A-P-053

Biological activities of stem extracts from Luvunga scandens

Prangchanok Sirinuta, Awanwee Petkongkeawb, Panumart Thongyoo*a

a
Department of Chemistry, bDepartment of Food Science, Faculty of Science and Technology,
Rangsit Campus Thammasat University, Pathumthani, Thailand 12120

*tpanumas@tu.ac.th phone +66-59410465 fax +66-25644483

ABSTRACT

Natural products have long been known as one of the most excellent sources of lead drug candidate in drug
discovery. Nowadays, a large number of Thai medicinal plants have been utilized for the treatment of a variety of
diseases. Luvunga scandens is commonly known as one of Thai folk medicinal plants applied for the treatment of
small pox, kidney diseases, wasting disease and urinal disorders. Stem bark of Luvunga scandens was extracted in
dichloromethane, ethyl acetate and methanol to evaluate anti-oxidative, anti-inflammatory and anti-bacterial
activities. The methanolic extract was found to reduce the level of cytokine (Tumor Necrosis Factor type-alpha
(TNF-Į) and interleukin-10 (IL-10) of monocyte), both of which are known as mediators for inflammation
responses. Interestingly, all crude extracts were also found to have very promising anti-bacterial activities against
Staphylococus aureus, Bacillus cereus, Enterococus faecalis, Escherichia coli and Methicillin-resistant
Staphylococcus aureus (MRSA) with MIC ranging from 3.125 to 100 mg/ml. Additionally, dichloromethane,
methanolic and ethyl acetate extracts were found to be moderately active as anti-oxidants in the DPPH radical
scavenging assay with IC50 = 94, 445 and 480 ȝg/ml, respectively.

Keywords: Luvungascandens, TNF-Į, Anti-oxidant, Antibacterial activity

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 1. INTRODUCTION
Inflammatory response is an important element in the pathogenesis of chronic inflammation-related disorders,
involving the sequential activation of various signaling pathways, including cyclooxygenase, nitric oxide synthase,
some proteases (such as tryptase) and cytokines [tumor necrosis factor-D (TNF-D) and interleukins (IL-6 and IL-
1E)]. The inappropriate production of pro-inflammatory mediators; such as interleukins (IL-6 and IL-1E) and tumor
necrosis factor-D (TNF-D) is implicated in several inflammation diseases; such as rheumatoid arthritis,
inflammatory bowel disease, osteoarthritis, psoriasis, endotoxemia and etc. Therefore, the inhibition of the over-
production of pro-inflammatory mediators IL-6, IL-1E and TNF-D should be an effective approach for treating the
diseases. Luvunga scandensis commonly known as South East Asian traditional herb, belonging to Rutaceae family,
and distributed widely in Eastern Bengal, Assam, Khasia hills, Burma and Thailand. The family Rutaceae has been
known to produce many compounds, ranging from triterpene, oxiranecarbamide to sesquiterpene, some of which
displayed anti-insecticidal activity, anti-tumor activity, antipyretic activity using for the treatment of gonorrhea. In
Thailand, L. scandenshas been used widely as folk medicine for the treatment of many diseases, such as abscess,
wasting disease, adrenocortical insufficiency, neurogenic bladder[1]. Presently, the roots and berries of L. scandens
have been prescribed for the treatment of snake-bite or scorpion sting (http://www.mpbd.info/plants). To this report
we focused on the evaluation ofthe inhibitory effect of the extract of L. scandens on lipopolysaccharide (LPS)
induced tumor necrosis factor-alpha (TNF-Į) secretion from isolated human peripheral blood mononuclear cells, and
anti-oxidative activity was also investigated via DPPH scavenging radical assay as well as an anti-bacterial activity
against a wide range of bacterial strains such as S. aureus, B. cereus, E. faecalis, Methicillin-resistant S. aureus
(MRSA) and E. coli.

2. MATERIALS AND METHODS

General experimental procedure

The NMR spectra were recorded in CDCl3 using BRUKER-NMR 400MHz spectrometer at 400 MHz for 1H
NMR and at 100 MHz for 13C NMR using TMS as internal standard, and chemical shifts are expressed in į values.
Analytical and preparative TLC was carried out on pre-coated silica gel 60F254 and RP-18F254 plates (Merck, 0.25
or 0.50 mm thickness).
Plant material
The stems and roots of L. scandens used in this experiment were collected at Hoey Sai National Park,
Prachuabkirikhun Province, Thailand, in May 2010. A voucher specimen (LS 150655) was deposited at Department
of Chemistry, Thammasat University, Thailand.
Plant preparation
The stems and roots of L. scandens (1 kg) were sliced into small pieces, and consecutively extracted with
dichloromethane (1000 ml), ethyl acetate (1000 ml), and methanol (1000 ml) for 2 weeks each. The decanted crude
CH2Cl2 extract was filtered by Whatman filter paper 1 and subsequently concentrated in vacuo to afford the crude
dichloromethane extract (1.920 g), ethyl acetate extract (2.578 g) and methanolic extract (4.695 g), respectively [2].
Anti-oxidative property
The antioxidant activity of plant extracts was assessed on the basis of the scavenging activity of the stable
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay[3, 4]. The stock solutions (1000 µg/mL) of crude extract
were prepared, and subsequently diluted to final concentrations of 500, 250, 125, 50, 25, 10 and 5 µg/mL in
methanol, respectively. To this solutions were added by 0.3 mM DPPH (1 mL) dissolved in methanol and kept in the
dark for 30 minutes. Absorbance was measured at 520nm. Methanol (1 mL) was used as blank and DPPH solution
(0.3 mM, 1 mL) with methanol (2.5 mL) was used as control. Ascorbic acid was used as a standard control. The
absorbance values were record and the percent of inhibition was calculated.
Anti-bacterial activity
The antibacterial activity of crude extracts was determined by resazur in microplate assay (REMA) by
modified method [5]. 50 µL of 0.85% normal saline was added into column 2-11 of sterile 96 wells plate. Then, 100
µL of crude extracts (100 mg/mL) was added into the first column serial dilution was performed and was trashed 50
µL of solution in column 11. 3.3x strength isosensitised nutrient broth (30 µL) was added to all wells to confirm the
final volume. 10 µL of bacteria (5 × 106cfu/mL) was added to each well. The plate was incubated at 37°C for 24 h.
After 24 h 10 µL of 0.1% resazurin was added to each well and incubated at 37°C for 3 h. After 3 h, the color of
resazurin wasn’t changed from blue to pink were recorded as positive. On the other hand, the color of resazurin was

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changed from blue to pink were recorded as negative. Cephalexin (1.5 mg/mL) was used as positive control and
0.85% normal saline was used as negative control.MBC values were determined by streak on nutrient agar plate.
Isolation of Human PBMC and Culture
PBMC (Polymorphonuclear cells) from healthy donors were isolated from EDTA blood by Ficoll-Hypaque
gradient centrifugation. Peripheral blood from the donors was diluted with sterile phosphate buffer saline and
overlaid on the Ficoll-Hypaque solution, and centrifugation was performed at 350 × g for 10 min at room
temperature. The recovered PBMC were rested in RPMI-1640 and incubated at 37°C 95% O2 + 5% CO2 for 30 min
before performing the experiments. Cell viability was determined by a Trypan blue dye exclusion assay. The
percentages of cell viability were calculated by the ratio of Trypan blue excluding cells to total cell number
LPS Stimulation and Incubation of the Luvunga scandens Extracts with PBMC
After the PBMC isolation and pre-incubation period, 200 l of 1×105 cells/ml PBMC were cultured in a 96-
well polypropylene plate in serum-free RPMI-1640 medium with LPS at a final concentration of 10 ng/ml and
various concentrations of the methanol extract of Luvunga scandens (0.0-3.0 mg/ml, final concentration) in DMSO.
In the control wells, cells were incubated with LPS and 0.01% DMSO vehicle. Cells in all conditions were incubated
at 37°C, 95% O2 + 5% CO2 for 6 h. Cell viability was determined by Trypan blue dye exclusion and culture medium
was collected and stored at -20°C until analysis for TNF-Į production
Determination of TNF-D Production
Supernatants were collected after an optimum incubation period and stored at -20°C until used. TNF-Į
ELISA was performed according to the manufacturer's instructions. Briefly, 100 PL of the collection medium and 50
PL of the detection antibody, conjugated with horseradish peroxidase, were added to 96-well plates pre-coated with
capture antibody for 2 h at room temperature. TMB substrate was added to the reaction for 30 min at room
temperature. The reactions were terminated by the addition of the stop solution before measuring the absorbance at
450 nm. The concentration of TNF-Į can be calculated from the standard curve produced by the serial-diluted
standard TNF-Į.

3. RESULTS
Anti-bacterial activity and anti-oxidative activity via DPPH assay of Luvunga scandens extracts
Ethyl acetate, dichloromethane and methanol extracts of L. scandens exhibited moderate anti-oxidative
activities as radical scavenger in the DPPH assays with IC50 values of 94, 445 and 480 µg/mL respectively, in
comparison with ascorbic acid (IC50 < 5 µg/mL) as a reference standard. All five bacterial strains were sensitive to
extracts from Lunvunga scandens.All of extracts, Ethyl acetate extract demonstrated anti-bacterial activity against
all bacterial strains used for this study with MIC value ranging from 3.125 to 25 mg/mL, and also methanol extract
displayed a moderate anti-bacterial activity against E. coli (TISTR 780), S. aureus (TISTR 1466), B. cereus (TISTR
687) and E. faecalis (TISTR 379) with MIC value of 25 mg/mL. Dichloromethane extract displayed an anti-bacterial
activity against S. aureus (TISTR 1466) and Methicillin-resistant S. aureus (MRSA) (ATCC 43300) with MIC value
of 6.25 and 12.5 mg/mL, respectively.

Table 1. Minimum inhibitory concentration (MIC) of stem extracts from Luvunga scandens

MIC and MBC values are in mg/mL

Bacterial strains Gram stain CH2Cl2 extract EtOAc extract MeOH extract
MIC MBC MIC MBC MIC MBC
Escherichia coli(TISTR 780) - 100 100 12.5 25 25 50
Bacillus cereus(TISTR 687 ) + 100 >100 25 50 25 100
Enterococusfaecalis(TISTR 379) + >100 >100 25 100 25 100
Staphylococusaureus (TISTR + 6.25 50 3.125 12.5 25 50
1466)
Methicillin-resistant S. aureus + 12.5 100 3.125 6.25 50 50
(MRSA)(ATCC 43300)

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 Effect of methanolic extract of Luvunga scandens on LPS-induced TNF-Į production
For activating TNF-Į-production, isolated human PBMCs were exposed to 10 ng/ml LPS for 6, 12 and 24 h.
The optimum condition for LPS-induced TNF-Į production was 6 h. Subsequently, the effect of the methanolic
extract of L. scandens on anti-inflammation was evaluated by the addition of various concentrations of the L.
scandens extracts. Exposure of the L. scandens extracts reduced the TNF-Į level in a dose-dependent manner
(Figure 1b). It was observed that at the final concentration of the methanolic extract at 5 mg/ml significantly reduced
the TNF-Į level. Furthermore, we tested whether the reduction of TNF-Į as treated with the extracts, as seen in the
previous results, was not due to the toxicity of the extract. Therefore, cell viability was performed by a Trypan blue
dye exclusion assay. Exposure of human PBMCs to the L. scandens extracts did not reduce the percentages of cell
viability at all concentrations as shown in Figure 1a.

(a) (b)

Figure 1. (a) Effect of Luvunga scandens extract on cell viability, (b) Inhibitory effect of the methanolic extract from
L. scandens on the production of TNF-D in LPS-activated monocytes. White blood cells were stimulated LPS in the
presence of the methanolic extract (0.01 mg/ml-5.0 mg/ml) for 6 h. Results are expressed as means ± SEM of three
experiments made in triplicate. (DMSO or blank = cells without treatment; LPS = cells previously treated with
LPS). *p < 0.05 vs control and # p < 0.05 vs LPS, LPS + 0.01 and LPS + 0.1.

4. CONCLUSIONS

To this research, we reported the investigation of anti-inflammatory anti-bacterial and anti-oxidative activities
of L. scandens extracts. It was found that the methanolic extract of displayed a markedly anti-inflammatory
inhibitory activity via reducing the level of TNF-Dsecreted by monocytes, and also showed anti-oxidative and anti-
bacterial activities against a variety of bacterial strains. This finding has paved the way of the identification of active
components from L. scandens with therapeutic applications.

ACKNOWLEDGEMENTS

This work was funded by The National Research University Project of Thailand’s Office of Higher Education
Commission and partially supported by Royal Chemical Society. We thank Dr. Sarawut Kum-Puan for anti-
inflammatory assay and Center of Scientific Equipment, Faculty of Science and Technology, Thammasat University
for mass spectrometer and NMR spectrometer.

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