Lantana camara: Phytochemical Analysis and Antifungal Prospective

Lantana camara: Análise Fitoquímica e Prospecção Antifúngica

ABSTRACT:

In the current study phytochemical analysis and in vitro antifungal potential of fruits, leaves and stem of Lantana camara L. were studied. The phytochemical analysis indicated the presence of alkaloids, flavonoids, tannins, saponins, glycosides and terpenoids in fruit, stem and leaves of L. camara. The in vitro antifungal activity of fruit, stem and leaves of L. camara was tested against Colletotrichum gloeosporioides Penz. Different concentrations (1-5%) of methanolic extract of all the selected parts of L. camara were applied in vitro against the test fungus. The results of in vitro experiment revealed that higher concentration of methanolic fruit extract (5%) significantly reduced the biomass C. gloeosporioides up to 66%. This effective extract of L. camara was partitioned with n-hexane, chloroform, ethyl acetate and n-butanol. The bioactivity of these fractions was tested against C. gloeosporioides. The trials showed that 0.5% concentration of n-hexane fraction of methanolic fruit extract caused the highest reduction (45%) in the radial colony growth of the test fungus. This effective n-hexane fraction was selected for GC-MS analysis to identify various possible antifungal compounds. Cyclopropane, carboxylic acid, 5-heptonic acid, 2,2-dimethyl1-4-pentenoate and 2-Propyloctahydro-1-benzothiophene were identified as major compounds. This study can be concluded that L. camara fruit comprised of bioactive compounds which possess antifungal activity against C. gloeosporioides.

Keywords:
anthracnose; bioactivity; Colletotrichum gleosporioides; GC-MS; phytochemicals

RESUMO:

Neste estudo, foi feita uma análise fitoquímica e do potencial antifúngico in vitro de frutos, folhas e caule de Lantana camara L. A análise fitoquímica indicou a presença de alcaloides, flavonoides, taninos, saponinas, glicosídeos e terpenoides em frutos, caule e folhas de L. camara. A atividade antifúngica in vitro de frutos, caule e folhas de L. camara foi testada contra Colletotrichum gloeosporioides Penz. Diferentes concentrações (1-5%) do extrato metanólico de todas as partes selecionadas de L. camara foram aplicadas in vitro contra o fungo de teste. Os resultados do experimento in vitro revelaram que a maior concentração do extrato metanólico da fruta (5%) reduziu significativamente a biomassa de C. gloeosporioides em até 66%. Esse extrato eficaz de L. camara foi dividido em seções com n-hexano, clorofórmio, acetato de etila e n-butanol. A bioatividade dessas frações foi testada contra C. gloeosporioides. Os testes mostraram que a concentração de 0,5% da fração de n-hexano do extrato metanólico da fruta causou a maior redução (45%) no crescimento de colónias radial do fungo de teste. Essa fração eficaz de n-heaxane foi selecionada por análise GC-MS para identificar vários compostos antifúngicos possíveis. Ciclopropano, ácido carboxílico, ácido 5-heptônico, 2,2-dimetil-1-4-pentenoato e 2-Propilocta-hidro-1-benzotiofeno foram identificados com os principais compostos. Esse estudo pode concluir que o fruto de L. camara compreendido de compostos de bioativos que possuem atividade antifúngica contra C. gloeosporioides.

Palavras-chave:
antracnose; bioatividade; Colletotrichum gleosporioides; GC-MS; fitoquímicos

INTRODUCTION

Mango (Mangifera indica L.) is an excellent and one of the most desirable fruits worldwide because of its delicious taste (Diedhiou et al., 2007). Mango is affected by a number of diseases at all stages of its development, right from the plants in the nursery to the fruits in storage or transit. The mango tree especially its fruit is the host of a large number of pathogens among which, fungi could be a major agent of fruit rot (Akem, 2006). Anthracnose disease, caused by Colletotrichum gloeosporioides Penz. belonging to Ascomycota is among the most destructive field and postharvest fungal pathogen of mango in the world (Arauz, 2000; Cannon et al., 2012). Synthetic fungicides are currently used as the primary means for the control of postharvest mango anthracnose. However, increasing public concern over the indiscriminate use of pesticides that leads to environmental hazards, as well as the occurrence of fungicide-resistant pathogen strains has stimulated research on alternative methods to control postharvest diseases (Yao and Tian, 2005). The plant world comprises a rich storehouse of biochemicals to be used as natural fungicides. Plant-based natural products emerging as safe alternatives to conventional fungicides for the control of plant diseases due to their ability to decompose rapidly (Tripathi and Shukla, 2007; Jabeen and Javaid, 2010; Karim et al., 2017; Hanif et al., 2017).

Lantana camara belongs to family Verbenaceae is an ornamental flowering plant and six hundred species of L. camara are available worldwide (Thakur et al., 1992). This plant is also known as, wild sage, tea plant and spanish flag. L. camara grows in un-shaded regions like wastelands, rainforest edges, beachfront and forests. Phytochemical investigation of L. camara showed the presences of flavones, isoflavones, flavonoids, anthocyanins, coumarins, lignans, catechins, isocatechins, alkaloids, tannin, saponins and triterpenoids (Ganjewala et al., 2009; Saraf et al., 2011; Singh and Srivastava, 2012; Mariajancyrani et al., 2014). The present study was therefore designed to study antifungal potential of L. camara against C. gloeosporioides.

MATERIALS AND METHODS

Phytochemical analysis of fruits leaves and stem of L. camara was carried out by using the protocols available to identify the categories of secondary metabolites present in the test plant species (Edeoga et al., 2005; Parekh & Chanda, 2007). In this analysis presence and absence of tannins, saponins, terpenoids, phenolics, alkaloids, glycoside & coumarin was tested.

In vitro antifungal activity of the experimental plant, L. camara against targeted fungus was performed by using the method of Waheed et al., (2016). Twenty grams of leaves, stem and fruit of test plant L. camara were soaked in 100 mL methanol separately. After 7 days soaked material was filtered with an autoclaved muslin cloth in pre-weighed beakers and the filtrate was evaporated at room temperature. The stock solutions for each test plant part were prepared by taking 5.144 g of each extract and diluted with 25.75 mL of distilled water to make 20% of the 60 mL stock solution. These stock solutions were stored at 4 oC. C. gloeosporioides was isolated from the infected inflorescence of mango plant, cultured and maintained on 2% MEA (Malt Extract Agar) medium and then re-cultured on 2% MEA and stored at 4 oC. The test fungus was identified on morphological basis using macroscopic and microscopic characters. In vitro antifungal activity of L. camara against C. gloeosporioides was tested. Various concentration viz. 1%, 2%, 3%, 4% and 5% of methanolic extract were prepared in 2% MEA medium (Sherazi et al., 2016). The control treatment was without any plant extract. Chloromycetin (anti-bacterial) capsules were added in each flask to avoid bacterial contamination. Each concentration (20 mL) was poured into 9 × 9 cm sterilized Petri plates. Three replicates were made for each treatment. Mycelia discs (5 mm) were taken from 7 days old culture as inoculum and were inoculated in all Petri plates with a sterilized cork borer. All these plates were sealed with parafilm strips and incubated at 25 oC for 7 days. After 7 days fungal growth was measured by using the formula:

$GrowthInhibition\%=\frac{Growthincontrol-Growthintreatment}{Growthincontrol}\times 100$

As the methanolic fruit extract exhibited maximum inhibition of fungal growth in initial screering therefore this extract was used for fractionation. For this purpose the fruit of L. camara (50 g) was extracted with 200 mL of methanol and 0.05 g gummy mass was obtained after evaporation. This gummy mass of methanolic fruit extract of L. camara (0.05 g) was partitioned with n-hexane, chloroform, ethyl acetate and n-butanol in separating funnel. These organic fractions were then evaporated in rotary evaporated at 40 oC. The in vitro antifungal activity of various isolated organic fractions and fungicide (Metalaxyl + Mancozeb 72% WP) was checked. Two concentrations (0.05% and 0.1%) for each fraction were prepared in MEA medium. Control media was without plant extract. Three replicates were made for each concentration and fungal inoculums were added in each plate of each concentration (Jabeen et al., 2013).

n-hexane fraction was selected for GC-MS analysis due to significant antifungal potential in the previous assay. n-hexane fraction was filtered with a nylon membrane filter (0.22 µm pore size and 47 mm diameter). Sample was analyzed by a GC-MS-QP2010 chromatograph and was separated on an DM-5MS capillary column (30 m, 0.25 mm, 0.25 µm) by applying the following temperature program 50 oC for 5 min, 40-70 oC at 2 oC min-1, 70 oC for 2 min, 70-120 oC at 3 oC min-1, 120-150 oC at 5 oC min-1, 150-220 oC at 10 oC min-1 and then 220 oC for 2 min. Helium was used as a carrier gas. The injector and detector temperatures were 200 oC and 250 oC respectively. Mass detector conditions were: ionization voltage 70 eV mass scanning range m/z 29-540 and source temperature 230 oC. The percentage composition of volatile compounds was computed from GC peak areas. Qualitative analysis was based on a comparison of retention times, indices and mass spectra with the corresponding data in the literature (NIST Library 2010 word software) (Sherazi et al., 2016)

All the data were statistically analyzed by using analysis of variance (ANOVA) followed by Duncan’s Multiple Range test (DMR) at Pd”0.05 (Steel et al., 1997).

RESULT AND DISCUSSION

In the present study, fruits, leaves and stem of L. camara were examined as a natural alternative of synthetic fungicide for the control of C. gleosporioides. Phytochemical analysis of L. camara was also performed instead of evaluated. The phytochemical analysis of L. camara extracts showed the presence of different secondary metabolites, like alkaloids, phenols, flavonoids, glycosides, tannins and terpenoids (Table 1). Bhakta and Ganjewala (2009) reported that flavonoids, carbohydrates, proteins, alkaloids, glycosides, saponins, steroids, triterpenes and tannin are the major phytochemicals groups present in L. camara. The presence of these compounds might be responsible for its antifungal activity as strengthened by literature. The flavones extracted from the methanol extract of dried leaves of L. camara also showed the antibacterial and antifungal properties (Boughalleb et al., 2005; Gujar and Talwankar, 2012).

Table 1
Phytochemical analysis of leaves, stem and fruit of Lantana camara

Phytochemical tests	L. camara seaves	L. camara stem	L. camara fruit
Tannins	+ve	+ve	+ve
Phlobtannins	-ve	-ve	-ve
Saponin	+ve	+ve	+ve
Flavonoids	-ve	+ve	+ve
Terpenoids	+ve	+ve	+ve
Glycoside	+ve	-ve	-ve
Alkaloids	+ve	+ve	-ve
Coumarins	-ve	-ve	-ve

• (+ve) present (-ve) absent.

Antifungal potential of fruits, stem and leaves of L. camara was assessed against C. gloeosporioides. Among all the applied extracts methanolic fruit extract of the test plant significantly inhibited the radial growth of C. gloeosporioides. Results showed that 5% concentration of methanolic fruit extract exhibited maximum inhibition (66%). While other concentrations 1%, 2% 3% and 4% also significantly reduced the test fungal growth up to 32-63% (Figure 1A). Stem methanolic extract of L. camara also significantly reduced the radial diameter of C. gloeosporioides. However, maximum inhibition was observed in 5% extract i.e. 29% as compared to control. Other applied concentration 1%, 2%, 3% and 4% were found comparatively less effective and retarded the growth of C. gloeosporioides up to 13-28% (Figure 1B). All the applied concentrations (1-5%) of methanolic extract of leaves of L. camara significantly retarded the growth of C. gloeosporioides. The maximum antifungal activity was shown by 5% leaves extract which caused 50% inhibition in the radial growth of test fungus as compared to control treatment. Other concentrations 1% - 4% caused 22% - 31% decline in the test fungal colony growth (Figure 1C). Similarly, Singh and Srivastava (2012) reported that L. camara leaves, likely possess biofungicidal activity against Alternaria alternata. Various literature reports also suggested that L. camara leaves displayed substantial antifungal properties (Boughalleb et al., 2005; Saraf et al., 2011; Gujar and Talwankar, 2012).

Figure 1
In vitro effect of methanolic fruit, stem and leaf extracts of Lantan camara on growth of Colletotrichum gloeosporioides.

The fruit methanolic extract was found to be highly effective in antifungal bioassay so this plant part was used in bioassay guided fractionation and further partitioned by using various organic solvents. Four organic fractions viz. n-hexane, chloroform, ethyl acetate and n-butanol were isolated. The bioactivity of all the isolated organic fractions and fungicide metalaxyl mancozeb was tested against C. gloeosporioides. The n-hexane fraction of fruit of L. camara showed the best results as its lowest applied concentration 0.05% inhibited the growth of test fungus 45% (Figure 2).

Figure 2
In vitro effect of different organic solvent fractions of fruit of Lantana camara extract on growth of Colletotrichum gloeosporioides.

The GC-MS analysis of n-hexane fraction of L. camara was performed and 16 bioactive compounds were identified. The identified compounds were Glutaraldehyde, 2- Decenal (E), 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, (R), 2-pentadecyn-1-ol, cis-α-Bergamotene, Benzene, (1,1-dimethyl butyl)-(1,1-dimethyl butyl)benzene, 2-Methyl-2-phenylpentane, 4-Hexadecen-6-yne, (z), Turmerone 2-Methyl-6-(4-methyl-1,3-cyclohexadien-1-yl)-2-hepten-4-one, Curlone, Hexadecanoic acid, 15-methyl-,methyl ester, Octadecanoic acid, 6-Methyl-2-tridecanone, Heptadecanoic acid, Nonadecane, 2-methyl-, Tetradecanoic acid and Pentadecanoic acid, 14-methyl-,methyl ester (Table 2). One of the major compound cyclopropane identified in the present study has known fungicidal activity against the rice blast disease caused by P. oryzae (Cartwright et al., 1977). A 5-Heptenoic acid compound also contains antifungal activity against Myrothecium verrucaria and Trichoderma viride (Gershon and Shanks, 1978). Similarly, the compound Glutaraldehyde i.e. is a dialdehyde also displayed bactericidal, fungicidal, mycobactericidal and sporicidal activity (Russell, 1994). Glutaraldehyde, a fungicide, is proved to be effective against the saprophytic fungi has been identified in GC-MS analysis of n-hexane fraction in the present study. 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7atrimethyl, a bioactive compound also possessed antifungal properties has also been identified in this GC-MS analysis. Curlone, a sesquiterpene compound and essential oil of turmeric, has been found in the current study of n-hexane fraction during GC-MS analysis. Previously Kumar et al. (2016) studied the antifungal properties of curlone (essential oil). Omoruyi et al., 2014 reported that n-hexadecanoic acid (Palmitic acid) which was identified in GC-MS analysis of n-hexane of L. camara fruit possesses antimicrobial properties. Heptadecanoic acid is an unsaturated fatty acid which has also been identified in the present study has antifungal potential against many fungal strains (Agoramoorthy et al., 2007). However, in the present study, the n-hexane extract of the fruit of L. camara is specifically tested against the anthracnose causing fungus C. gloeosporioides. So, the results of the present study depicted that L. camara fruit contains potential antifungal constituents against C. gloeosporioides. Any of these identified compounds alone or in combination might be responsible for the strong fungicidal potential of Lantana camara. These compounds can be exploited in future for the synthesis of nature-friendly antifungal compounds against C. gloeosporioides.

Table 2
GC-MS analysis of n-Hexane fraction of Lantana camara fruit

Sr#	R. time	Compound	Molecular formula	Mol. weight	Peak area
1.	6.650	Glutaraldehyde	C5H8O2	100	4.02
2.	29.120	2- Decenal (E)	C10H18O	154	0.89
3.	40.880	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl, (R)	C11H16O2	180	2.23
4.	42.525	2-pentadecyn-1-ol	C15H28	224	2.67
5.	42.658	cis-α-Bergamotene	C15H24	204	2.23
6.	43.092	Benzene, (1,1-dimethylbutyl)-(1,1-dimethylbutyl)benzene,2-Methyl-2-phenylpentane	C12H18	162	8.03
7.	44.258	4-Hexadecen-6-yne, (z)	C16H28	220	3.57
8.	44.792	Turmerone 2-Methyl-6-(4-methyl-1,3-cyclohexadien-1-yl)-2-hepten-4-one	C15H22O	218	8.48
9.	45.517	Curlone	C15H22O	218	3.12
10.	45.925	Hexadecanoic acid, 15-methyl-,methyl ester	C18H36O2	284	6.25
11.	46.750	Octadecanoic acid	C18H36O2	284	8.03
12.	47.770	6-Methyl-2-tridecanone	C14H28O	212	3.13
13.	48.090	Heptadeanoic acid	C17H34O2	270	3.13
14.	48.500	Nonadecane, 2-methyl-	C20H42	282	2.23
15.	49.850	Tetradecanoic acid	C14H28O2	228	21.00
16.	48.870	Pentadecanoic acid, 14-methyl-,methyl ester	C17H34O2	270	21.00

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Publication Dates

• Publication in this collection
  02 Dec 2019
• Date of issue
  2019

History

• Received
  28 Mar 2018
• Accepted
  07 June 2018