ABSTRACT

The known triterpenoids lantic acid, camaric acid,camarinic acid and lantanilic acid were isolated fromLantana camara (L.) cultivated in Egypt. The antibac-terial activity of lantic acid was carried out usingbioautography assays for Gram-positive and Gram-negative bacteria. Lantic acid was found to possessstrong antibacterial activity against Escherichia coliand Bacillus cereus, in which 0.08 and 0.1 �g were theminimum inhibition doses, respectively, compared to0.05 and 0.005 �g for chloramphenicol, respectively.The results indicate that lantic acid has broad spec-trum antibacterial activity and may hold potential as anon-selective antimicrobial agent.

INTRODUCTION

Lantana camara L. are erect herbs or shrubs thatbelong to the Verbenaceae. They are generally native totropical and subtropical America, however, a few existin Africa and Asia (Lundell, 1942). L. camara hasreceived attention due to its role in economy and ecol-ogy. It is a serious weed in several countries that causestoxicity in grazing animals and is rapidly disturbing theecological balance due to its luxuriant growth (Sharmaet al., 1988; Sharma & Sharma, 1989). The major tox-ins of L. camara are triterpenoids of the oleananeseries, only a few of which have been assayed forantibacterial activity (Hart et al., 1976). L. camara isalso cultivated as an ornamental plant which has beenused in traditional medicine. Its leaves are used as an

antiseptic and for treating wounds (Acchireddy &Singh, 1984; Siddiqui et al., 1995).

The present study deals with the isolation and iden-tification of the antibacterial triterpenoids present in theplant as well as carrying out antibacterial bioassays forthe determination of the minimum inhibition concen-tration (MIC) compared to the known antibioticchloramphenicol.

MATERIALS AND METHODS

Antimicrobial AssaysUsing bioautography assays (Hamburger & Cordell,1987) with a modified procedure, the crude extract andpure compounds were assayed for antibacterial activ-ity against Bacillus cereus ATCC 11778, Bacillus sub-tilis ATCC 6633, Micrococcus luteus ATCC 9341,Staphylococcus aureus ATCC 6538P, Streptococcusfaecalis ATCC 8043, Esherichia coli ATCC 25922,Pseudomonas aeruginosa ATCC 25619 and Proteusmirabilis ATCC 14153. Bacteria were grown in nutri-ent broth (Difco, 0003–01) at 37°C for 48 hr withshaking at a speed of 70 rpm. The cultures were cen-trifuged (2500 rpm for 15 min), the supernatant dis-carded, and the bacteria were suspended in a smallvolume of fresh nutrient broth. The suspensions werethen diluted to ca. 109 bacteria/ml (turbidity at 560 nm:0.84 Au), dispensed in cryotubes and stored at �78°Cuntil used.

The TLC chromatograms (Whatman silica gel 60,250 �m, 20 � 20 cm, without fluorescent indicator)were developed with CH2Cl2:MeOH:H2O (90:10:1)and dried. A sample (7–8 ml) of the diluted bacterialsuspensions was sprayed evenly over the TLC plates.The TLC plates were incubated overnight at 33°C inplastic boxes lined with wet chromatography paper.The plates were then sprayed with 5 ml of an aqueoussolution of p-iodonitrotetrazolium violet (INT) (2mg/ml at pH 5). The TLC plates were again incubated

Pharmaceutical Biology 1388-0209/99/3701-0063$15.001999, Vol. 37, No. 1, pp. 63–66 © Swets & Zeitlinger

ANTIBACTERIAL TRITERPENOIDS ISOLATED FROM LANTANA CAMARA

Mahmoud Saleh, Alaa Kamel, Xiaoyang Li and James Swaray

Department of Chemistry, Texas Southern University, Houston, Texas 77004, USA

Keywords: Lantana camara, Verbenaceae, antibacterial activ-ity, lantic acid, triterpenoids

* Address correspondence to: Mahmoud Saleh, Departmentof Chemistry. Texas Southern University, 3100 CleburneAve., Houston, TX 77004, USA. Fax: +1 713 313–7824,email: aascaskamel@tsu.edu

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in the plastic boxes at 35°C for 2–4 hr to reveal bacte-ria as pink colonies. Antibacterial compounds weredetected as white zones of inhibition.

Extraction and IsolationAir-dried and powdered leaves and stems of 200 g ofL. camara, purchased from the local market in Cairo,Egypt, were macerated in 2 liters of a mixture ofCH2Cl2:MeOH (1:1) and further washed with 500 mlof MeOH. The extract was partitioned between hexaneand MeOH:H2O (6:4). The aqueous fraction was thensubjected to liquid chromatography on a silica gel col-umn (silica gel 60, 250–630 mesh, Sigma, St. Louis,Missouri, USA) and eluted with CH2Cl2 containingincreasing amounts of MeOH (1–10%). Fractionsshowing antibacterial activity were combined andpurified by HPLC (Pharmacia LKB) on a RP-18reversed phase column (Whatman, ODS-2, 50 cm � 9mm) with MeOH:H2O (9:1) monitored at 205 nm(Pharmacia LKB). Four active compounds were iso-lated and identified as lantic acid, camaric acid,camarinic acid, and lantanilic acid. TLC chro-matograms were carried out on Si gel plates (Whatmansilica gel 60, 100 �m, with fluorescent indicator)developed with CH2Cl2:MeOH:H2O (90:10:1) andvizualized by spraying with vanillin/H2SO4 reagentwith heating at 120°C for 5 min. All solvents usedwere HPLC grade (EM Science, Gibbstown, New Jer-sey, USA) and all chemicals were of the highest purityavailable (Sigma, St. Louis, Missouri, USA).

RESULTS AND DISCUSSION

The structures of the isolated triterpenoids were deter-mined on the basis of their IR, MS, 1H- NMR, 2D-NMR (COSY), and 13C-NMR spectral data being iden-tical to those recently published (Siddiqui et al., 1995).The CH2Cl2:MeOH extract of L. camara was tested forantibacterial activity on a developed TLC plate. Itshowed an inhibited growth of some Gram-positive andGram-negative bacteria. After partitioning the extractbetween hexane and MeOH:H2O (6:4), only the aque-ous fraction showed antibacterial activity (Fig. 1). Col-umn chromatography of the aqueous fraction of theCH2Cl2:MeOH extract of L. camara on Si gel followedby reversed phase HPLC showed four antibacterialcompounds in which lantic acid possessed the strongestantibacterial activity. A bioautographic assay of lanticacid with chloramphenicol as a positive control showedthat it possessed about one tenth the activity of

chloarmphenicol against the tested Gram-positive andGram-negative bacterial strains. The antibacterial activ-ity was closest to chloramphenicol when tested againstE. coli and B. cereus in which 0.08 and 0.1 �g were theleast amount of bacterial growth inhibition, respec-tively, compared to 0.05 and 0.005 �g of chloram-phenicol respectively (Table 1). These results indicatethat lantic acid has broad spectrum antibacterial activ-ity and it may hold promise as a non-selective anti-microbial agent. It seems that the carboxylic group atC-17 is essential for the antibacterial activity, especiallysince it is in the �-position of a �-cloud of the C-12/C-13 double bond. This explains the strong activity oflantic acid compared to lantanilic, camrinic andcamaric acids, whose C-17 contains an ester grouprather than a carboxylic acid group. The antibacterialtriterpenoids isolated from Lantana species so far areoleanolic and ursolic acids (Hart et al., 1976). Althoughseveral triterpenoids have been previously reported tobe of the toxic constituents of L. camara, no informa-tion indicating the toxicity of lantic acid was reported.Moreover, L. camara plants that do not contain lan-tadenes A and B are non toxic and are characterized bytriterpene acids which have a C-3/C-25 oxide bridge asin lantic acid (Hart et al., 1976). Lantic acid and lan-tanilic acid were earlier known triterpenoids from L.camara (Barua et al., 1969, 1972, 1985), but their com-plete spectral data were presented recently with thoseof camaric and camarinic acids (Siddiqui et al., 1995).

REFERENCES

Achhireddy NR, Singh M (1984): Allelopathic effects of lan-tana (Lantana camara) on milkweed vine (Morreniaodorata). Weed Sci 32: 757.

Barua AK, Chakrabarti, P, Chowdhury MK, Basak A, BasuK, Ray S, Saha SK (1985): The structure and stereo-chemistry of lantanilic acid. A new triterpene isolatedfrom Lantana camara. J Indian Chem Soc 57: 298–305.

Barua AK, Chakrabarti P, Sanyal PK, Basu K, Nag K (1972):Triterpenoids: XL. The structure of lantic acid- A newtriterpene from Lantana camara. J Indian Chem Soc 49:19–20.

Barua AK, Chakrabarti P, Sanyal PK, Das BC (1969): Triter-penoids: XXXII. The structure of lantic acid-A new triter-pene from Lantana camara. J Indian Chem Soc 46:100–101.

Hamburger MO, Cordell GA (1987): A direct bioautographicTLC assay for compounds possessing antibacterial activ-ity. J Nat Prod 50: 19–22.

Hart NK, Lamberton JA, Sioumis AA, & Suares H (1976):New triterpenes of Lantana camara. A comparative studyof the constituents of several taxa. Aust J Chem 29:655–71.

64 M. SALEH ET AL.

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TRITERPENOIDS FROM LANTANA CAMARA 65

Table 1. Minimum growth inhibition of tested Gram-positive and Gram-negative bacterial strains with lantic acid*.

Test organism Lantic acid Chloramphenicol

Bacillus cereus 0.1 0.005Bacillus subtilis 0.3 0.03Micrococcus luteus 0.3 0.02Staphylococcus aureus 0.3 0.07Streptococcus faecalis 0.5 0.05Esherichia coli 0.08 0.05Pseudomonas aeruginosa 0.5 0.04Proteus mirabilis 0.3 0.05

*Amount in �g on TLC plates.

Fig. 1. Bioautogram using Bacillus cereus as indicator organism (a) and TLC chromatogram (b) of the aqueous fraction (1) and theCH2Cl2:MeOH extract (2) of L. camara.

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Lundell CL (1942): Flora of Texas, Vol. 3, Dallas, UniversityPress, p. 48.

Sharma OP, Makkar HP, Dawra RK (1988): A review of thenoxious plant Lantana camara. Toxicon 26: 975–987.

Sharma OP, Sharma PD (1989): Natural products of the Lan-tana plant: The present and prospects. J Sci Ind Res 48:471–478.

Siddiqui BS, Raza SM, Begam S, Siddiqui S, Firdous S(1995): Pentacyclic triterpenoids from Lantana camara.Phytochem 38: 681–685.

Accepted: December 29, 1998

66 M. SALEH ET AL.

Fig. 2. Structure of Lantic acid.

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