonogalactosylglycerol and sulfurcontaining glucosides [7-9].

There are various skin diseases which are caused by bacterial colonisation particularly by the normal microflora of the skin. Among these, acne is one of the most common disorders of the skin [10]. Antibiotics such as erythromycin and clindamycin are topically used against acne. Tetracyclines are used as oral antibiotics against acne. Propionibacterium acnes is the bacterium which causes inflammation of sebaceous glands and cause acne [11-13]. The emergence of antibioticresistant P. acnes bacteria represents a growing problem worldwide [14]. Also, Staphylococcus epidermidis causes biofilms to grow on plastic devices placed within the body [15,16]. S. epidermidis shows resistance to various antibiotics [17]. Similarly, infections caused by bacteria such as S. aureus, B. cereus and E. coli are also difficult to treat with current antibiotics [18]. Hence, development of newer antibacterial agents is required and it is the need of the time. Since, C. nutans has been used in Thai traditional medicine against various skin infections [1], we hypothesized that C. nutans may possess antibacterial activity against skin pathogens such as Propionibacterium acnes and Staphylococcus epidermidis. Therefore, our study evaluated the effectiveness of C. nutans against these skin pathogens and other selected clinically important pathogens.

Materials and Methods

Preparation of Plant Extract

Clinacanthus nutans was obtained from Yik Poh Ling Herbal Farm, Seremban, Malaysia. Authentication of the plant was done by Negeri Sembilan Forestry Department, Seremban, Negeri Sembilan, Malaysia. The leaves were separated, dried and powdered. The powder (255 g) was macerated with 1 litre methanol in a conical flask under room temperature for three days with frequent stirring using a magnetic stirrer. After three days, the mixture was filtered and fresh methanol was added to the residue. This process was repeated several times until a clear colourless solution was obtained. The filtrates were combined and evaporated under reduced pressure at 40°C using a rotary evaporator (BUCHI Rotavapor R-200, Switzerland). After evaporating the methanol, the dried extract was lyophilized with a freeze dryer (CHRIST Alpha 1-4 LD plus Freeze Dryer, Germany) at -50 °C to remove all traces of moisture.

Preliminary Phytochemical Screening

The crude methanolic extract of C. nutans was tested for the presence of phytochemicals by using specific reagents as described previously [19].

Estimation of Total Phenolic Content

The dried methanolic extract of C. nutans was dissolved in methanol and made to the concentration of 10 mg/ml. Six concentrations of gallic acid standard solution ranging from 0.1 to 0.6 mg/ml were prepared in methanol. The total phenolic content in extracts was determined by using Folin-Ciocalteu reagent (Merck, Germany) and external calibration with gallic acid (GA) as per the previously reported method [20]. Briefly, 0.2 ml of extract solution or 0.2 ml of gallic acid solution in a test tube and 0.2 ml of Folin-Ciocalteu reagent were added and the contents mixed thoroughly. After 4 min, 1 ml of 15% Na2CO3 was added, and then the mixture was allowed to stand for 2 h at room temperature. The absorbance was measured at 760 nm by using LAMBDA 25 UV-vis spectrophotometer (Perkin- Elmer, USA). The content of total phenolics was calculated by using gallic acid (GA) calibration curve. The results were expressed as mg gallic acid equivalents / g extract.

Estimation of Total Flavonoid Content

The dried methanolic extract of C. nutans was dissolved in methanol and made to the concentration of 10 mg/ml. Six concentrations of quercetin (Sigma-Aldrich, USA) standard solution ranging from 0.2 to 0.8 mg/ml were prepared in methanol. Determination of total flavonoid content was done by following previously reported method [21] by mixing 0.1 ml of 10 mg/ml extract or 0.1 ml of quercetin solution with 0.3 ml of deionised water followed by 0.3 ml of 5% sodium nitrite, NaNO2, (Acros Organics, USA). After 5 min, 0.3 ml of 10% aluminium chloride was added. At sixth min, 2 ml of 1M NaOH (Merck, Germany) was added and the final volume was made up with deionised water. Absorbance of each sample was measured at 510 nm against the prepared reagent blank using LAMBDA 25 UV-vis spectrophotometer (Perkin Elmer, USA). All tests were performed three times and averaged. Total flavonoid content of C. nutans methanolic extract was expressed as milligrams of quercetin equivalents per 1 g of plant extract (mg quercetin equivalents / g extract).

Microbroth Dilution Test

The antibacterial effect was tested by microdilution assay based on the method published by Zgoda and Porter with modifications [22]. The strains of Propionibacterium acnes (ATCC 11827), Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Bacillus cereus (ATCC 10876) and Escherichia coli (ATCC 25922) were obtained from All eights (M) Sdn Bhd. Based on CLSI guideline, microorganisms were suspended in saline and the turbidity is adjusted to 0.5 McFarland standard which corresponds to 1 × 108 CFU/ml [23,24]. The dried crude extract of C. nutans was reconstituted with 10 %v/v dimethyl sulfoxide (DMSO) in sterile water and tween 80 up to 2% to give a stock solution of 100 mg/ml. From this stock solution, dilutions of 10 and 1 mg/ml were prepared. From these solutions, concentrations ranging from 0.1 to 12.5 mg/ml were prepared in the wells of microplate by diluting them with Mueller-Hinton broth based on CLSI guideline [25]. The antibiotic positive controls (erythromycin and chloramphenicol) were prepared by dissolving the antibiotic powder in solvents recommended by CLSI [24]. Erythromycin and chloramphenicol powder were separately dissolved in 95% ethanol and subsequently diluted with Mueller-Hinton broth to make the dilutions ranging from 0.125 to 256 μg/ml and added to the wells of the microplate. The negative control wells are filled with only 100 μl of broth. Growth control wells are filled with 100 μl of broth and 100 μl of inoculum. Then, 100 μl of inoculum was added to the wells which contained positive controls and extract. The microplates were covered and incubated at 37 °C for 24 hr [26]. The microplates were then read with an ELISA plate reader (BioTek ELx800 absorbance microplate reader, United States) at 620 nm. The concentration at which sharp decrease of absorbance or zero absorbance occurs was considered as minimum inhibitory concentration (MIC) [27,28].

Results

The yield of methanolic extract of C. nutans leaves was found to be 15.29 %w/w with respect to the dry weight of leaf powder. The results of phytochemical screening are summarized in Table 1. Total phenolic content was found to be 1.77±0.008 mg gallic acid equivalent/g. Total flavonoid content was found to be 0.04±0.001 mg quercetin equivalent/g.

	Crude methanolic extract of
	C. nutans leaf
Alkaloids - Mayer’s reagent - Modified Dragendorff’s reagent	? ?
Saponins - Foam test	+
Phenolic compounds - FeCl3 test	+
Tannins - Gelatin test	?
Flavonoids - Alkaline reagent test - Lead acetate test	+ +
Diterpenes - Copper acetate test	+
Phytosterols -Salkowski’s test -Liberman Burchard’s test	+ +

? indicates absence; + indicates presence

Table 1: Phytochemical screening data of crude methanolic extract of Clinacanthus nutans leaf

The MIC’s of erythromycin, chloramphenicol and methanolic extract of C. nutans were recorded and presented in Table 2.

Bacteria	Erythromy cin (µg/ml)	Chlorampheni col (µg/ml)	Methanol ic extract of C. nutans leaf (mg/ml)
Propionibacterium acnes	2.000	n.t.	> 12.50
Staphylococcus aureus	0.250	n.t.	12.50
Staphylococcus epidermidis	0.250	n.t.	> 12.50
Bacillus cereus	0.125	n.t.	> 12.50
Escherichia coli	n.t.	2.000	12.50

n.t. – not tested

Table 2: MIC’s of erythromycin, chloramphenicol and methanolic extract of C. nutans leaf against selected bacteria

Discussion

MIC of erythromycin against P. acnes was found to be 2 μg/ml which is higher than the breakpoints (0.5 μg/ml) set by EUCAST [29]. Hence, P. acnes used in our study can be considered as nonsusceptible to erythromycin. In our study, MIC of erythromycin against S. aureus was 0.25 μg/ml and MIC of erythromycin against S. epidermidis was 0.25 μg/ml. These results are in agreement with the clinical breakpoints (1 μg/ml) set by EUCAST and CLSI [25,29]. MIC of chloramphenicol against E. coli was found to be 2 μg/ml which is consistent with the clinical breakpoints set by CLSI [25]. MIC of erythromycin against B. cereus was found to be 0.125 μg/ml, which is within the previously reported range of 0.032-3 μg/ml, as reported by Turnbull et al [30]. DMSO was used to reconstitute the C. nutans extract. In our experiment (data not shown), DMSO showed inhibitory action at concentrations of ≥ 5 %v/v. However, the final concentration of DMSO in all the microdilution wells was less than 0.125 % v/v. Hence, the antibacterial effect observed in this study can be attributed to the plant extract.

The C. nutans extract did not exhibit any significant inhibitory activity against P. acnes at the concentrations employed (MIC > 12.5 mg/ml). In a previous study by Chomnawang et al, C. nutans exhibited MIC and MBC of >5 mg/ml against the strain of P. acnes (ATCC 6919) used in their study [31] . However, this study did not include information on the part of the plant used and solvent used for extraction. Hence, a direct comparison cannot be made. In this study C. nutans leaf extract exhibited MIC on S. aureus at 12.5 mg/ml. The results appear similar to a study conducted by Hussain et al on antibacterial activity of methanol extracts of various plants with agar dilution and agar well diffusion methods. In their study, the antibacterial activity of various plant extracts on S aureus was observed at concentrations of 6.25 mg/ml and above [32].

Chomnawang et al has also studied the antibacterial effects of ethanolic extracts of various Thai medicinal plants, one of them being C. nutans, against S. aureus and methicillin resistant S. aureus (MRSA) [33]. Their study reported the MIC of 5 mg/ml and above for S. aureus and MRSA respectively. Although the ATCC strain of S. aureus used in their study differed from our study, the MIC of 12.5 mg/ml observed in our study is similar to their findings.

In our study, the crude extract of C. nutans exhibited MIC’s of 12.5 mg/ml and above for all the organisms studied. Amongst the five organisms tested in our study, S. aureus and E. coli showed higher susceptibility to C. nutans extract. The higher MIC value may be attributed to the reason that the extract is a crude extract. Further fractionation of extract and isolation of compounds may give better MIC values.

In our study, preliminary phytochemical screening indicated the presence of saponins, phenolic compounds, flavonoids, diterpenes and phytosterols. Total amount of phenolics and flavonoids were also quantitated. These results are in agreement with the compounds isolated from this plant [7-9]. Lupeol and β-sitosterol were reported to exhibit inhibitory action against S. aureus [34]. Also, few studies showed that lupeol possessed inhibitory activity against E. coli [34-36]. Similarly, flavonoids and terpenoids were reported to possess antibacterial property [34,37,38]. All these compounds were reported to be present in C. nutans [7-9]. Hence, the observed antibacterial activity may be attributed to the presence of these compounds. However, this cannot be concluded from this study. Further studies are required to identify the compounds responsible for antibacterial activity.

Conclusion

The study explored the potential antibacterial effect of C. nutans against selected skin pathogens and other organisms. Our study found out that methanolic extract of C. nutans leaf possessed antibacterial effect against S. aureus, E. coli, P. acnes, S. epidermidis and B. cereus with MIC’s ≥ 12.5 mg/ml.

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References

1. Roosita K, Kusharto CM, Sekiyama M, Fachrurozi  Y, Ohtsuka R. Medicinal plants used by the villagers of a Sundanese community in West Java, Indonesia. J Ethnopharmacol. 2008; 115: 72-81.
2. Cheeptham N, Towers GHN. Light-mediated activities of some Thai medicinal plant teas. Fitoterapia. 2002; 73: 651-662.
3. Wanikiat P, Panthong A, Sujayanon P, Yoosook C, Rossi AG, Reutrakul V. The anti-inflammatory effects and the inhibition of neutrophil responsiveness by Barleria lupulina and Clinacanthus nutans extracts. J Ethnopharmacol. 2008; 116: 234-244.
4. Jayavasu C, Dechatiwongse T, Balachandra K. Virucidal activity of Clinacanthus nutans Lindau extracts against herpes simplex virus type-2: In vitro study. Bull Dept Med Sci. 1992; 34: 153-158.
5. Thawaranantha D, Balachandra K, Jongtrakulsiri S, Chavalittumrong P, Bhumiswasdi J, Jayavasu C. In vitro antiviral activity of Clinacanthus nutans on varicella-zoster virus. Siriraj Hosp Gaz. 1992; 44: 285-291.
6. Pannangpetch P, Laupattarakasem P, Kukongviriyapan V, Kukongviriyapan U, Kongyingyoes B, Aromdee C. Antioxidant activity and protective effect against oxidative hemolysis of Clinacanthus nutans (Burm.f) Lindau. Songklanakarin J Sci Technol. 2007; 29: 1-9.
7. Dampawan P, Huntrakul C, Reutrakul V. Constituents of Clinacanthus nutans and the crystal structure of LUP-20(29)-ene-3one. J Sci Soc Thailand. 1977; 3: 14-26.
8. Teshima K-I, Kaneko T, Ohtani K, Kasai R, Lhieochaiphant S, Picheansoonthon C, et al. Sulfur-containing glucosides from Clinacanthus nutans. Phytochemistry. 1998; 48: 831-835.
9. Tuntiwachwuttikul P, Pootaeng-on Y, Phansa P, Taylor W. Cerebrosides and a Monoacylmonogalactosylglycerol from Clinacanthus nutans. Chem Pharm Bull. 2004; 52: 27-32.
10. Tortora GJ, Funke BR, Case CL. Microbiology: An introduction . 9th ed. San Francisco: Pearson Education Inc, 2007, pp.
11. James J L. The evolving role of Propionibacterium acnes in acne. Semin Cutan Med Surg. 2001; 20: 139-143.
12. Leyden JJ. Current issues in antimicrobial therapy for the treatment of acne. J Eur Acad Dermatol Venereol. 2001; 15: 51-55.
13. Toyoda M, Morohashi M. Pathogenesis of acne. Med Electron Microsc. 2001; 34: 29-40.
14. Tzellos T, Zampeli V, Makrantonaki E, Zouboulis CC. Treating acne with antibiotic-resistant bacterial colonization. Expert Opin Pharmacother. 2011; 12: 1233-1247.
15. O'Gara JP, Humphreys H. Staphylococcus epidermidis biofilms: importance and implications. J Med Microbiol. 2001; 50: 582-587.
16. Otto M. Staphylococcus epidermidis--the 'accidental' pathogen. Nat Rev Microbiol. 2009; 7: 555-567.
17. Romling U, Balsalobre C. Biofilm infections, their resilience to therapy and innovative treatment strategies. J Intern Med. 2012; 272: 541-561.
18. Furuya EY, Lowy FD. Antimicrobial-resistant bacteria in the community setting. Nat Rev Microbiol. 2006; 4: 36-45.
19. Tiwari P, Kumar, B., Kaur, M., Kaur, G., Kaur, H. Phytochemical screening and Extraction: A Review. Internationale Pharmaceutica Sciencia. 2011; 1: 98-106.
20. Akowuah GA, Mariam A, Chin JH. The effect of extraction temperature on total phenols and antioxidant activity of Gynura procumbens leaf. Phcog Mag. 2009; 5: 81-85.
21. Hazra B, Biswas S, Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complement Altern Med. 2008; 8: 63.
22. Zgoda JR, Porter JR. A Convenient Microdilution Method for Screening Natural Products Against Bacteria and Fungi. Pharm Biol. 2001; 39: 221-225.
23. Şahin F, Karaman İ, Güllüce M, Öğütçü H, Şengül M, Adıgüzel A, et al. Evaluation of antimicrobial activities of Satureja hortensis L. J Ethnopharmacol. 2003; 87: 61-65.
24. Stohs SJ, Ray SD. A review and evaluation of the efficacy and safety of Cissus quadrangularis extracts. Phytother Res. 2012; In Press: DOI: 10.1002/ptr.4846.
25. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty Second Informational Supplement. M100-S22. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute, 2012, pp.
26. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. M07-A9. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute, 2012, pp.
27. Salie F, Eagles PFK, Leng HMJ. Preliminary antimicrobial screening of four South African Asteraceae species. J Ethnopharmacol. 1996; 52: 27-33.
28. Devienne KF, Raddi MSG. Screening for antimicrobial activity of natural products using a microplate photometer. Braz J Microbiol. 2002; 33: 166-168.
29. EUCAST. European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 2.0.: European Society of Clinical Microbiology and Infectious Diseases, 2012, pp.
30. Turnbull PC, Sirianni NM, LeBron CI, Samaan MN, Sutton FN, Reyes AE, et al. MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest. J Clin Microbiol. 2004; 42: 3626-3634.
31. Chomnawang MT, Surassmo S, Nukoolkarn VS, Gritsanapan W. Antimicrobial effects of Thai medicinal plants against acne-inducing  bacteria. J Ethnopharmacol. 2005; 101: 330-333.
32. Hussain T, Arshad M, Khan S, Sattar H, Qureshi MS. In vitro screening of methanol plant extracts for their antibacterial activity. Pak J Bot. 2011; 43: 531-538.
33. Chomnawang MT, Surassmo S, Wongsariya K, Bunyapraphatsara N. Antibacterial Activity of Thai Medicinal Plants against Methicillin-resistant Staphylococcus aureus. Fitoterapia. 2009; 80: 102-104.
34. Singh B, Sahu PM, Sharma MK. Anti-inflammatory and antimicrobial activities of triterpenoids from Strobilanthes callosus Nees. Phytomedicine. 2002; 9: 355-359.
35. Kuete V, Kamga J, Sandjo L, Ngameni B,  Poumale H, Ambassa P, et al. Antimicrobial activities of the methanol extract, fractions and compounds from Ficus polita Vahl. (Moraceae). BMC Complement Altern Med. 2011; 11: 6.
36. Kuete V, Wansi JD, Mbaveng AT, Kana Sop MM, Tadjong AT, Beng VP, et al. Antimicrobial activity of the methanolic extract and compounds from Teclea afzelii (Rutaceae). S Afr J Bot. 2008; 74: 572-576.
37. Dorman HJD, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol. 2000; 88: 308-316.
38. Cushnie T, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005; 26: 343- 356.