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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 1  |  Page : 5-10

Estimation of invitro antifungal activity of ethanolic crude extract of punicagranatum peel and woodfordiafruticosa leaves on isolates of candida species


1 Department of Oral Medicine and Radiology, Drs Sudha and Nageswararao Siddhartha Institute of Dental Sciences, Gannavaram Mandal, Krishna District, Andhra Pradesh, India
2 Department of Oral Medicine and Radiology, Mamata Dental College, Khammam, Telangana, India
3 Department of Oral Medicine and Radiology, Govt Dental College, Vijayawada, Andhra Pradesh, India
4 Department of Oral Medicine and Radiology, SVS Institue of Dental Sciences, Mahaboobnagar, Telangana, India

Date of Submission13-Jul-2021
Date of Decision11-Oct-2021
Date of Acceptance27-Nov-2021
Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Ramesh K Koothati
Department of Oral Medicine and Radiology, Govt Dental College, Vijayawada, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_198_21

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   Abstract 


Introduction: The Candida species which are opportunistic fungi especially the species of C. albicans, C. glabrata, C. tropicalis have been causing oral candidiasis. Due to resistance of conventional antifungal agents, the scientists are in verge of exploring novel antifungal drugs such as traditional medicinal plants. Hence we aimed in studying the antifungal effect of Woodfordia fruiticosa and Punicagranatum which are plants of Lythraceae on Candida species. Materials and Methods: The ethanolic extracts of both the plants were prepared using soxhlet extraction method and the obtained metabolites were confirmed by Thin Layer Chromatography. The Paper discs were prepared by Whatman filter paper No. 1 The disc was moistened with 10 μL from the prepared stock solution. Were kept at 4°C for 3 hours for diffusion and incubated for 48 hours. Then the diameters of the circular inhibition zones were measured. Results: The obtained data was expressed as mean and standard deviation. Chloroform fraction of Woodfordia fruticosa showed superior antifungal effect when compared to Punicagranatum peel. Conclusion: The invitro antimicrobial activity of Woodfordia leaf depicted superior antifungal activity against Candida isolates with effective anticandidal effect when compared to Punica peel. In future both of these plants could serve as basis for the new anti-candidal agents.

Keywords: Antifungal agents, Candida, Punica granatum, Woodfordia fruticosa


How to cite this article:
Bhavana S, Maloth KN, Koothati RK, Chintamaneni R, Kumar K N, Anusha NV. Estimation of invitro antifungal activity of ethanolic crude extract of punicagranatum peel and woodfordiafruticosa leaves on isolates of candida species. J Indian Acad Oral Med Radiol 2022;34:5-10

How to cite this URL:
Bhavana S, Maloth KN, Koothati RK, Chintamaneni R, Kumar K N, Anusha NV. Estimation of invitro antifungal activity of ethanolic crude extract of punicagranatum peel and woodfordiafruticosa leaves on isolates of candida species. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Jul 2];34:5-10. Available from: https://www.jiaomr.in/text.asp?2022/34/1/5/340736




   Introduction Top


Oral candidiasis is an opportunistic fungal infection, affecting the oral mucosa which often presents in both acute and chronic forms clinically. Although more than 150 species of Candida exist 95% of oral candidiasis is caused by C. albicans. While other species, such as C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, etc: Can further complicate the management of this oral and systemic candidiasis due to sporadic infections caused by them.[1]

C. albicans is a ubiquitous commensal organism which is well adapted to human host however alterations in the host microenvironment can provide opportunity to the pathogen and help in invading virtually at any site.[2] The colonization of Candida is favored by some of the etiological factors such as xerostomia, use of removable dentures, antibiotics, corticosteroids, chemotherapy and patients with endocrine disorders such as diabetes mellitus, hospitalized patients and human immunodeficiency virus (HIV) infected individuals.[3]

The emergence of resistance and toxicity of conventional antifungals has caused imperative need for alternate sources, and there is utmost need for safe, nontoxic, affordable and potent antifungal drug. In this perspective the plant based antifungals has got potential therapeutic applications in the current scenariowhich can be attributed to either systemic and topical use with minimum or no side effects.[4] The medicinal importance of a plants are due to some special substances like tannins, terpenoids, flavonoids, alkaloids, glycosides, resins, volatile oils, gums etc.[5]

The plants of Lythraceae, the loosestrife family, has 31 genera and 620 species, both cultivated and wild type of which, most of them are perennial herbs, shrubs or trees, and are widely distributed in tropics.[6]

Among them Woodfordiafruticosa which is an evergreen shrub popularly grown in tropical and subtropical region of India has got numerous pharmacological activities like antimicrobial, antioxidant, immunostimulant, anticancer, antiinflammatory, antifertility activities[7] etc. The earlier research and literature on Woodfordia has revealed that phytoconstituents such as tannins glycosides, phytosterols, hydrocarbons, octacosanol and β-sitosterol. etc: Have been responsible for their versatile pharmacological effects.[8] Various researchers have reported the antimicrobial potential of Woodfordia, although the inventories regarding its antifungal potential has been very limited.Several investigators like Parekh et al. (2007),[9] Kumaraswamy et al. (2008),[10] Dubey et al. (2014),[11] Kharate et al. (2018),[12] Joshi et al. (2019)[13] have assessed the antibacterial potential on different strains. In particular when we tried to search the literature for the antifungal potential of Woodfordia using popular research engines such as Embase, Medline, Science Citation index, NIH public access, pubmed and Cochrane Database of systematic reviews only two articles were found particularly on the assessment of antifungal potential on candida sp.

Similarly the other plant of lythraceae we chose was Punicagranatum which is well grown and cultivated in India, China and other regions such as Mediterranean, South Africa, America and Mexico.[14] The different chemical components of Punica like alkaloids, polyphenolics, ellagic acid, gallic acid and tannins possess various pharmacological activities such as antimicrobial, antifungal, antimutagenic, antioxidant, antiinflammatory, antidiarrheal, immunomodulatory and antitumor activities.[14] Furthermore several studies like Al-Zorcky et al. (2009),[15] Duman et al. (2009),[16] Anibal et al. (2013),[14] Bassir Jahromi et al. (2014),[17] Saeed et al. (2018),[18] have demonstrated the antifungal activity of Punica against yeast cells of Candida.

Henceforth we aimed to investigate the antifungal potential of Woodfordiafruticosa leaf and Punicagranatum peel against three strains of Candida (C. albicans, C. tropicalis, C. glabrata) and find out the active substance in both the plants and isolate it as well.


   Materials and Methods Top


Materials

The chosen plants were collected from precious herbals Pvt Ltd in July 2019. The chosen plants were taxonomically authenticated and identified at Deccan branch, Botanical survey of INDIA Hyderabad, with herbarium voucher no. D-95.

This study procedure was continuation of our previous study published in the journal of clinical and diagnostic research in 2016 bearing Clinical Trial Registry (CTRI) number REF/2016/03/11053, where pilot study was carried out on the same chosen Candida isolates.

Methods

Extraction isolation and purification of bioactive compounds

The dried leaves of Woodfordiafruticosa and peel of Punicagranatum 30 gm each were put in mechanical grinder and the grounded powder was subjected to extraction using 200 ml of 70% ethanol by soxhlet extractor at a temperature of 60°C for 4 hrs. The obtained extract was filtered using Whatman filter paper No. 3 and concentrated under reduced pressure in a rotary evaporator to remove the solvent. The yield percentage was calculated using the following formula

Extract yield% = R/S × 100

(Where R; weight of extracted plants residues and S- weight of plant raw sample).

For the phytochemical analysis the extracts were dissolved in ethanol (1 mg/ml), Approximately 10 μl of each extract obtained from leaves and peel of Woodfordia and Punica were subjected to qualitative phytochemical analysis to determine the presence of secondary metabolites such as alkaloids, flavonoids, steroids, terpenoids, proteins and amino acids, carbohydrates. The secondary metabolites like glycosides, carbohydrates were found in Woodfordiafruticosa leaf (Wfl) and flavonoids, steroids and terpenoids in Punicagranatum peel (Pgp). To confirm the presence of these phytoconstituents (HPTLC) – High performance thin layer chromatography was performed where 10 μl of alcoholic extract dissolved in ethanol was applied in the form of band of width 6 mm with a 100 μl, sample syringe on precoated silica gel aluminum plate 60 F254 (5 × 10) with 250 μm thickness using a CAMAG Linomat 5 sample applicator. The composition of mobile phase was ethanol: Isopropyl alcohol: Triethyl amine (5:4:1).

Plate was observed in the day light, under UV light (254 and 366 nm). After each observation on the central point of spots were marked with needle. Retention factor (Rf) was calculated by following formula and the results of HPTLC bands confirmed the same phytoconstituents in both the plants.

Rf = A/B: A = distance between point of application and central point of spot being examined.

B = distance between the point of application and the mobile phase point.

The activity of each extract obtained by column chromatography using silica gel was eluted with ethanol: Distilled water (1:1). It was prepared by dissolving specific solvents like chloroform and aqueous. Further of these fractions were subjected to antifungal activity against three fungal isolates of Candida with MTCC CODES namely MTCC 3017- Candida albicans, MTCC- 3019 Candida glabrata, MTCC 3421 Candida tropicalis were ordered from INDIAN INSTITUTE OF MICROBIAL TECHNOLOGY, Chandigarh. In this study, three fungal strains were used 3 of each species were taken and the SDA broth was prepared from the obtained freeze dried cultures.

The identification of Candida species was done using Hi-Chrome Candida differential agar (Hi media lab, Mumbai INDIA), which was categorized Green color denoting C. albicans, Metallic blue Candida tropicalis, and Cream or white as Candida glabrata [Figure 1].
Figure 1: Chloroform and Aqueous extract of Punica granatum peel

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The fractions for column chromatography were selected based on fraction which showed maximum antifungal activity. Finally two chloroform and aqueous fractions of Pgp and Wfl were obtained namely Pgp Aq &Pgp Cl, Wfl Aq &Wfl Cl [Figure 2] and [Figure 3]
Figure 2: Antimicrobial effect of Woodfordia, Punica aqueous and chloroform extracts on Different candida species

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Figure 3: Chloroform and Aqueous extract of Woodfordia fruticosa leaf

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The potency of various extracts was tested against fungal strains using filter paper disc method and ethanol was put as control for accurate results. The plates were prepared with Sabaroud's dextrose agar. Initially the blank paper discs of 3 to 5 mm were taken and pre weighed. They were sterilized under UV light for 15 to 20 min and were soaked in different preparations so a total of 10 μl of each compound was absorbed on filter disc and it was dried under laminar flow. They were incubated at 37°C for 48 hrs and zone of inhibition was calculated.


   Results Top


SPSS 21.0 for windows was used for the purpose of data analysis. The data were expressed as means ± standard deviation. The probability value was assessed as P ≤ 0.05, which was considered statistically significant, and P ≤ 0.01 as highly significant and P ≥ 0.05 as not significant.

The antifungal activity of chloroform and aqueous fractions of Pgp were tested against three fungal isolates of Candida where Pgp chloroform fraction showed more zone of inhibition and was more effective with C. glabrata. There was mean decrease in number of fungi colonies with Pgp chloroform extracts. [Table 1]
Table 1: Mean difference of antifungal activity of chloroform and aqueous fractions of obtained compounds of Pgp10 μg/ml

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The antifungal activity of chloroform and aqueous fractions of Wfl were tested against three fungal isolates of Candida where the chloroform fraction of Wfl showed more antifungal activity. There was mean decrease in the number of fungal colonies with Chloroform fraction of Wfl and among the three species it was effective against C. tropicalis. [Table 2]
Table 2: Results of antifungal activity of chloroform and aqueous fractions of obtained compounds of Wfl 10 μg/ml

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The chloroform fraction of Wfl showed more antifungal efficacy when compared to Pgp and there was mean decrease in the number of fungal colonies. Among the three species of Candida the chloroform fraction of Wfl was more effective with C. albicans. [Table 3] and [Figure 3]
Table 3: Comparison of chloroform fraction of Punicagranatum peel and Woofordia fruticosa leaf

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   Discussion Top


The incidence of C. albicans from the oral cavity has been reported to be 45-65% in healthy children, where as in neonates it is 45%, 30-45% in healthy adults and 50-65% in denture wearers, 65-88% in debilitating individuals and 90-95% of patients undergoing chemotherapy and HIV.[19] In the current study the chloroform fraction of Woodfordia leaf showed superior antifungal activity compared to Punicagranatum peel. Some of the invitro studies were reported supporting our study where Bhattaraie et al.[20] 2011 conducted in vitro study prepared from leaf and flower samples of Wff for antimicrobial activity against Candida isolates by disc diffusion method, using methanol, chloroform and hexane and the tested plant had effective antimicrobial activity. Dabur et al.[21] did an invitro study on the antimicrobial potential of various bacteria and four pathogenic fungi, and found superior antimicrobial activity. In another study Kumar et al.[8] conducted an invitro study to determine the antibacterial activity of leaves of Woodfordia flower on different microorganisms using ethanol, methanol, aqueous solvents and found superior activity with ethanolic extracts, however in our study chloroform extract showed good activity probably due to its property for extraction of nonpolar compounds. Some of the reported in vitro studies have revealed that bio constituents like alkaloids, glycosides, tannins, flavonoids, sterols, proteins, and amino acids could be responsible for the antimicrobial activity of Woodfordia.[21] In a study done by Khera et al.[22] the antimicrobial activity of different plants against bacterial and fungal isolates were evaluated, where Woodfordia showed potent broad-spectrum activity where both methanolic and ethanolic extract of flavonoids in Wfl contributed to antimicrobial activity owing to their ability to complex with bacterial cell wall causing cell death, however, in our study glycosides showed strong inhibitory effect. Mitscher et al.[23] stated that the isolation and characterization of a hydrolysable tannin dimer, woodfordin C, from the leaf extract of W. fruticosa has known to exhibit antitumor and antimicrobial effects through the inhibition of DNA topoisomerase enzyme II which is important for DNA replication and has been considered as one of the chemo-taxonomic markers originated in the Lythraceae family. Similarly Velanganni et al.[24] has conducted The GCeMS analysis of the n-butanol fraction of Woodfordia fruticosa and revealed that The monocyclic phenolic compound, 5- methyl-2-(1-methylethyl) phenol has antibacterial, antifungal properties, and these isolated compounds may help in bringing herbal antifungal drug. The other plant was punicagranatum, which also showed effective antifungal activity, Duman et al.[16] has done his research work on the six varieties of Punicagranatum, to find out the antimicrobial activity correlating the responses and the phytonutrient properties, such as total phenolic and anthocyanin compounds, where positive results were seen regarding the inhibition of Gram-positive and Gram-negative microorganisms, and C. albicans. De Souza Vasconcelos et al.[25] against various bacteria and fungi pomegranate gel showed good antifungal efficacy against C. albicans, and their findings are in accordance with our study. Shafighi et al.[26] has done a study to evaluate the antifungal effect of methanolic extract of pomegranate peel, flower, leaf and stem against C. albicans, where maximum inhibition zones were seen in flower extract, although in the current study peel extracts were taken. Some of the metabolites such as phenols and tannin derivatives like punicalgin, ellagic acid isolated from Pgp extract have shown antimicrobial efficacy reason being their ability to precipitate the proteins on the cell surface, while phenol derivatives interact with proteins and inhibit microbial aggregation by depriving the substrates and exhibit direct action on microbial metabolism, causing cell lysis as documented in the literature with studies done by various researchers. Rongai et al.[27] has compared different genotypes of Punica granatum to evaluate the antifungal properties by assessing the inhibition of mycelial growth and reported that wild genotypes CREA-FRU6, CREAFRU11 and CREAM-FRU76 have potent antifungal activity. Likewise Mbatha TF has reported from his study on pomegranate skin and concluded that apart from the fungicidal action it also in addition inhibits germ tube formation at sub-therapeutic levels.[28] The toxicological studies were also taken into consideration while we selected the plants, Bhujbal et al.[29] conducted toxicological studies on Wf and proved to be safe up to 2000 mg/kg. Patel et al.[30] carried out an acute toxicity study of pomegranate peel extract and found that it is safe up to 5000 mg/Kg body wt. Based on the above findings, there is further need for considerable number of studies to evaluate antifungal effect invivo along with spectral characterization of chosen phytochemical in future.

Limitations

Isolation of obtained metabolites would further help the other researchers in conducting the invivo studies on these two plants. Antifungal effect of selected phytochemicals on each Candida isolate individually would further help in obtaining future prospects for this type of research.

Future study prospects The determination of the mechanism of action of the obtained compounds from each plant on the Candida isolate could contribute to the management of alternative therapies rather than the conventional antifungal agents.


   Conclusion Top


In summary, the in vitro antimicrobial activity of Woodfordia leaf depicted superior antifungal activity against Candida isolates with effective anti-candidal effect when compared to Punica peel. In future both of these plants could serve as basis for the new anti-candidal agents. However it would be interesting for future researches to verify in vitro and vivo activity along with chemical characterization and spectral analysis of these Phytochemicals and confirm the safety of natural products by performing clinical trials using standard protocols.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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