EVALUATION OF ANTIFUNGAL PROPERTY AND PHYTOCHEMICAL ANALYSIS OF UNDEREXPLOITED LEAFY VEGETABLES

EVALUATION OF ANTIFUNGAL PROPERTY AND PHYTOCHEMICAL ANALYSIS OF UNDEREXPLOITED LEAFY VEGETABLES

Sona Peter *, P. Sainamole Kurian and S. Karthika Menon

Department of Botany, Vimala College, Thrissur, Kerala, India.

ABSTRACT: The adverse environmental impact of fungicides used in plant disease control is escalating day by day. Generally, plant diseases are controlled by synthetic fungicides, chemical methods, and bio-control agents. Chemical methods lead to deterioration of soil fertility and cause pollution. Being highly stable and resistant to biodegradation, chemical fungicides also enter the food chain and are responsible for many harmful effects on human health and the environment. Hence, eco-friendly alternative methods are of great urgency. One of the prominent approaches in this direction includes utilizing plant extracts with natural antifungal constituents. So, a study was attempted to determine the antifungal and phytochemical activity of underexploited leafy vegetables. Methanol and water extracts of the leaves were tested against two economically important plant pathogens Alternaria solani and Pythium aphanidermatum and the antifungal activity of plant extracts was determined. All the plant species under study showed inhibitory activity against the plant pathogens. However, B. diffusa and C. frutescens exhibited maximized efficiencies. Moreover, the water extracts of B. diffusa and C. frutescens (1%) were tested against Phytophthora fruit rot of Brinjal under open field conditions. Reduced disease severity was observed in C. frutescens water extract. Phytochemical analysis revealed that alkaloids, carbohydrates and flavonoids are the active components present in the water and methanol leaf extracts; among them, saponins, tannins, alkaloids, flavonoids and terpenoids are major components responsible for antifungal activity. Results of the present study indicate that plant extracts can be effectively used as a botanical fungicide as an alternative management method to tackle plant disease.

Keywords: Antifungal activity, Poisoned food technique, Accelerated solvent extraction and phytochemical analysis

INTRODUCTION: The annual crop losses of the world as a result of plant diseases have been increasing day by day, in which fungal disease contributes a major share ¹.  Existing control measures are not enough to deal with the emergence of outbreaks of plant fungal diseases.

Chemical methods lead to deterioration of soil fertility and cause pollution. Being highly stable and resistant to biodegradation, chemical fungicides also enter the food chain and are responsible for many harmful effects on human health and the environment.

Therefore, the focus is shifting towards alternative strategies for the control of fungal diseases of plants. Based on the knowledge that plants have recently developed their defense against fungal pathogens ² in different parts of the world, attention has been paid to the exploitation of higher plant products as novel chemotherapeutics in plant protection. Similarly, traditional medicinal knowledge on the use of plant extracts for the treatment of fungal infection has also increased the interest of researchers all over the world for developing an alternative control strategy to reduce dependency on synthetic fungicides. Many workers have tried extracts of medicinal plants for plant disease management, but they are costly and less available. However, the demand for economically viable and environmentally safe strategies for plant disease control has amplified the use of botanical fungicides. In this context, biodegradable materials like fresh plant leaf extracts serve as suitable alternatives.

Plants can produce secondary metabolites like phenols, phenolic acids, quinones, flavones, flavonoids flavonols, tannins, coumarins, carvacrol, eugenol, and thymol which were noted to be highly active against the pathogen. These groups of compounds exhibit antimicrobial activity and serve as plant defense mechanisms against pathogenic microorganisms. Vegetables contain a wide variety of biologically active, non-nutritive compounds known as phytochemicals ³. Antifungal substances obtained from plants have no side effect on the environment, thus giving a significant advantage. So, we mainly consider the leaf of underexploited vegetables, which serve as suitable alternatives for fungal infection. These are inexpensive and widely available compared to medicinal plants.

Antifungal agents based on natural products have always been promising in the control of fungi. Moreover, these agents are not toxic and are decomposed easily. Complete elimination of chemical fungicides for controlling plant diseases in modern agriculture may be impossible, but a logical reduction in their application is feasible. There is no doubt that the use of biological control agents is one of the safest solutions for disease control and it leads us towards a sustainable agricultural system in near future. The battle to protect plant health is ongoing, and plant disease management is essential for our continued ability to feed a growing human population. Numerous works of literature have highlighted the ability of the under-exploited vegetables to control fungal diseases ^{4, 5, 6} effectively. Among them, some of the underexploited leafy vegetables include Boerhavia diffusa, Capsicum frutescens, Cnidoscolus aconitifolius, Sauropus androgynus, Talinum portulacifolium, Moringa oleifera, Basella alba, Pisonia grandis, Fleurya interrupta, Coccinia grandis. In this light, the present study is an attempt to investigate the phytochemical and antifungal effects of selected underexploited leafy vegetables using two important plant pathogens namely Alternaria solani and Pythium aphanidermatum test organisms.

MATERIALS AND METHODS: Healthy, disease-free plant leaves were collected separately and brought to the laboratory, and washed thoroughly under the tap water to remove dirt. The leaves were then dried under shade for two to three days until the weight was reduced to half. Extract the leaf (10%) was prepared by soaking 1.5 g of powdered dry leaves in 15 ml distilled water or methanol.  It was then mixed thoroughly and kept undisturbed for 24 hrs at room temperature. The supernatant was filtered through Whatman No. 1 filter paper, and the filtrate was preserved. Accelerated solvent extraction was also carried out.

In-vitro Evaluation by Poisoned Food Technique: The sterilized, poisoned PDA was melted and cooled to 40 °C. PDA devoid of the leaf extract but with the same amount of distilled water or methanol was plated to serve as the control. Mycelial disc (5 mm) taken from the seven-day-old culture of P. aphanidermatum and A. solani was placed on the center of the plates and incubated at 27+/-2 °C. Mycelial growth was measured daily till there was full growth in control. The percent inhibition (PI) was calculated using the formula given by Vincent ⁷:

PI = C – T × 100/ T

PI = percent inhibition, C= Growth of the pathogen in control (mm), T = Growth of pathogen in treatment (mm).

In-vivo Evaluation of Leaf Extracts against Phytophthora Fruit rot of Brinjal: The field experiment for managing fruit rot of brinjal caused by phytophthora sps was carried out superimposing the treatment on an existing brinjal field with Surya variety. The experimentation was designed using randomized block design and with three treatments and seven replications, in which each plot consists of three Brinjal. The spacing between each plant is 60 cm × 45 cm. The experiment was carried out from August to October 2019.

Preliminary Phytochemical Screening of Vegetable Leaves: Phytochemical analysis of water and methanol extracts was conducted to identify the presence of carbohydrates, alkaloids, saponins, phenol, tannins, sterols, proteins, reducing sugar, terpenoids ⁸ and flavonoids ⁹.

RESULTS: The leaves were dried at 50 °C to constant dry weight. Fresh weight and percent of the dry weight of leaves were calculated. The percent dry weight varies from seven to 50 percent, and it was highest in M. oleifera and the lowest in B. alba. Leaves with more water content showed less percent of dry weight.

The percent dry weight of leaves such as B. diffusa, C. frutescens, C. aconitifolius, S. androgynus, T. portulacifolium, P. grandis, F. interrupta and C. grandis varies between nine to 34 per cent. The moisture content of leaves has a direct relationship with antifungal property ¹⁰. In the present study highest moisture content was observed in B. alba followed by T. portulacifolium.

In-vitro Evaluation of Leaf Extracts using Poison Food Technique: All plant species showed significant inhibitory activities in both water and methanol extracts. However, B. diffusa and C. frutescens were noted to be superior. In water extract, percent inhibition varied from 84 (B. diffusa) to 0 (F. interrupta and S. androgynous) against P. aphanidermatum, and in the case of A. solani, more than 80 percent inhibition was exhibited by leaf extract of T. portulacifolium, C. frutescens, C. grandis, B. alba, P. grandis and B. diffusa.  Whereas, the cent per cent inhibition of P. aphanidermatum was recorded in methanol extract of C. frutescens, C. grandis, T. portulacifolium, B. alba, P. grandis and F. interrupta.  In the case of A. solani more than 80 per cent inhibition was recorded by methanol extract of T. portulacifolium, S. androgynous, M. oleifera, C. frutescens, C. grandis, B. alba, P. grandis, C. aconitifolius and B. diffusa. The results are depicted in Fig. 1 and 2.

Leaves of plants that showed more than 80 per cent inhibition in water and methanol extracts viz., C. frutescens, C. grandis, B.alba and T. portulacifolium were used for accelerated solvent extraction study. When subjected to accelerated solvent extraction, in methanol, the per cent inhibition of A. solani increased slightly (up to 87 per cent) in T. portulacifolium.

However, in the case of C. frutescens, the per cent inhibition decreased to 41 against A. solani and there was no inhibition in P. aphanidermatum. The reduction in inhibition may be due to the degradation of some of the secondary metabolites in high temperatures (200 psi), to which samples were exposed during the extraction. The details are furnished in Fig. 3. Accelerated solvent extraction offers a better alternative to several thermal extraction methods in terms of less solvent consumption, shorter operational time, high recoveries, good reproducibility, cost-effectiveness and minimal sample manipulation for the extraction process.

FIG. 1:  IN-VITRO EVALUATION OF LEAF EXTRACTS AGAINST ALTERNARIA SOLANI                    

FIG. 2:  IN-VITRO EVALUATION OF LEAF EXTRACTS AGAINST PYTHIUM APHANIDERMATUM

FIG. 3:  IN-VITRO EVALUATION OF ACCELERATED SOLVENT EXTRACT OF VEGETABLE LEAVES AGAINST PYTHIUM APHANIDERMATUM AND ALTERNARIA SOLANI IN METHANOL EXTRACT

In-vivo Evaluation of Leaf Extracts against Phytophthora Fruit Rot of Brinjal: The water extracts of B. diffusa and C. frutescens (1%) were tested against Phytophthora fruit rot of Brinjal under open field conditions. The lowest disease severity was observed in C. frutescens water extract; this was followed by B. diffusa. The details are furnished in Fig. 4.

FIG. 4:  EFFECT OF PLANT EXTRACTS ON PHYTOPHTHORA FRUIT ROT OF BRINJAL

Preliminary phytochemical analysis of vegetable leaves: The selected ten vegetable plants are the source of the secondary metabolites i.e., alkaloids, flavonoids, terpenoids and reducing sugars. Leafy vegetables play a vital role in preventing various diseases. Leafy vegetables play a vital role in preventing various diseases. The antidiuretic, anti-inflammatory, antianalgesic, anticancer, anti-viral, anti-malarial, anti-bacterial and anti-fungal activities are due to the presence of the above-mentioned secondary metabolites. Carbohydrates, alkaloids and flavonoids are the common compounds present in all plants. Among them, saponins, tannins, alkaloids, flavonoids and terpenoids are major components responsible for antifungal activity shown in Tables 1 and 2. Water extraction of C. grandis contain Carbohydrate (+++), protein (+), amino acid (+), Flavonoid (++), Alkaloids (+++) and steroids (++). Whereas in methanol extract it contains carbohydrate (+++), protein (+), amino acids (+), alkaloids (+++), flavonoids (++) and steroids (++) are present. Carbohydrate and alkaloids are higher in both water and methanol extracts, while amino acids and proteins are only seen in methanol extract. Kaviya and Shukla ¹¹ found that carbohydrates were absent in methanol extract, and water phenol, tannin, and saponins were present. In water and methanol extract of C. frutescens, the concentration of carbohydrate (+++), alkaloids (+++), flavonoids (+++) and steroids (+++) are higher. Saponin (+) is only present in methanol extract of C. frutescens.  The most abundant and pungent constituents of chilies, capsaicinoid, belong to the alkaloid class. Gurnani et al. ¹² found that alkaloids, flavonoids, polyphenols, steroids, and tannins were observed in methanolic extract of C. frutescens. A similar result was found in Koffi-Nevry et al. ¹³ and Vinayaka et al. ¹⁴. Roa et al. ¹⁵ found that in C. annuum, flavonoids, saponins, tannins, and phenols were absent. portulacifolium contain carbohydrate (+++), protein (+), flavonoids (++), alkaloids (+++), steroids (++), phenol (+), tannins (+) and saponins (++) in water extracts. Phenols and tannin were only present in water extracts of T. portulacifolium. Saponin was moderately present in T. portulacifolium were as in C. frutescens, B. alba, S. androgynous and F. interrupta extracts their presence is very low in concentration. Whereas in methanol extract carbohydrate (++), alkaloids (++), flavonoids (+), and steroids (+) are mainly present. Aja ¹⁶ showed the same result that T. portulacifolium possesses alkaloids, flavonoids, phenol, tannin, and saponin in the water extract. In C. aconitifolius carbohydrate (++), alkaloids (++), flavonoids (++), steroids (++), and triterpenoids (+) are the main compounds existing in methanol extract.

In water extract, it contains the same component as in methanol, but their concentration was high they are carbohydrate (+++) and alkaloids (+++), flavonoids (+++), steroids (+++) the additional compound are protein (++) and amino acid (+) while triterpenoids were absent. Flavonoids are the main component in C.aconitifolius leaves extract exhibited a wide range of biological activities like antimicrobial, anti-inflammatory, analgesic, anti-allergic, cytostatic, and antioxidant properties ¹⁷.

In water extraction of B.alba it contain carbohydrate (+++), alkaloids (+++), flavonoids (++), steroids (++) and saponnins (+). B. alba is only one species that contain triterpenoids (++), but in methanol extract, it is present but in moderate concentration. In methanol extract it contain carbohydrate (+), alkaloids (+++), flavonoids (++), steroids (++) and triterpenoids (++). Murakami ¹⁸ and Maisuthisakul ¹⁹ reveal that B. alba contains basellasaponins and peptide, phenolic compounds.

In M. oleifera, carbohydrates (+) and alkaloid (+) are the compound present in methanol extract. But in water extracts, the carbohydrate (+++) alkaloid (+++) and flavonoid (++) are mainly present.

In leaf extract of S. androgynous alkaloids (+++), saponins (+), steroids (++), carbohydrates (+), and flavonoids (++) are major compounds in the water extract. The same result was found by Hegde and Divya ²⁰. In methanol extract, the component was the same as in water, but their concentration was low.

In F. interrupta the amount of secondary metabolite is very low i.e., in methanol, it contains only carbohydrate (+) and alkaloids (++), whereas in water, it contains carbohydrate (+), alkaloids (+++), flavonoids (++), steroids (+++) and saponins (+) were present. Thamizh Selvam et al. ²¹ found the same result that the amount of alkaloids is very low in methanol extract.

Water extract of P. alba contain carbohydrate (++), alkaloids (++), flavonoids (++), steroids (+). Whereas protein, amino acid, phenol, tannins, triterpenoids and saponins remain completely absent.

In methanol extract, it contains only carbohydrate (+), flavonoids (+) and steroids (+). Poongothai and Shubashini ²² have reported that the methanol extract of P. alba leaf possesses flavonoids and resins, but tannins, alkaloids, terpenoids, glycosides, saponins, and proteins were absent.

In B. diffusa water extract, the key component was carbohydrate (+), protein (+), amino acids (+), alkaloids (+++), flavonoids (+), triterpenoids (+) and steroids (++). A vital component is the presence of alkaloids. The results were different in the case of methanol extract it comprises only the presence of carbohydrate (++) all others are absent.

TABLE 1: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF VEGETABLE LEAVES USING WATER EXTRACT

* ‘+ ’low, ‘++’ average, ‘+++’high,   ‘–’ nil

TABLE 2: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF VEGETABLE LEAVES USING METHANOL EXTRACT

* ‘+ ’low, ‘++’ average, ‘+++’high, ‘ –’ nil

DISCUSSION: Results of the study suggest that leaf extracts of vegetables can be potential botanical fungicides. The organic solvent dissolves more in bioactive compounds and makes it available for antifungal action, as reflected by the enhanced inhibition of pathogens by methanol extract compared to water extract. The laboratory observations could be reflected under field conditions as well.

The major bioactive components in vegetable leaf extract belong to alkaloids, flavonoids, steroids, and saponins. Phytochemical analysis revealed that antifungal action by plant secondary metabolites is rather complex. Variable efficacy recorded by accelerated solvent extracts may be due to the variable heat sensitivity of the biochemical compounds present in leaves.

There is a correlation between the kind and content of secondary metabolites and the antifungal action of the leaf extract. However, further studies and analyses are needed to decipher the specific and synergistic effects of different subgroups of compounds and to identify novel compounds and activities.

CONCLUSION: There were several reports regarding utilizing plant-based products as alternatives for insecticides; however, their application as fungicides is scanty. Complete elimination of chemical fungicides for controlling plant diseases in modern agriculture may be impossible, but a logical reduction in their application is feasible. There is no doubt that botanical fungicides from the leaves of vegetables could be the cheapest and safest solution against plant diseases shortly.

ACKNOWLEDGEMENT: The authors are grateful to the Department of Plant Pathology, Department of vegetable Science and Department of Soil Science and Agricultural Chemistry College of Horticulture, Kerala Agricultural University (KAU), Vellanikkara and DST – FIST Laboratory, Vimala College (Autonomous), Thrissur and Central Instruments Laboratory (CIL) Kerala Veterinary and Animal Science University (KVASU) Mannuthy for providing institutional facilities.

CONFLICTS OF INTEREST: There is no conflicts of interest among the authors.

REFERENCE:

1. Fisher MC, Henk DA, Briggs CJ, Brownstein LC, Madoff LC, McCraw SL and Gurr SJ: Emerging fungal threats to animal, plant and ecosystem 590 health. Nature 2012; 484: 186-194.
2. Gurgel LA, Sidrim JJC, Martins DT, Cechinel Filho V and Rao VS: In-vitro antifungal activity of dragon's blood from Croton urucurana against dermatophytes. Journal of Ethnopharmacology 2005; 97: 409-412.
3. Gupta S and Prakash J: Studies on Indian green leafy vegetables for their antioxidant activity. Plant Foods for Human Nutrition 2009; 64: 39-45.
4. Farooq MA, Iqbal U, Iqbal SM, Afzal R and Rasool A: In-vitro evaluation of different plant extracts on mycelial growth of Sclerotium rolfsii the cause of root rot of sugar beet. Mycopath 2010; 8: 81-84.
5. Nunez YO, Salabarria S, Collado IG and Hernandez-Galan Rosario: Antifungal activity of extracts and terpene constituents of aerial parts of Juniperus lucayana. The Revista Latinoamericana de Quimica 2010; 38: 145-152.
6. Gupta SK and Tripathi SC: Fungitoxic activity of Solanum torvum against Fusarium sacchari. Plant Protection Science 2011; 47: 83-91.
7. Vincent JM: Distortion of fungal hyphae in the presence of certain inhibitors. Nature 1947; 159: 850-850.
8. Kokate CK, Purohit AP and Gokhale SB: Pharmacognosy. Nirali Prakashan 2005; 619.
9. Harborne JB: Phenolic compounds. In Phytochemical Methods 1973; 33-88.
10. Ogle M, Ha Thi Anh Dao, Generose Mulokozi and Leif Hambraeus B: Micronutrient composition and nutritional importance of gathered vegetables in Vietnam. Inter J of Food Sciences and Nutrition 2001; 52: 485-499.
11. Kaviya K and Shukla P: Physiochemical analysis and phytochemical screening of ivy gourd, Coccinia grandis (L.) Voigt leaves. Journal of Pharmacognosy and Phytochemistry 2019; 8: 1091-1094.
12. Gurnani N, Gupta M, Mehta D and Mehta BK: Chemical composition, total phenolic and flavonoid contents, and in vitro antimicrobial and antioxidant activities of crude extracts from red chilli seeds (Capsicum frutescens). J of Taibah University for Science 2016; 10: 462-470.
13. Koffi-Nevry R, Kouassi KC, Nanga ZY, Koussemon M and Loukou GY: Antibacterial activity of two bell pepper extracts: Capsicum annuum and Capsicum frutescens. International journal of food properties 2012; 15: 961-971.
14. Vinayaka KS, Prashith-Kekuda TR., Nandini KC, Rakshitha MN, Ramya M, Shruthi J and Anitha B: Potent insecticidal activity of fruits and leaves of Capsicum frutescens (L.) var. longa (Solanaceae). Der Pharm Lett 2010; 2: 172-6.
15. Rao P, Barik SK, Pandey HN and Tripathi RS: Community composition and tree population structure in a sub-tropical broad-leaved forest along a disturbance gradient. Vegetatio 1990; 88: 151-162.
16. Aja PM, Okaka ANC, Onu PN, Ibiam U and Urako AJ: Phytochemical composition of Talinum triangulare (water leaf) leaves. Pakistan Journal of Nutrition 2010; 9: 527-530.
17. Haslam E: Natural polyphenols (vegetable tannins) as drugs: possible modes of action. Journal of natural products 1996; 59: 205-215.
18. Murakami T, Hirano K and Yoshikawa M: Medicinal Foodstuffs. XXIII. 1) Structures of New Oleanane-Type Triterpene Oligoglycosides, Basellasaponins A, B, C and D, from the Fresh Aerial Parts of Basella rubra Chemical and pharmaceutical bulletin 2001; 49: 776-779.
19. Maisuthisakul P, Pasuk S and Ritthiruangdej P: Relationship between antioxidant properties and chemical composition of some Thai plants. Journal of Food Composition and Analysis 2008; 21: 229-240.
20. Hegde K and Divya KV: Evaluation of anti-stress activity of hydro-alcoholic extract of sauropus androgynus leaves. International Journal of Pharmaceutical Sciences and Research 2015; 6:
21. Thamizh Selvam N, Surabhi KR, Vasantha Kumar KG, Deep VC and Acharya MV: Physico-chemical, Phytochemical and Spectroscopic Characteristics of Aqueous and Methanolic Extracts of Laportea interrupta Chew Leaf. Phytochemical Analysis 2016; 9: 13.
22. Poongothai G and Shubashini K: HPTLC method of quantitation of bioactive marker constituent pinitol in extracts of Pisonia grandis. International Research Journal of Pharmacy 2012; 3: 207-12.How to cite this article:Peter SP, Kurian SP and Menon SK: Evaluation of antifungal property and phytochemical analysis of underexploited leafy vegetables. Int J Pharm Sci & Res 2022; 13(4): 1708-14. doi: 10.13040/IJPSR.0975-8232.13(4). 1708-14.
    

    ---

    All © 2022 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.