Investigation of essential oil and biological activities of · · 2015-05-31Investigation of...

Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013 13

Address for correspondence:Prof. Dr. Salwa F. Farag, Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut-71526, Egypt. E-mail: [email protected]

Department of Pharmacognosy,

Faculty of Pharmacy, Assiut University,

Assiut-71526, Egypt

Investigation of essential oil and biological activities of Lantana montevidensis (Spreng.) Briq. cultivated in Egypt

Makboul A. Makboul, Ahmed A. Attia, Salwa F. Farag, Nesma M. Mohamed

ABSTRACT

Background: Lantana montevidensis (L. montevidensis) is native to Brazil, the tea and infusions of the dried leaves have been used in folk medicine. Previous study of L. montevidensis leaves has resulted in the isolation of ten anti-proliferative fl avones. Also, the essential oil of the Brazilian L. montevidensis leaves was investigated for its chemical composition and antimicrobial activity. Furthermore, the ethanolic extracts of the leaves and roots demonstrated anti-bacterial activity. Materials and Methods: The essential oils extracted by hydrodistillation from the Egyptian L. montevidensis leaves and fl owers were investigated for their chemical composition. Also, a qualitative phytochemical analysis of 70% aqueous methanolic extract of L. montevidensis leaves was performed for the detection of alkaloids, tannins, triterpenoids, steroids, fl avonoids, anthraquinones, saponins, iridoids, carbohydrates and/or glycosides. Furthermore, the extracts from the leaves were assessed for their anti-infl ammatory, anti-pyretic, analgesic, antioxidant and antibacterial activities. Results: The composition of the essential oils included large amount of sesquiterpenes, mainly caryophyllene (33.74%), ß-selinene (43.95%), germacrene-D (3.79%) and nerolidol (6.32%) in the oil of the leaves, while caryophyllene (42.38%), γ-terpinene (22.64%), D-germacrene (9.10 %) and nerolidol (7.09%) are major in the oil of the fl owers. Phytochemical screening of 70% aqueous methanolic extract of L. montevidensis leaves revealed the presence of carbohydrates and/or glycosides, unsaturated sterols, triterpenoids and fl avonoids in addition to traces of saponins and iridoids. Furthermore, the extracts from the leaves exhibited anti-infl ammatory, anti-pyretic, analgesic, antioxidant and antibacterial activities. Conclusion: The obtained results may provide a support to explore the plant for isolation of the active constituents accountable for these activities.

Key words: Analgesic, antibacterial, anti-infl ammatory, antioxidant, antipyretic, Lantana montevidensis, sesquiterpenes

Original Article

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Website:Website: www.jnatpharm.orgDOI:DOI: 10.4103/2229-5119.110342

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INTRODUCTION

The genus Lantana (Verbenaceae) comprises 150 species, most of which are native to tropical or sub-tropical regions.[1] Extensive phytochemical studies on Lantana species have led to the isolation of triterpenoids, steroids, flavonoids, iridoid glycosides, ol igosaccharides, phenylpropanoid glycosides and naphthoquinones.[1-3] Several species of the genus Lantana are used in folk medicine in gastrointestinal and respiratory affections.[4] Lantana montevidensis (Spreng.) Briq. is a shrub known as Weeping or Trailing Lantana. It was introduced in many countries as an ornamental plant and considered as an invasive species in many parts of the world. The tea and infusion of

the dried leaves are used in folk medicine to treat fever, infl uenza, asthma, bronchitis and other broncho-pulmonary diseases, headaches and sunstroke. The methanolic extract of the leaf of the plant showed an anti-proliferative activity against tumor cells and the fl avonoid rich fraction was effective against human gastric adenocarcinoma, human uterine carcinoma and melanoma cell lines. The ethanolic extracts of the leaves and roots presented antibacterial activity against clinically relevant pathogens (gram positive and gram negative) the extracts were active against Pseudomonas aeruginosa, Pseudomonas vulgaris and Escherichia coli. Also, it was reported that the leaves essential oil exhibited promising antimicrobial activity.[5-9]

Makboul, et al.: Investigation of essential oil and biological activities of Lantana montevidensis

Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 201314

This work describes the chemical composition of essential oils from the leaves and fl owers of L. montevidensis (Spreng.) Briq. cultivated in Egypt. Also, we report the qualitative phytochemical analysis and different biological activities of the extracts from the leaves of this plant.

MATERIALS AND METHODS

PlantLeaves and fl owers of L. montevidensis (Spreng.) Briq. were collected during the fl owering stage in the period of February to November 2009 from the gardens of Assiut University, Assiut, Egypt. The plant was kindly identifi ed and authenticated by Prof. Dr. Naeem E. Keltawy, Professor of Ornamental Horiculture and Floriculture, Faculty of Agriculture, Assiut University. A voucher sample (no. 2009 LM) has been deposited in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt.

Animals and drugsAdult male albino rats (each 100-120 g) and mice (each 25-35 g) were used. The animals were bred and housed under standardized environmental conditions in the Pre-clinical Animal House, Pharmacology Department, Faculty of Medicine, Assiut University, Assiut. They were fed with standard diet and allowed free access to drinking water.

Indomethacin was used as a standard antipyretic and anti-infl ammatory drug (El-Nile Co., Egypt). Tramadol was used as a standard analgesic drug (October Pharma Co., Cairo, Egypt). Gentamicin was used as reference antibiotic drug (Memphis Co, Egypt). 1,1-Diphenyl-2-Picryl-Hydrazyl radical (DPPH) was obtained from Sigma and quercetin (reference antioxidant) was obtained from El-Nasr Pharmaceutical and Chemical Co., Egypt (Adwic).

Experimental procedureInvestigation of essential oilsThe fresh leaves and flowers (500 g of each) of L. montevidensis were collected separately and the oils were extracted by using hydrodistillation method for 4 h using Clevenger-type apparatus. The resulting oils were separated, dried over anhydrous Na2SO4, weighed and their yields calculated with respect to the weight of the fresh material. Oils were kept in sealed vials at 6°C until analysis. The analysis of the oil samples was performed using GC-MS by injection onto a DB-5 column (Agilent; 30m ´ 0.25mm i.d., 0.25 mm fi lm thickness) on a 6890N gas chromatograph (Agilent) coupled to a 5975B mass spectrometer (Agilent). The injection port temperature was 250°C. The oven temperature was at 50°C for 1min

then raised from 50 to 250°C at 3°C/min for 5 min and from 250 to 280°C at 2°C/min for 10 min. The gas carrier was helium at a fl ow rate of 1 ml/min. The ionization of the sample components was performed in the EI mode (70 eV). The oil components were identifi ed by comparison of their retention times and mass spectral data with mass spectra library.

Phytochemical analysisDried and pulverized leaves were exhaustively extracted with 70% aqueous MeOH and the extract was concentrated under reduced pressure to afford a dark green viscous residue which was partitioned between water and organic solvents of increasing polarities to afford the new extracts: n-hexane, CHCl3, EtOAc, n-BuOH and water-soluble remaining extract. The dried extracts were fi rst reconstituted in the respective solvents used for their extraction and then tested by standard phytochemical methods[10-12] for the presence of alkaloids, fl avonoids, anthraquinones, tannins, saponins, iridoids, steroids, triterpenoids, carbohydrates and/or glycosides.

Anti-inflammatory, antipyretic and analgesic activitiesPreparation of extracts for administrationA specifi c weight (5g) of each dried test extract (total MeOH, n-hexane, CHCl3, EtOAc, n-BuOH and water-soluble remaining extract) was prepared as an emulsion in 0.9% NaCl solution containing 2% (v/v) Tween 80. A control solution was prepared using the same amount of Tween 80 in normal saline (placebo).

Acute toxicityThe acute toxicity (LD50) was determined according to the procedure described by Lorke, 1983.[13] The experiment was carried out in two phases; the fi rst phase involved an initial dose fi nding procedure, in which rats were divided into three groups, each containing three animals. The test extracts were intra-peritoneally (i.p.) injected at doses of 10, 100 and 1000mg/kg, one dose for each group. The treated animals were monitored for 24 h for mortality and general behavior. In the second phase, based on the result of the above step, four different doses were chosen and administered (i.p.) respectively to four groups of one mouse per group. The treated animals were again monitored for 24 h. The LD50 was then calculated as the geometric mean of the lowest dose showing death and the highest dose showing no death.

Anti-infl ammatory activityThe anti-infl ammatory activity consisted of measuring the inhibition of edema produced acutely by injection of an irritant (phlogistic agent) into the tissues of the plantar surface of the hind paw of the rat.[14,15] The pedal infl ammation was induced in rat paws by intradermal injection of 0.1 ml 1% freshly prepared carrageenin



suspension in 0.9% NaCl solution into the plantar side of the right hand paw of each rat. Male rats were divided into eight groups of six animals. At the beginning of the experiment, the paws thickness was measured in mm using Smiec Stainless Varnier caliper. The fi rst group injected intra-peritoneally by 2% v/v Tween 80 in normal saline (negative control) and the second group injected by indomethacin (8 mg/kg) (positive control). The other groups of rats were intra-peritoneally injected with the test extracts at a dose of 400mg/kg. After 30 min from administration, the pedal infl ammation was induced in rat paws by intradermal injection of 0.1ml, 1% carrageenin suspension in 0.9% NaCl solution into the plantar side of the right hand paw of rats while, the left one was injected by an equal volume of saline solution. The difference between the thicknesses of the two paws was taken as a measure of edema. The anti-infl ammatory effi cacy was estimated by comparing the magnitude of paw swelling in the pretreated animals with those induced in control animals receiving saline. The measurements were carried out at 1, 2, 3, 4 and 5 h after injection of the infl ammatory agent. The percentage of inhibition was calculated as follows:

% Variation (edema) =

% Inhibition =

While:Vo = The average paw thickness of the control groupVt = The average paw thickness of the treated group

Antipyretic activityRats were divided into eight groups of six rats each. Initial rectal temperatures were recorded with a thermometer. Pyrexia was induced by injecting 10ml/kg of a 20% (w/v) yeast aqueous suspension subcutaneously. The fi rst group was injected intraperitoneally by 2% (v/v) Tween 80 in 0.9% NaCl solution (negative control) while, the second group injected by indomethacine (8mg/kg) (positive control). The other groups were injected intra-peritoneally with the test extracts at doses of 400 mg/kg body weight. Rectal temperatures were taken after 1, 2, 3, 4 and 5 h from administration of test extracts.[14,16]

Analgesic activityThe analgesic activity of the test extracts was studied in mice by the hot plate method.[17] In this method, the time taken by the mouse to lick its feet or jump within a plexiglass cylinder placed on a hot surface (55 + 1°C) was determined. This reaction time was taken as the end point.[18] Male rats were divided into eight groups of six

animals. The fi rst group (control animals) received vehicle 3% PEG 600 intra-peritoneally and the second group (reference group) was intra-peritoneally injected with tramadol 10 mg/kg. The latency was recorded before and after 30 min following intra-peritoneal administration of 400 mg/kg of each of the total MeOH, n-hexane, CHCl3, EtOAc, n-BuOH and aqueous extracts to the other groups. Average reaction times were then calculated and the percentage protection calculated using the following ratio:

% Protection =

Statistical AnalysisResults were expressed as mean ± S.E. Student’s t-test was applied to the results to evaluate the signifi cance. P values less than 0.05 were considered.

Antioxidant activityThe antioxidant activity of the extracts from the leaves of L. montevidensis was measured by spectrophotometric method using DPPH. One ml of ethanolic solutions of each test extract of various concentrations (50-1000 μg/ml)were mixed with 1ml of ethanolic solution of DPPH (200 μM). Similarly, 1ml of ethanolic solutions of quercetin (50 and 100μg/ml) were mixed with 1ml DPPH solution and used as a positive control. One ml of ethanol was used instead of the sample solution as a blank. After mixing, all the solutions were incubated in dark for 20min and absorbance was measured at 517nm.[19] The experiments were performed in triplicate and percent scavenging activity was calculated as follows:

Scavenging % =

Antibacterial activityPreparation of extractsThe dried extracts of L. montevidensis leaves were sterilized by overnight UV-irradiation and sterility checked by plating the reconstituted extract on nutrient agar. Two hundred mg of each test extract was separately reconstituted in the least amount of Dimethylformamide (DMF) and the volume was completed to 2ml with DMF (100mg/ml). DMF served as a negative control.

Test organismsGram-positive bacteria namely, Staphylococcus aureus AUMC No. B-54 and Bacillus cereus AUMC No. B-52 and Gram-negative bacteria namely, Escherichia coli AUMC No. B-53, Salmonella typhimurium AUMC No. B-62, Klebsiella pneumonia AUMC No. B-77, Pseudomonas aeruginosa AUMC No. B-73 and Serratia marcescen AUMC No. B-55s were obtained from Assiut University, Mycological Center.



Screening extracts for antibacterial activityThe inoculum size of each test strain was standardized according to the national committee for the clinical laboratory standard (NCCLS, 1993). The tested bacteria strain was inoculated in Muller Hinton broth (MHB) medium and incubated for 3-6 h at 35ºC in a shaker water bath until the culture attained the turbidity of 0.5 McFarland unit. The fi nal inoculum was adjusted to 5 ´ 105 cfu/ml. Antibacterial screening was done by a modifi ed agar well diffusion method.[20] One ml of the standard suspension (5 105 cfu/ml) of each test bacterial strain was spread evenly on Muller Hinton Agar plates using sterile glass rod spreader and the plates allowed to dry at room temperature. Subsequently, 10mm wells were bored in the agar using the sterile cork borer and 100μl of each plant extract was pipetted into the wells to allow diffusion of the extract to the agar, they were incubated at 37ºC for 24 h and the bacterial growth inhibition zone diameter (IZD) was measured to the nearest mm. Gentamicin (5μg/ml) was included as a positive control while DMF served as a negative control.

Determination of the minimal inhibitory concentration (MIC)The MIC was determined by the modifi ed agar-well diffusion method.[20] In this method, a two fold serial dilution of each extract in DMF was prepared to obtain

a 0.1-100mg/ml concentration range. A 100 μl volume of each dilution was introduced in wells in Muller Hinton Agar (MHA) plates pre-inoculated with test bacterial cells. Similarly, treated Gentamicin preparations (concentration range, 0.123-512μg/ml) were included as a positive control. All test plates were incubated at 37ºC for 24 h. The least concentration of each extract showing a clear zone of inhibition was taken as the MIC.

RESULTS

Investigation of essential oilsThe yields of volatile oil from the leaves and fl owers were 0.20% and 0.46%, respectively. The identified components of the oils, their retention times and their relative percents are presented in Table 1. The GC-MS analysis of the volatile oil of the leaves permitted the identifi cation and quantifi cation of 14 constituents (98.4%) with predominance of sesquiterpenes (88.51%) and a little amount of monoterpenes (9.59%). The major components in the oil of the leaves are caryophyllene (33.74%), -selinene (43.95%), germacrene-D (3.79%) and nerolidol (6.32%).

The GC-MS analysis of the fl owers volatile oil permitted the identifi cation and quantifi cation of 26 constituents

Table 1: Compositi on of essenti al oil of the fl owers and leaves of L. montevidensis

Flowers essen al oil Leaves essen al oil

Cons tuents RT % Cons tuents RT %

α-Pinene 15.401 0.26 α-Pinene 7.426 1.15Sabinene 17.292 7.94 Sabinene 8.856 5.34-Pinene 17.493 0.30 -Pinene 9.036 0.97-Myrcene 17.948 1.48 1-Octen-3-ol 9.147 1.65α-Terpinene 19.341 1.64 -Myrcene 9.508 0.66dl-Limonene 19.945 0.04 3-Octanol 9.825 0.27-Phellandrene 20.003 0.04 dl-Limonene 11.117 0.28-Terpinene 21.523 22.64 (Z)-Sabinene hydrate 12.886 0.61(Z)-Sabinene hydrate 21.846 0.19 Caryophellene 28.306 33.74α-Terpinolene 22.894 0.10 -Selinene 29.815 43.95Linalool 23.291 0.50 D-Germacrene 30.795 3.79α-Terpineol 27.909 0.11 Bicyclogermacrene 31.382 0.71δ-Elemene 34.856 0.10 Nerolidol 34.120 6.32α-Copaene 36.678 0.01 Camphene 35.428 0.58β-Elemene 37.292 2.07Caryophyllene 38.875 42.38-farnesene 39.770 0.15D-Germacrene 41.311 9.10δ-Cadinene 42.566 0.13Nerolidol 44.171 7.09(-)-Caryophyllene oxide 45.458 2.30-Selinene 46.019 0.08δ-Cadinene 47.459 0.741-Naphthalenol 47.629 0.10α-Cadinol 47.989 0.20Farnesyl acetone C 56.800 0.04Total iden fi ed 99.73 Total iden fi ed 98.10Monoterpenes 35.24 Monoterpenes 9.59Sesquiterpenes 64.49 Sesquiterpenes 88.51



(99.73%) with predominance of sesquiterpenes (64.49%) and some of monoterpene constituents (35.24%). The major components of the fl owers essential oil are caryophyllene (42.38%), -terpinene (22.64%), D-germacrene (9.10 %) and nerolidol (7.09%).

Phytochemical analysisThe phytochemical analysis showed that the 70% aqueous methanolic extract of L. montevidensis leaves contained carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes and fl avonoids in addition to traces of saponins and iridoids [Table 2]. Cardenolides, alkaloids and/or basic nitrogenous compounds, anthraquinones (free or combined) and coumarins were not detected.

Biological activitiesAcute toxicityToxicity of the test extracts from L. montevidensis characterized by irritability, writhing, hypothermia, loss

of motor coordination, sedation and deep sleep, followed by death. Toxicity appears at different dose levels 2.25, 3.30, 2.25, 1.95, 2.25 and 1.95g/kg orally for total MeOH, n-hexane, CHCl3, EtOAc, n-BuOH and aqueous extracts, respectively.

Anti-infl ammatory activityThe extracts from L. montevidensis leaves exhibited a signifi cant inhibition of the edema induced by carrageenin when the animals were pre-treated with the different extracts one hour before injecting the phlogistic agent. The effects begin within the fi rst hour and become highly signifi cant in the 3rd and 4th hours and continue till the 5th hour. The n-hexane, CHCl3 and EtOAc extracts showed higher percentage of anti-infl ammatory activity than the total MeOH, n-BuOH and aqueous extracts [Tables 3 and 4].

Antipyretic activityThe n-hexane, CHCl3 and EtOAc extracts from L. montevidensis leaves exhibited signifi cant antipyretic activity against yeast-induced pyrexia while, the polar extracts (n-BuOH and aqueous) showed no signifi cant antipyretic activity. The effect reached its maximum at the 3rd hour and continued till the 5th hour [Table 5].

Analgesic activityThe results of the hot plate test indicated that all the test extracts from L. montevidensis leaves showed a signifi cant analgesic activity against thermal stimuli as evidenced by increase in the latency time in seconds. The n-hexane, CHCl3 and EtOAc extracts showed higher percentage of protection than the total MeOH, n-BuOH and aqueous extracts [Table 6].

Table 2: Results of phytochemical screening of L. montevidensis leaves

Plant cons tuent Rela ve level

Carbohydrates and/or glycosides +++Cardenolides -Unsaturated sterols and/or triterpenes +++Flavonoids +++Saponins +Alkaloids and/or basic nitrogenous compounds -Tannins ++Anthraquinones -Coumarins -Iridoids ++++ = major, ++ = moderate, + = traces, - = absent

Table 3: Eff ect of test extracts from L. montevidensis leaves on carrageenin induced paw edema in rats

Treatment Dose(mg/kg)

Thickness of the right hind paw (mm)

1 h 2 h 3 h 4 h 5 h

Nega ve control 8.00 ± 0.044 8.26 ± 0.022 8.51 ± 0.025 8.48 ± 0.027 8.43 ± 0.0163Indomethacin 8 5.30 ± 0.07*** 4.80 ± 0.08*** 4.30 ± 0.06*** 4.30 ± 0.030*** 4.20 ± 0.006***Total MeOH 400 7.61± 0.019*** 7.85 ± 0.037*** 8.06 ± 0.08*** 7.88 ± 0.013*** 8.15 ± 0.019***n-Hexane 400 6.96 ± 0.011*** 6.40 ± 0.0322*** 5.66 ± 0.036*** 5.33 ± 0.0103*** 7.50 ± 0.044***CHCl3 400 6.60 ± 0.089*** 6.31 ± 0.0183*** 5.70 ± 0.022*** 5.80 ± 0.013*** 7.06 ± 0.016***EtOAc 400 6.65 ± 0.012*** 6.40 ± 0.0322*** 6.68 ± 0.014*** 6.90 ± 0.086*** 7.50 ± 0.017***n-BuOH 400 7.81± 0.132 7.90 ± 0.172* 7.91 ± 0.263* 8.28 ± 0.147 8.25 ± 0.207Aqueous 400 7.85 ± 0.333 8.10 ± 0.363 8.23 ± 0.233* 8.25 ± 0.137* 8.36 ± 0.260

Data are expressed as mean ± S.E, n = 6, Diff erences with respect to the control group were evaluated using the student t-test, (*P < 0.05, ** P < 0.01, *** P < 0.001)

Table 4: Inhibitory eff ects of extracts from L. montevidensis leaves on carrageenin-induced paw edema in rats

Treatment Dose (mg/kg) Edema inhibi on %

1 h 2 h 3 h 4 h 5 h

Indomethacin 8 33.75 41.88 49.41 49.39 50.18Total MeOH 400 4.87 4.96 5.28 7.07 3.32n-Hexane 400 13.00 23.97 33.40 37.10 11.03CHCl3 400 17.50 22.50 33.01 31.6 16.25EtOAc 400 16.87 23.60 21.50 18.63 11.03n-BuOH 400 2.37 4.35 7.05 1.22 2.13Aqueous 400 1.88 1.93 3.29 2.70 1.12



Antioxidant activityThe EtOAc extract showed strong antioxidant activity (88.4%), while the total MeOH, CHCl3 and n-BuOH extracts demonstrated moderate activity (63.6%, 67.3% and 65.7%, respectively) relative to the positive control (quercetin). The aqueous and n-hexane extracts showed poor activity (51.1% and 36.8%, respectively) [Figure 1].

Antibacterial activityThe anti-microbial affect of plant extracts against the different strains are illustrated in Table 7. Gentamicin showed bactericidal activity against all tested bacterial strains at concentration of 5 μg/ml and caused inhibition zone of 15-30 mm diameter after 24 h of incubation at 37C (MIC; 0.6 μg/ml). All the tested bacterial strains showed susceptibility to each extract with the exception of K. pneumonia and S. marcescens. The aqueous extract demonstrated no activity against any of the strains tested. The n-BuOH extract exhibited mild bactericidal activity against S. aureus, B. cereus and E. coli relative to the positive control (Gentamicin) and caused inhibition zone of 3-5mm diameter (MIC; 100 mg/ml). The n-hexane extract showed mild bactericidal activity against S. aureus, B. cereus and P. aeurginosa relative to the positive control and caused

Table 5: Eff ect of test extracts from L. montevidensis leaves on yeast-induced pyrexia in rats

Treatment Dose(mg/kg)

Rectal temperature (C) a er yeast injec on

1 h 2 h 3 h 4 h 5 h

Nega ve control 39.86 ± 0.050 39.90 ± 0.080 39.60 ± 0.081 39.70 ± 0.090 39.70 ± 0.070Indomethacin 8 37.17 ± 0.012*** 36.10 ± 0.015*** 36.07 ± 0.042*** 36.07 ± 0.012*** 36.23 ± 0.023***Total MeOH 400 39.73 ± 0.029* 39.76 ± 0.016* 39.34 ± 0.028** 39.44 ± 0.017** 39.49 ± 0.014**n-Hexane 400 38.70 ± 0.015*** 37.54 ± 0.012*** 36.72 ± 0.016*** 36.31 ± 0.011*** 37.92 ± 0.023***CHCl3 400 38.11 ± 0.020*** 37.43 ± 0.012*** 36.43 ± 0.025*** 36.14 ± 0.012*** 38.36 ± 0.029***EtOAc 400 38.84 ± 0.014*** 37.82 ± 0.013*** 36.94 ± 0.037*** 36.53 ± 0.013*** 38.36 ± 0.021***n-BuOH 400 39.74 ± 0.041* 39.82 ± 0.022 38.84 ± 0.013 39.75 ± 0.027 39.92 ± 0.020Aqueous 400 39.76 ± 0.049 39.84 ± 0.030 39.53 ± 0.027 39.75 ± 0.021 39.72 ± 0.028Data are expressed as mean ± S.E, n = 6, Diff erences with respect to the control group were evaluated using the student t-test, (*P < 0.05, ** P < 0.01, *** P < 0.001)

Table 6: Eff ect of extracts from L. montevidensis leaves on the latency of mice exposed to the hot plate

Treatment Dose (mg/kg) Average reac on me (sec.) % protec on

Nega ve control b 12.10 ± 0.109a 12.23 ± 0.103

–

Tramadol HCl 10 b 12.50 ± 0.075a 21.31 ± 0.160

70.40

Total MeOH 400 b 12.20 ± 0.178a 14.20 ± 0.116

16.39

n-Hexane 400 b 12.48 ± 0.116a 16.38 ± 0.098

31.25

CHCl3 400 b 12.20 ± 0.126a 18.31 ± 0.117

50.08

EtOAc 400 b 12.58 ± 0.091a 17.36 ± 0.163

37.99

n-BuOH 400 b 11.90 ± 0.116a 13.80 ± 0.122

15.96

Aqueous 400 b 12.10 ± 0.154a 13.31 ± 0.141

10.00

b: average reac on me before treatment, a: average reac on me a er treatment, Data are expressed as mean ± S.E, n=6; Diff erences with respect to the control group were evaluated using the student t-test, (*P < 0.05, ** P < 0.01, *** P < 0.001)

inhibition of 4.2-6.0 mm diameter (MIC; 50-100 mg/ml). The CHCl3 and EtOAc extracts demonstrated strong bactericidal activity against S. aureus, B. cereus, E. coli, S. typhi and P. aeurginosa relative to positive control and exhibited inhibition zones of 4.1-15.0 and 7.5-11.0mm diameter, respectively (MIC; 6.25-50 and 6.25-100mg/ml, respectively). The total MeOH extract showed moderate bactericidal activity against S. aureus, B. cereus, E. coli,

Figure 1: Free radical scavenging ac vity of the extracts from L. montevidensis leaves



S. typhi and P. aeurginosa relative to the positive control and caused inhibition zone of 3.1-10.8 mm diameter (MIC; 25-100 mg/ml).

DISCUSSION

Investigation of essential oilsThe yield of volatile oil from the fl owers is higher than that of the leaves. Analysis of both oils showed that the oil of the fl owers is a complex mixture of numerous compounds with more monoterpene constituents and less sesquiterpenes. The results of the present study indicated that the major components in the oil of the leaves are caryophyllene (33.74%), -selinene (43.95%), germacrene-D (3.79%) and nerolidol (6.32%). Thus, the composition of the oil from the leaves of L. montevidensis cultivated in Egypt differs from species from Brazil, in which caryophyllene, germacrene-D and bicyclcogermacrene are major constituents.[6,9]

Phytochemical analysisPhytochemical analysis of 70% aqueous methanolic extract of L. montevidensis leaves showed the presence of carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes and fl avonoids in addition to traces of saponins and iridoids. Different phytochemicals have been found to possess a wide range of activities, which may help in protection against chronic diseases. Among the great variety of secondary compounds found in plants, phenolics and terpenoids represent the main antimicrobial agents while glycosides, flavonoids and tannins have anti-infl ammatory activities. Steroids and triterpenoids showed the analgesic properties and central nervous system activities.[1,2,17,21]

Biological activitiesAcute toxicityThe determination of acute toxicity described is an

important parameter, as the doses used in the investigation of pharmacological activities must be lower than the LD50 of test extracts of the plant.

Anti-infl ammatory, antipyretic and analgesic activitiesThe present study demonstrated the signifi cant anti-inflammatory, antipyretic and narcotic analgesic activities of the n-hexane, CHCl3 and EtOAc extracts of L. montevidensis leaves. The phytochemical constituents of this plant mainly sterols/triterpenes and fl avonoids may contribute to these activities.[21] Also, it has been reported that different Lantana species exhibited anti-infl ammatory, antipyretic and analgesic activities due to the presence of fl avonoids and triterpenoids.[1,2]

Free radical scavenging activityThe results indicated scavenging activity of the EtOAc, total MeOH, CHCl3, and n-BuOH extracts of L. montevidensis leaves towards DPPH radicals in comparison with quercetin. Phenolics including fl avonoids are the major contributors to the antioxidant activity of these extracts.[22]

Antibacterial activityThe results of our study demonstrated the antibacterial activity of the extracts of L. montevidensis leaves against Gram-positive bacteria and three strains of Gram-negative bacteria namely E. coli, P. aeurginosa and S. typhi. Previous studies using the ethanolic extract of L. montevidensis leaves showed that it was able to inhibit the growth of Gram-positive bacteria and two strains of Gram-negative bacteria namely E. coli and P. aeurginosa.[5]

The phytochemical metabolites detected in the plant of this study, namely sterols, triterpenes and fl avonoids have been associated with the antimicrobial activities of several herbs.[20] Several sterols, triterpenes and fl avonoids isolated from different Lantana species were reported to have antimicrobial properties.[1-3,23]

Table 7: Inhibiton zones diameter (IZD) and MICs (Given in brackets) of test extracts from L. montevidensis leaves against the tested bacterial strains

Test bacterial strains Gentamicin5μg/ml

Total MeOH 100 mg/ml

n-Hexane 100 mg/ml

CHCl3 100 mg/ml

EtOAc 100 mg/ml

n-BuOH 100 mg/ml

Aqueous 100 mg/ml

S. aureus 17.0(0.6)

10.8(25.0)

6.0(50.0)

12.3(12.5)

11.0(25.0)

3.0(100.0)

–

B. cereus 15.0(0.6)

7.5(50.0)

4.8(50.0)

10.3(50.0)

8.3(50.0)

6.0(100.0)

–

E. coli 17.0(0.6)

9.3(25.0)

5.8(100)

14.0(6.25)

10.9(6.25)

5.0(100.0)

–

S. typhi 20.0(0.6)

3.1(100.0)

– 4.1(50.0)

7.5(100.0)

– –

K. pneumonia 16.0(0.6)

– – – – – –

P. aeurginosa 22.0(0.6)

7.2(50.0)

4.2(100.0)

15.0(12.5)

10.1(25.0)

– –

S. marcescens 30.0(0.6)

– – – – – –

− = no eff ects; IZD (inhibi on zone diameter) in mm; MIC given in g/ml for the posi ve control and mg/ml for the test extracts



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Cite this article as: Makboul MA, Attia AA, Farag SF, Mohamed NM. Investigation of essential oil and biological activities of Lantana montevidensis (Spreng.) Briq. cultivated in Egypt. J Nat Pharm 2013;4:13-20.

Source of Support: Nil. Confl ict of Interest: None declared.

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