*Corresponding Author:
E. S. J. Nidiry
ICAR-Indian Institute of Horticultural Research,
Bengaluru,
Karnataka 560089,
India
E-mail: nidiry@yahoo.co.in
Date of Received 01 March 2020
Date of Revision 24 August 2021
Date of Acceptance 22 February 2022
Indian J Pharm Sci 2022;84(1):224-227  

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Abstract

In vitro antifungal activity of thirty two extractives of seven plants obtained using hexane, ethyl acetate and methanol were tested against the mycelial growth of Colletotrichum gloeosporioides. Among these extractives, ethyl acetate extractives of onion seeds, chilli seeds and chilli pericarps and methanol extractives of chilli seeds and chilli pericarps exhibited more than 20 % mycelial growth inhibition at a concentration of 0.5 % level. Methanol extractives of Solanum viarum berries and ethyl acetate extractive of soybean leaves exhibited more than 20 % mycelial growth inhibition at a concentration of 1.0 % level. Thin layer chromatographic bioautography showed that hexane and ethyl acetate extractives of onion seeds, chilli seeds and chilli pericarps exhibited spore germination inhibition of Cladosporium cucumerinum at a dose of 1.0 mg.

Keywords

Antifungal activity, plant extractives, Colletotrichum gloeosporioides, Cladosporium cucumerinum

Investigations regarding the antifungal activity of plant extractives are important because they give insight into the defence mechanism evolved by plants against the attack of fungal pathogens. Such plant extractives and compounds with antifungal activity can be used against human, animal and plant pathogens and for post-harvest preservation, provided they possess suitable properties and sufficient amounts can be extracted from natural sources. Alternatively, structure elucidation of natural antifungal compounds helps in the synthesis of the compounds per se or analogues with improved physical and biological properties. Quantitative evaluation of such compounds in crop plants can facilitate the screening of plants for the development of resistant varieties.

In this paper the evaluation of thirty-two extractives of plant materials namely onion seeds, onion leaves, Gloriosa superba seeds, Bougainvillea spectabilis (green leaves, white bracts and pink bracts), chilli seeds, chilli pericarps, Solanum viarum berries Albizia amara leaves and soybean leaves obtained using hexane, ethyl acetate and methanol for their antifungal activity against the mycelial growth of Colletotrichum gloeosporioides is being discussed. Evaluation of the extractives of onion seeds, chilli seeds and chilli pericarps against the spore germination of Cladosporium cucumerinum by Thin Layer Chromatography (TLC) bioautography is also being discussed.

The plant materials except soybean leaves were collected from the farms of ICAR-Indian Institute of Horticultural Research (IIHR), Hessaraghatta, Bangalore, India or purchased from the local market. Soybean leaves were obtained from ICAR-Indian Institute of Soybean Research, Indore, Madhya Pradesh, India. The plant materials were dried at 60° and subsequently powdered.

The powdered plant materials were extracted using a Soxhlet apparatus first with hexane, then with ethyl acetate and finally with methanol. In the case of Gloriosa superba, methanol extraction was done immediately after hexane extraction. The respective extractives were obtained by completely distilling out the solvents on a water bath and were tested for antifungal activity.

Colletotrichum gloeosporioides ITCC 4573 was used for the poisoned food technique and Cladosporium cucumerinum IMI 249540 was used for TLC bioautography. Antifungal activity of hexane, ethyl acetate and methanol extractives was studied by observing the mycelial growth inhibition of Colletotrichum gloeosporioides by poisoned-food technique[1]. Per cent mycelial growth inhibition was calculated using the formula P=(C-T/C)×100, where C is the mycelial diameter of the control and T that of treated ones, after giving due adjustment of the mycelial diameter of the inoculum. The values given in Table 1 are the average of two replications. For TLC bioautography, conidial suspension of Cladosporium cucumerinum in the nutrient solution suggested by Homan and Fuchs[2].

Plant Plant part Solvent used for preparation of extractive Concentration (%) Percent mycelial growth inhibition
Onion (Allium cepa) Seeds Hexane 0.5 0
Onion (Allium cepa) Seeds Ethyl acetate 0.5 22.5
Onion (Allium cepa) Seeds Methanol 0.5 10
Onion (Allium cepa) Leaves Hexane 1 0
Onion (Allium cepa) Leaves Ethyl acetate 1 10
Onion (Allium cepa) Leaves Methanol 1 6
Chilli (Capsicum annuum) Seeds Hexane 1 0
Chilli (Capsicum annuum) Seeds Ethyl acetate 0.5 46.4
Chilli (Capsicum annuum) Seeds Ethyl acetate 1 54.6
Chilli (Capsicum annuum) Seeds Methanol 0.5 25
Chilli (Capsicum annuum) Seeds Methanol 1 38.1
Chilli (Capsicum annuum) Pericarps Hexane 1 0
Chilli (Capsicum annuum) Pericarps Ethyl acetate 0.5 21.4
Chilli (Capsicum annuum) Pericarps Methanol 0.5 20.7
Chilli (Capsicum annuum) Pericarps Methanol 1 43.6
Gloriosa superba Seeds Hexane 0.5 0
Gloriosa superba Seeds Methanol 0.5 0
Bougainville spectabilis Green leaves Hexane 0.5 0
Bougainville spectabilis Green leaves Ethyl acetate 0.3 10
Bougainville spectabilis Green leaves Methanol 0.5 0
Bougainville spectabilis White bract Hexane 0.5 0
Bougainville spectabilis White bract Ethyl acetate 0.3 7
Bougainville spectabilis White bract Methanol 0.5 0
Bougainville spectabilis Pink bract Hexane 0.5 0
Bougainville spectabilis Pink bract Ethyl acetate 0.3 7
Bougainville spectabilis Pink bract Methanol 0.5 0
Solanum viarum Berries Hexane 1 0
Solanum viarum Berries Ethyl acetate 1 31.2
Solanum viarum Berries Methanol 1 31.2
Albizzia amara Leaves Hexane 1 0
Albizzia amara Leaves Ethyl acetate 1 0
Albizzia amara Leaves Methanol 1 0
Soybean (Glycine max) Leaves Hexane 1 0
Soybean (Glycine max) Leaves Ethyl acetate 1 25
Soybean (Glycine max) Leaves Methanol 1 20

Table 1: Antifungal Activity of Plant Extractives For The Mycelial Growth Inhibition of Colletotrichum gloeosporioides

The results of in vitro antifungal activity of thiry-two extractives of seven plants against the mycelial growth of Colletotrichum gloeosporioides are presented in Table 1. Among these extractives, ethyl acetate extractives of onion seeds, chilli seeds and chilli pericarps and methanol extractives of chilli seeds and chilli pericarps exhibited more than 20 % mycelial growth inhibition at a concentration of 0.5 % level. Methanol extractives of Solanum viarum berries and ethyl acetate extractive of soybean leaves exhibited more than 20 % mycelial growth inhibition at a concentration of 1.0 % level. Hexane extractives of onion seeds, chilli seeds and chilli pericarps did not show activity against the mycelial growth of Colletotrichum gloeosporioides. The extractives of onion leaves, Albizia amara leaves, Bougainvillea spectabilis (green leaves, white bracts and pink bracts) and seeds of Gloriosa superba also did not show any significant activity.

Chemical nature of some antifungal compounds present in the presently investigated plants has been reported by earlier workers. An antimicrobial protein has already been isolated from onion seeds[3]. But it is doubtful whether the antifungal activity of ethyl acetate extractive presently reported is due to this protein since ethyl acetate extractive has only moderate polarity. Antifungal activity of capsaicin present in chilli has been established[4]. Antifungal activity of glycoalkaloids of Solanum viarum has been reported[5]. Antifungal activity of isoflavones[7] in soybean leaves and glyceollins[6] in infected soybean leaves also has been reported.

The results presented in Table 1 shows that the hexane extractives of onion seeds, chilli seeds and chilli pericarps do not show activity against the mycelial growth of Colletotrichum gloeosporioides even at a high concentration of 1 % level. But Table 2 shows that these extractives show activity against the spore germination of Cladosporium cucumerinum on TLC plate. A similar trend was observed in the earlier studies reported from this institute in the cases of watermelon seeds and tomato seeds[8,9]. Most probably the fatty acid esters present in the hexane extractives of these vegetable seeds (watermelon, tomato, onion and chilli) have an antagonistic effect on the antifungal compounds as far as mycelial growth inhibition of Colletotrichum gloeosporioides is concerned. But this antagonistic effect of fatty acid esters is not effective in case of activity against the spore germination of Cladosporium cucumerinum. It is noteworthy that the ethyl acetate extractives of onion seeds, chilli seeds and chilli pericarps exhibit activity against the mycelial growth of Colletotrichum gloeosporioides and spore germination of Cladosporium cucumerinum on TLC plate. Direct bioautography of the methanol extractives of onion seeds, chilli seeds and chilli pericarps on TLC plate without prior elution with ethyl acetate did not exhibit any inhibition. This is most probably due to the presence of some carbohydrates or peptides present in the methanol extractives which promote the growth of Cladosporium cucumerinum. But if TLC plates spotted with methanol extractives are previously eluted with ethyl acetate and subsequently sprayed with the inoculum, inhibition zones are seen upon incubation.

Plant Plant part Solvent used for preparation of the extractive Inhibition at a dose of 1.0 mg (Direct bioautography without prior elution) Rf value of inhibition spot after elution with EtOAc
Onion (Allium cepa) Seeds Hexane + 0.49
Onion (Allium cepa) Seeds Ethyl acetate + 0.49
Onion (Allium cepa) Seeds Methanol - 0.79
Chilli (Capsicum annuum) Seeds Hexane ++ 0.72
Chilli (Capsicum annuum) Seeds Ethyl acetate + 0.62, 0.72
Chilli (Capsicum annuum) Seeds Methanol - 0.72 (faint)
Chilli (Capsicum annuum) Pericarps Hexane ++ 0.72
Chilli (Capsicum annuum) Pericarps Ethyl acetate + 0.62, 0.72
Chilli (Capsicum annuum) Pericarps Methanol - 0.72 (faint)

Table 2: Antifungal Activity of Extractives of Onion Seeds, Chilli Seeds and Chilli Pericarps Against The Spore Germination of Cladosporium cucumerinum By TLC Bioautography

References