Keywords

Mailkara zapota, Averrhoa carambola, Phytochemical, Antifungal

Introduction

Infectious diseases caused by bacteria, fungi, viruses and parasites are still a significant risk to public health, due to evolvement of microbial resistance to the synthetic drug. Their impact is particularly large in developing countries due to the unavailability of medicines or due to development of side effects by indiscriminate use of drug [1].

About 80% people from developed countries use traditional medicines, which has compound obtained from medicinal plant. Plants contain some organic compounds which show some physiological effects on human body [7]. Phytochemicals or the bioactive compounds are secondary metabolites with extremely diverse compound and obscure function. Phytochemicals can be derived from any part of the plant but accumulation of bioactive compounds varies in various parts. Leaves are known to have highest accumulation of bioactive compounds [2].

Manilkara zapota belongs to the family Sapotaceae. The plant is evergreen, smooth tree with a milky juice. It is cultivated throughout India. The bark is antibiotic, astringent and febrifuge. It is used as tonic and the decoction is given in diarrhoea and peludism [3]. Averrhoa carambola belongs to family Oxalidaceae. It is a small, slow-growing evergreen tree with a short-trunk or a shrub. www.advancejournals.org Open Access Scientific Publisher International Journal of Ethnobiology & Ethnomedicine PM 16|Volume 3|Issue 1|2017 2 The leaves are used for chicken pox, ringworm and headache and a decoction of leaves is used to arrest vomiting [4].

Materials and Method

Collection of Plant material and identification

Leaves of Manilkara zapota and Averrhoa carambola was collected from natural habitat from the Aksharwadi of Surat [3]. Identification of plant material was done by Department of Botany, P. T. science college, Athwalines Surat and was documented.

Leaves of Manilkara zapota before and after drying

Fig. 1: Leaves of Manilkara zapota before and after drying

Leaves of Averrhoa carambola before and after drying

Fig. 2: Leaves of Averrhoa carambola before and after drying

Preparation of crude plant extract

The collected leaves were washed thoroughly 2-3 times with running water and with distilled water. The leaves were air-dried under shade [6]. The leaves were crushed to make possible fine powder with the help of electric grinder and stored for further analysis [5].

Aqueous extractions

20 grams of dried plant material was extracted in 100 ml water for 1 hour at 400 C on magnetic stirrer [5]. After 1 hour it was filtered through whattman filter paper-1 and centrifuged at 5000 rpm for 15 minute [3]. The supernatant was collected and used for further phytochemical analysis [6].

Solvent extractions

20 grams of dried plant material was extracted in 100 ml of Ethyl acetate and Petroleum ether. The flasks were covered with aluminium foil and kept on rotatory shaker at 191 rpm for 24 hour at room temperature. After 24 hour the solution was filtered through whattman filter paper-1 [3, 5]. The filtrates were collected in Falcon tubes and were concentrated up to dryness by keeping it in incubator at 350 C. The stock solution (0.5 mg./ml) of each extract was prepared in Dimethyl sulfoxide (DMSO) [5].

Leaf Extract of Manilkara zapota

Fig. 3: Leaf Extract of Manilkara zapota

Leaf Extract of Averrhoa carambola

Fig. 4: Leaf Extract of Averrhoa carambola

Phytochemical screening of leaves crude extract

Phytochemical analysis for screening and identification of bioactive chemical constituents of extracts were performed with the standard methods with modification as mentioned in the Table 1 [7, 8, 9].

Table 1

List of Phytochemical test performed

Sr. No. Phytochemicals Tests
1 Alkaloids Wagners test
Hagers test
Mayers test
2 Carbohydrates Molish’s test
Benedict test
Fehling’s test
3 Saponin Frothing test
4 Glycosides Modified Brontrager’s Reagent test
5 Phytosterols Salkowski’s test
6 Resins Acetone test
7 Phenol Ferric chloride test
8 Taninn Gelatin test
9 Diterpenes Copper acetate test
10 Flavonoids Alkaline reagent test
Lead acetate test
Zinc hydrochloride test
Ferric chloride test
11 Protein and amnio acid Xanthroproteic test
Biuret test
12 Terpenoids Salkowski test
13 Quinones Borntrager’s test
Ammonia test
Conc. Hydrochloric test
14 Phlobatannin 1% hydrochloric test

Microbial strains and culture condition

The Fungal strains used to assess the antifungal properties includes Mucor hiemalis (MTCC No.157), Rhizopus stolonifera (MTCC no.162), Alternaria alternate (MTCC No.1362), Fusarium eumartii (MTCC No.399), Curvularia lunata (MTCC No. 283), Penicillum chrysogenum (MTCC No.160), Aspergillus niger (MTCC No.281), Saccharomyces cerevisiae (MTCC No. 36) and Candida albicans (MTCC No.183). The investigated fungal and yeast strains were obtained from Department of Microbiology, Shree Ramkrishna Institute of Comp. education & App. Sciences, Surat, India. The organisms were maintained on Sabouraud dextrose agar (Hi-media), at 4˚C and sub cultured before use. The organisms studied are clinically important ones causing several infections, food borne diseases, spoilage, and skin infection [2].

Antimicrobial Assay

In vitro antimicrobial activity of the different solvent extracts of the two screened plants was studied against 9 pathogenic microbial strains by the agar well diffusion method [10, 11]. Sabouraud dextrose agar (Hi-media) was used for antifungal susceptibility test. The extracts were prepared in 100% DMSO at a concentration of 0.5 mg/ml. The Sabouraud dextrose agar was melted and cooled to 48- 50 °C and a standardized inoculum (1.5 × 108 CFU/ml, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a solid plate. Wells were prepared (8.5 mm) in the seeded agar plates. The wells were filled with different concentration of the extract to know the minimum inhibitory concentration (MIC value). The plates were incubated overnight 28 °C for 48 h. The antimicrobial spectrum of the extract was determined in terms of zone sizes around each well. Fluconazole was used as positive control and DMSO was used as negative control [2, 12].

Results

Phytochemical analysis

The preliminary phytochemical screening of different extracts was done to ascertain the presence of bioactive components. The result is shown in Table-2.

Table 2

Results of phytochemical analysis

Phytochemicals Tests Manilkara zapota Averrhoa carambola
Aqueous Ethyl acetate Petroleum ether Aqueous Ethyl acetate Petroleum ether
Alkaloids Wagners test + + + + + +
Hagers test + + + + + +
Mayers test + + + + + +
Carbohydrates Molish’s test + + + + + +
Benedict test + + + + + +
Fehling’s test + + + + + +
Saponin Frothing test
Glycosides Modified Brontrager’s Reagent test + + + + + +
Phytosterols Salkowski’s test + + + + + +
Resins Acetone test + + + + +
Phenol Ferric chloride test + + + +
Taninn Gelatin test + +
Diterpenes Copper acetate test + + +
Flavonoids Alkaline reagent test + + + + + +
Lead acetate test + + + + + +
Zinc hydrochloride test + + + + + +
Ferric chloride test + + + + + +
Protein and amnio acid Xanthroproteic test + + + + + +
Biuret test + + + + + +
Terpenoids Salkowski test + + + +
Quinones Borntrager’s test + + + + + +
Ammonia test + + + + + +
Conc. Hydrochloric test + + + + + +
Phlobatannin 1% hydrochloric test + + +

‘+’= Presence and ‘-’ = Absence

Antimicrobial compounds that are alkaloids, flavonoids and phenolic component are present in leaf extract of Manilkara zapota and Averrhoa carambola.

Antimicrobial assay

The result of antifungal assay of various extract of Manilkara zapota and Averrhoa carambola and their minimum inhibitory concentration are as shown in Fig.5 to Fig. 10

Manilkara zapota (aqueous extract)

Fig. 5: Manilkara zapota (aqueous extract)

Manilkara zapota (ethyl acetate extract)

Fig. 6: Manilkara zapota (ethyl acetate extract)

Manilkara zapota (petroleum ether extract)

Fig. 7: Manilkara zapota (petroleum ether extract)

Averrhoa carambola (ethyl acetate extract)

Fig. 8: Averrhoa carambola (ethyl acetate extract)

Averrhoa carambola (ethyl acetate extract)

Fig. 9: Averrhoa carambola (ethyl acetate extract)

Averrhoa carambola (petroleum ether extract)

Fig. 10: Averrhoa carambola (petroleum ether extract)

From the above Figure 5 to 10 it can be concluded that the above leaf extract of Manilkara zapota and Averrhoa carambola shows antifungal activity against Mucor hiemalis, Alternaria alternate, Fusarium eumartii, Candida albicans and Sacchromyces cerevisiae.

figure-11

Discussion

The present study reveals that the phytochemical constituents of leaf extract of Manilkara zapota includes alkaloids, flavonoids, terpenoinds, glycosides, carbohydrates and phytosterols which are similar to the result of Islam, M. R., et al. (2013) [6] except for tannin and saponin; and the phytochemical contituents of leaf extract of Averrhoa carambola showed presence of alkaloids, flavonoids, terpenoinds, glycosides, carbohydrates, phloatannin and phytosterols. The result of antifungal inhibition against Candida albicans (MTCC No.183) of petroleum ether extract are same as result of Sumitra et al. (2011) [2], Whereas on the same species Ethyl acetate extract shows greater activity compared to results of Sumitra et al. (2011) [2]. Various extract of Manilkara zapota and Averrhoa carambola shows antifungal activity against Mucor hiemalis (MTCC No.157), Alternaria alternate (MTCC No.1362), Fusarium eumartii (MTCC No.399), Saccharomyces cerevisiae (MTCC No. 36) and Candida albicans (MTCC No. 183).

Conclusion

The present study revealed that petroleum ether extract of Manilkara zapota and ethyl acetate extract of Averrhoa carambola shows maximum antifungal activity compared to other against Mucor hiemalis (MTCC No.157), Fusarium eumartii (MTCC No.399) and Candida albicans (MTCC No. 183). The activities might be due to the presence of bioactive compounds or certain inhibitory compounds or due to synergist effect of bioactive compounds.

Since a variety of constituents are present in the extract studied; so, further investigations are necessary to find out the active antimicrobial compound present in the plant leaves.