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Antifungal, anti-inflammatory and cytotoxicity activities of three varieties of labisia pumila benth: from microwave obtained extracts
© Karimi et al.; licensee BioMed Central Ltd. 2013
Received: 10 October 2012
Accepted: 22 January 2013
Published: 24 January 2013
Labisia pumila, locally known as Kacip Fatimah, is a forest-floor plant that has tremendous potential in the herbal industry. It is one of the five herbal plants identified by the government as one of the national key economic areas to be developed for commercial purposes. There are three varieties of L. pumila namely, L. pumila var. pumila, L. pumila var. alata and L. pumila var. lanceolata and each has its own use.
The leaves and roots of the three varieties of L. pumila Benth. were extracted using microwave assisted extraction (MAE). Antifungal activity of all plant extracts were characterized against Fusarium sp., Candida sp. and Mucor using the agar diffusion disc. Anti-inflammatory assays were performed using NO production by macrophage RAW 264.7 cell lines induced by LPS/IFN-g and cytotoxic activity was determined using several cancer cell lines and one normal cell line.
The overall result demonstrated that leaf and root extracts of all three varieties of L. pumila exhibited moderate to appreciable antifungal activity against Fusarium sp., Candida sp. and Mucor compared to streptomycin used as positive control. Leaf and root extracts of all varieties significantly decreased NO release. However, the root extracts showed higher activity compared to the leaf extracts. Cytotoxic activity against MCF-7, MDA-MB-231 and Chang cell lines were observed with all extracts.
These findings suggest the potential use of L. pumila Benth. as a natural medicine and indicated the possible application of this medicinal plant such anti inflammatory activity and cytotoxic agents.
Currently, there is growing interest in the application of plants as a medicinal agent since synthetic drugs have shown several side effects on the human body. Experimental investigations demonstrated that many naturally occurring agents in plant extracts have shown antioxidant, antimicrobial and anticancer potential in a variety of bioassay systems and animal models, having relevance to human disease . Medicinal plants are known to have weak or strong therapeutic abilities and contribute in reducing risk of diseases of various etiologies such as inflammatory and cancer. This is attributed to the large amounts of phytoconstituents such as flavonoids, phenolics and saponins  found in medicinal plants. These bioactive compounds have received considerable attention due to their therapeutic potential for antimicrobial, anti-inflammatory, anticancer and antioxidant activities . Burda and Oleszek  had demonstrated high antiradical activity of some flavonoids like rutin, kaempferol, morin and fustin as strong scavengers with radical scavenging activity of more than 90%. Faried et al. indicated that gallic acid isolated from Phaleria macrocarpa induced cancer cell death in various cancer cells such as breast cancer (MCF-7), gastric cancer (MKN-28) and colon cancer (HT-29, colon 201 and colon 26). Labisia pumila Benth. (Myrsinaceae) popularly known as Kacip Fatimah, is a herbaceous plant with creeping stems used by Malay women to induce and facilitate childbirth as well as a post-partum medicine. Recently, it was found that the bioactive compounds of L. pumila consisted of resorcinols, flavonoids and phenolic acids. These compounds have been identified as natural bioactive compounds with high biological activity . Stone  had categorized three varieties of this herb in Malaysia namely L. pumila (Blume) var. alata, L. pumila (Blume) var. pumila and L. pumila var. lanceolata. Each of these varieties has different applications. Uses of this herb include treatment for dysentery, dysmenorrheal, flatulence, and gonorrhea . The results of our previous experiments revealed that all three varieties of L. pumila Benth. consisted of bioactive compounds including phenolics, flavonoids and saponins which might be responsible for the enhanced antioxidant activity observed in this plant . This present study was carried out to evaluate antifungal, anti-inflammatory and cytotoxic activity of leaf and root extracts derived by microwave extraction from the three varieties of Labisia pumila Benth.
Seedlings of the three Labisia pumila varieties were collected from places of origin at Hulu Langat (Selangor), Sungkai (Perak) and Kota Tinggi (Johore), respectively, and raised under glasshouse conditions for 18 months before use in the study. Voucher specimens were identified by the Herbarium unit, Institute of Bioscience, University Putra Malaysia (alata (Stone 6030 (KLU)), pumila ((Stone 7233 (KLU)), and lanceolata ((Stone 8385 (KLU)). Healthy and uniform seedlings in terms of leaf numbers were selected from the three varieties. The leaves and roots of selected seedlings were cleaned, separated, freeze dried and stored until further analysis.
Microwave assisted extraction (MAE)
MAE was performed using a microwave apparatus with a closed vessel system under pressure (ETHOS® T Microwave digestion/extraction system, Milestone Co., Italy) based on the method described by Xiao et al. with some modifications. One gram of leaf and root samples of the three varieties of L. pumila was weighed in a clean aluminum container, and then transferred into the vessel of the Ethos E Microwave Extraction System and extracted with 30 ml of methanol for 2 min (p = 750 w). The extraction temperature was 60°C. After extraction, the vessels were allowed to cool to room temperature, and the extracts were filtered and stored in a refrigerator.
Antifungal activity assay
The antifungal assay was carried out by the agar well diffusion method  with slight modifications. Briefly, a suspension of the tested fungi was prepared (105 spore/mL) and dispensed (100 μL) uniformly on the surface of the agar plate. Small wells were cut in the agar plate using a cork borer (6 mm). A fixed volume of the different extracts or amphotericin B (PAA Lab., Germany) as positive control (at 450 μg/well), were loaded into the wells. The plates were incubated at 29°C for 72 h. The diameter of the inhibition zone around each well was then recorded in four different directions.
The murine monocytic macrophage cell line RAW 264.7 was cultured in Dulbecco’s Modified Eagle Media (DMEM) (2mM L-glutamine, 45 g/L glucose, 1 mM sodium pyruvate, 50 U/ml penicillin; 50 μg/ml streptomycin) with 10% foetal bovine serum (FBS). The cells were cultured at 37°C with 5% CO2 and were split twice a week. Approximately 1 × 106 cells/ml of RAW 264.7 cells were seeded in 96-well tissue culture plates and incubated for 24 h at 37°C with 5% CO2. The cells were then incubated in prepared DMEM medium containing 100 μl of test extract in DMSO and serially diluted, to give a final concentration of 100 μg/ml in 0.1% DMSO. Cells were then stimulated with 200 μ/ml of IFN-γ and 10 μg/ml LPS for 17 h. The presence of nitrite in the cell culture media was determined using Griess reagent and absorbance was read at 550 nm using the microplate reader (Spectra Max Plus 384, Molecular Devices Inc., USA). Nitrite concentration in the supernatants was determined by comparison with a sodium nitrite standard curve. The cell viability was detected by MTT cytotoxicity assay. L-NAME was used as iNOS inhibitor (control) at a concentration 250 μM .
Anti cancer activity assay
Human cancer cell lines (MCF-7; MDA-MB-231) and Human hepatocytes (Chang liver cells) cell lines obtained from the American Type Culture Collection (ATCC) were used in this study. Cells were grown at 37°C in humidified 5% CO2 and 95% air atmosphere in Dulbecco’s Modified Eagle Media (DMEM) (2mM L-glutamine, 45 g/l glucose, 1 mM sodium pyruvate, 2g/l sodium bicarbonate and 10% fetal bovine serum). Monolayers of cells (5 × 103/100 μl) were grown in 96-well microlitre plates and exposed to two fold serial dilutions of the extracts from 200 μg to 3.1 μg/100 μl. After 3 days incubation at 37°C, the cytotoxicity of extracts was determined using the MTT assay according to Ahmad et al.. Tamoxifen, an anticancer drug, was used as the positive control in the present study.
Data were analyzed using the analysis of variance procedure in the Statistical Analysis System (SAS) Version 9 (SAS Institute, Cary, NC). Significant differences between means from triplicate analyses (p < 0.05) were determined by Duncan’s Multiple Range Test. GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA) was also used in data analyses.
Results and discussion
Antifungal activity of the three varieties of Labisia pumila Benth
Inhibition zones of leaf and root extracts of three varieties of Labisia pumila against pathogenic fungi at a concentration of 450 μg/well
Inhibition Zone (diameter in cm)
Anti-inflammatory activity of the three varieties of Labisia pumila Benth
Effect of leaf and root extracts of three varieties of L. pumila Benth. on NO production and cell viability of RAW 264.7 cells
Level of Activity
NO inhibition (%)a
Cell viability (%)a
45.66 ± 1.29
93.64 ± 11.59
46.64 ± 2.10
72.75 ± 17.06
55.17 ± 1.96
98.12 ± 3.71
66.45 ± 1.84
44.57 ± 6.06
75.68 ± 1.70
71.32 ± 4.26
64.81 ± 2.22
60.26 ± 4.56
78.43 ± 0.97
97.67 ± 4.40
Anticancer activity of the three varieties of Labisia pumila Benth
IC 50 values of leaf and root extracts of L. pumila Benth on Chang liver cells and MCF-7 and MDA-MB-231 cell lines
IC50 Value (μg/ml)
Chang liver cell
48.19 ± 2.59
55.80 ± 1.83
53.96 ± 3.41
62.26 ± 2.92
59.88 ± 2.63
66.39 ± 2.74
192.16 ± 1.48
81.92 ± 2.28
98.93 ± 1.93
160.90 ± 2.15
86.14 ± 2.11
110.30 ± 1.52
176.68 ± 0.97
92.94 ± 2.54
116.87 ± 2.41
34.97 ± 1.55
36.54 ± 0.79
35.46 ± 0.46
Medicinal plants are now increasingly getting more attention than ever before. The medicinal value of these plants lies in the bioactive phytochemical constituents such as phenolics, flavonoids and saponins that result in various biological activities . The results of this study showed the potential of Labisia pumila plants and in particular the leaves for use in the development of anti fungal, anti-inflammatory and anti cancer drugs using microwave extraction. These activities might be due to the presence of various phenolic and flavonoid compounds in the leaves compare to root in all three varieties of L. pumila Benth [9, 13]. It should be noted that the results obtained were based on in vitro screening tests. Therefore isolation of bioactive compounds responsible for the observed activities is necessary and in vivo studies are needed for further confirmation.
The authors would like to thank the Ministry of Higher Education Malaysia, and the Research Management Centre, Universiti Putra Malaysia (UPM) for sponsoring this work. Authors also wish to acknowledge the support given by the Faculty of Agriculture, and the Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia in terms of laboratory facilities.
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