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ORIGINAL ARTICLE
Ahead of print publication  

Broad-spectrum antifungal activity of Phyllanthus niruri leaves tested against Candida species


1 Department of Microbiology, Era's Lucknow Medical College, Lucknow, U.P, India
2 Department of Botany, Shia P.G. College, Lucknow, U.P, India
3 Department of Biochemistry, Era's Lucknow Medical College, Lucknow, U.P, India
4 Department of Microbiology, King George's Medical University, Lucknow, U.P, India

Date of Submission07-Jul-2022
Date of Acceptance14-Oct-2022
Date of Web Publication21-Jan-2023

Correspondence Address:
Saher Khan,
Department of Microbiology, Era's Lucknow Medical College, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/aihb.aihb_131_22

  Abstract 


Introduction: The emergence of resistant pathogenic microorganisms against conventional antimicrobials has become a global concern. To combat new and re-emerging infectious illnesses, new antimicrobial agents with different chemical structures and novel modes of action are required. Therefore, this study evaluated ethanolic extracts of Phyllanthus niruri for their antimicrobial activities against Candida species isolated from different clinical samples. Materials and Methods: This involved the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of Candida species. The ethanolic extraction of P. niruri leaves was examined. The agar well diffusion method was used to determine the antifungal activity of the leaf extracts against standard American Type Culture Collection strains as well as clinical isolates of Candida species. Results: Using PCR-RFLP, Candida tropicalis was found to be the most prevalent species of Candida, followed by Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The ethanol extract of P. nirui leaves showed good activity against all the clinical and standard strains of Candida which were comparable to the standard drug fluconazole. The activity of P. niruri against C. albicans was highest followed by C. parapsilosis, C. tropicalis, C. glabrata and C. krusei at a concentration of 100 mg/ml. Conclusion: The findings of this study support the use of P. niruri plant to treat Candida infections, particularly fluconazole-resistant Candida species.

Keywords: Agar well diffusion, fluconazole and Candida, polymerase chain reaction-restriction fragment length polymorphism, Phyllanthus niruri



How to cite this URL:
Khan S, Singh M, Khare V, A.Ali Khan MM, Raza T, Gupta P. Broad-spectrum antifungal activity of Phyllanthus niruri leaves tested against Candida species. Adv Hum Biol [Epub ahead of print] [cited 2023 Feb 2]. Available from: https://www.aihbonline.com/preprintarticle.asp?id=368368




  Introduction Top


Candida species are thought to be a major source of human opportunistic infections. Despite advances in healthcare and therapeutic methods, the frequency of invasive systemic candidiasis has risen dramatically in recent years. This is thought to be the result of the increase in the size of populations at risk, such as transplant recipients, cancer patients, HIV-infected patients and those receiving immunosuppressive and broad-spectrum antibiotic therapy.[1] Candida species are commonly found as commensals on mucosal membranes in healthy people, and they can be found in a non-virulent form in around half of the population. However, if the host's natural flora is disrupted or immunity is compromised, these species can become pathogenic. This genus has at least 30 medically significant species that are implicated in human candidiasis.[2] Candida albicans has been the most often isolated species, but non-albicans Candida (NAC), particularly Candida tropicalis, Candida parapsilosis, Candida krusei and Candida glabrata, have emerged as key causes of Candida infection. Due to their therapeutic effectiveness against several Candida species, azole antifungal medicines are becoming increasingly popular globally.

Fluconazole, among the azole medications, has demonstrated excellent tolerance, and antifungal drug resistance is rapidly becoming a serious problem, particularly in immunocompromised individuals. Thus, there is a rising interest in discovering new, natural chemicals with anti-candidal efficacy.[3] Nowadays, the plant-derived antimicrobial is a hot topic of research due to their ease of application, and broad antimicrobial-spectrum antimicrobial effects are usually attributed to a number of substances with multiple target sites, which reduces the likelihood of the development of microorganisms resistance.[4]

In this contribution, we studied the anti-candidal impact of medicinal herbs Phyllanthus niruri leaves (Bhumi Amla) by the well diffusion method and calculate its minimum inhibitory concentration. P. niruri is a member of the Phyllanthaceae family. The P. niruri species is known by a variety of names, including 'stone breaker', 'pigeon weed' and 'burrow wall', among others. However, 'stone breaker' is the most well-known common name. This species is native to South America, India and China. P. niruri has been widely used in treating a number of traditional ailments.[5] This plant was selected based on good activity found against some bacterial infections and has been shown to have anti-inflammatory, hypoglycaemic, antiviral, antifungal antioxidant and hepatoprotective action. Acidic diterpenes, hypophyllanthin, arabinogalactan, alkaloids, flavonoids, lignans, terpenes, tannins and Phyllanthus are some of the compounds found in the plant.[6]


  Materials and Methods Top


Sample collection

Urine, blood, sputum, pus and vaginal swab samples were collected from 70 clinically suspected cases of fungal infections at Era's Lucknow Medical College in Lucknow. Samples were collected in sterile collecting devices and containers, which were labelled appropriately, and delivered to the laboratory for further processing.

Inclusion criteria

All the clinically known and suspected cases of fungal infections were admitted to various wards of the hospital.

Exclusion criteria

Patients on antifungal therapy were excluded from the study.

Chemicals

Sabouraud Dextrose Agar (SDA), antifungal discs, fluconazole (25 μg/disc) and standard strains were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Dimethyl sulfoxide (DMSO), ethanol and other chemicals and reagents used for the study were of analytical grade.

Processing of specimen

Samples were collected using aseptic precautions and inoculated on SDA (HiMedia) screw-capped bottles and incubated at 37°C for 48–72 h. After growth, species identification was done by molecular method – polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism

Extracting DNA

Using a Fungal DNA isolation kit, (Qiagen), DNA was extracted from all clinical isolates and standard strains.

Polymerase chain reaction assay

The master mix was made up of 25 ul of Qiagen PCR mix, 1 ul of Internal transcribed spacer (ITS-1) forward primer (forward 5' TCC GTA GGT GAA CCT GCG G-3') and ITS-4 reverse primer (reverse 5 TCC TCC GCT TAT TGA TAT GC-3'), 1 ul of template DNA and 50 ul of sterile nuclease-free water.

Restriction fragment length polymorphism analysis

The restriction enzyme MspI (cat N0-R01065) (New England BioLabs USA) was used to digest the amplified products.[1]

Antifungal susceptibility testing

Collection and identification of plant materials

P.niruri therapeutic plants were obtained from the CSIR-CIMAP, Lucknow. The plant that was healthy and disease-free was chosen for the antifungal studies.

Preparation of extracts

To eliminate dirt and soil, freshly acquired plant materials were rinsed twice with distilled water. The plant materials were dried at room temperature in the shade and coarsely ground in a blender. Specific amounts of ethanol were added to them for 5 days at room temperature. Regular infusions were used to blend the combinations. The extracts were filtered using Whatman filter paper No. 1 after 5 days. The filtrates were dried in a rotator evaporator at 40°C. The dried extracts were kept at −20°C in sterile glass vials until needed.[7]

Antifungal susceptibility testing by well diffusion methods

Inoculums were created and adjusted according to the standards of 0.5 McFarland turbidity. The suspension of inoculums was uniformly inoculated on Mueller–Hinton agar medium. With a sterile cork borer, wells (10 mm diameter and 2 cm apart) were drilled into the lawn cultures. Each well in each plate was filled with 100μl of each extract in an aseptic manner. A control group was likewise inoculated with no plant extract. For 1 h, the plates were kept at room temperature to allow the extract to diffuse into the agar. It was then incubated at 37°C for 24 h. Triplicates of the trials were carried out. The activity was determined by the presence of a defined circular zone surrounding the well. At the end of 24–48 h, the results were documented using callipers to measure the diameter of the zones in millimetres.[8] A drug susceptibility test was performed using the M27-S4 standard protocol presented by the Clinical and Laboratory Standards Institute (CLSI).[8]

Control

C. albicans American Type Culture Collection (ATCC) 90028, C. tropicalis ATCC 750, C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as assay controls. Fluconazole was used as a positive control, whereas DMSO medium without the herbal extracts was taken as a negative control.

Statistical analysis

The SPSS 24.0 version was used to analyse the data. The frequency and percentages were used to depict categorical data. Graphs were also used to present the data. The mean and standard deviation were used to depict continuous data. For the antifungal susceptibility testing of Candida isolates, zone diameters around the disc were measured and classified as sensitive or resistant based on the CLSI breakpoint system.[8] For the antimicrobial activity evaluation of the plant extracts using the agar well diffusion method, the zone diameters around the wells were measured in millimetres and recorded.


  Results Top


Out of 70 clinical samples, the majority of the isolates were obtained from urine 25 (35.71%) followed by sputum 15 (21.42%), blood 12 (17.14%), vaginal swab 10 (14.28%) and pus 8 (11.42%) [Figure 1].
Figure 1: Type of sample

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The maximum number of samples were from female 42 (60%) to male 28 (40%) patients [Figure 2]. Patients in the adult age group (45.71%) had the highest prevalence of Candida, followed by those in the neonates (14.28%) [Figure 3].
Figure 2: Patient demographics by age and gender

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Figure 3: Species-wise distribution of Candida isolates

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All the Candida isolates were identified up to species level by PCR-RFLP method. The ITS-1 and ITS-4 universal primers were successful in amplifying the ITS15.8S rDNA ITS-2 region of their rDNA, giving PCR products of approximately 510-875 bp. The ITS region was amplified and digested using MspI restriction enzyme, which created two-band patterns for C. tropicalis (182,340), C.albicans (238, 297), C. glabrata (316,559) and C. krusei (261,249) However, the ITS region of C. parapsilosis lacked a recognition site for this enzyme, and its PCR and digestion products were identical (520 bp) [Table 1].
Table 1: Size of ITS region before and after endonuclease digestion with MspI

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As a result, the strains were identified as five different species using an ITS-RFLP with MspI and ITS-amplicon size combination. Among the isolates, C. albicans accounted for 28.57% and NAC species for 71.42% of all the Candida colonisation cases. Among the 71.42% NAC species isolates, the most common was C. tropicalis 25 (35.71%) followed by C. glabrata 10 (14.28%) C. krusei 8 (11.42%) and C. parapsilosis 7 (10%) respectively [Figure 3].

Plant extract yield and properties:-

The percent yields of the ethanolic extracts of P.niruri and its properties are given in [Table 2].
Table 2: Plant extract yield and properties

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The following formula was used to compute the extract rates:

Yield = m0/m1 × 100

Where m0 is the mass of evaporated extract, and m1 is the mass of vegetal matter.[9]

Antimicrobial activity of ethanolic extract of Phyllanthus niruri against standard American type culture collection strains of Candida

The antifungal activities of the ethanol extracts of P. niruri leaves were tested against a variety of ATCC strains. In vitro antifungal activity was tested for the presence or absence of a zone of inhibition in diameter in contrast with the reference antifungal drug (fluconazole).

P. niruri demonstrated a comparatively high level of antimicrobial activity with a zone of inhibition ranging from 28 to 18 mm against Candida species. The most sensitive standard strains were C. albicans ATCC 90028 (27.3 ± 0.3) with a zone of inhibition similar to fluconazole (27.66 ± 0.5) followed by C. tropicalis ATCC750 (24.3 ± 1.15), C. parapsilosis ATCC 22019 (21 ± 0.0) and C. krusei ATCC 6258 (18.33 ± 0.5). Fluconazole showed the least or no activity against C. krusei (2 ± 0), whereas P. niruri showed an 18–19 mm zone (18.33 ± 0.5) against C. krusei [Table 3].
Table 3: Mean zone of inhibition and standard deviation of the standard American Type Culture Collection strains of Candida species

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Antimicrobial activity of ethanolic extract of Phyllanthus niruri and fluconazole against clinical isolates

Results for mean zones of inhibition against clinical Candida isolates for plant extract and fluconazole are shown in [Table 4]. According to this study, C. albicans (23.05 ± 1.75) showed a maximum mean zone of inhibition followed by C. tropicalis (21.36 ± 1.4), C. parapsilosis (19.85 ± 1.6), C. glabrata (18.2 ± 1.03) and C. krusei (14.62 ± 0.77) against plant extract. Fluconazole shows maximum activity against C. albicans (22.7 ± 2.2) followed by C. parapsilosis (20.42 ± 1.39), C. tropicalis (21.76 ± 1.83), C. glabrata (17.1 ± 1.28), whereas no activity was shown against C. krusei [Table 4].
Table 4: Mean zone of inhibition and standard deviation of test strains of Candida species

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  Discussion Top


Invasive mycoses caused by opportunistic fungal diseases have become more common over the past two decades. The most prevalent opportunistic fungal infections in humans are members of the Candida genus.[10]

As shown in the table. Seventy clinical samples were collected from clinically fungal suspected patients. Out of the 70 different clinical samples, the majority of samples were from female 42 (60%) to male 28 (40%) patients, suggesting that women have a high risk for developing Candida infection. In a prior study, Guru and Raveendran also reported the majority of clinical specimens for Candida were from women (54.5%).[11] In a similar vein, Prabhakaran et al. and Pawar et al. reported a larger proportion of females than males in the numerous specimens they analysed.[12],[13] This could be due to some physiological reasons such as age, use of oral contraceptives and pregnancy are more prone to Candida infections. In the present study, the majority of strains were recovered from urine followed by sputum, blood, vaginal swab and pus [Figure 1]. The large percentage of urine samples (35.71%) supports the predominance of vulvovaginal sources of infection. According to Singh et al.'s study, the most prevalent location or specimen of fungal isolation in medical and surgical critical care section patients was urine (74.7%), followed by blood (20.8%).[14]

In this study, gel electrophoresis separates bands specific for particular species, identifying them to the species level after the amplicon was treated with the enzyme MspI. Using primers ITS-1 and ITS-4, we were able to successfully amplify the ITS-1 and ITS-2 regions of 70 isolates and got a single PCR product of approximately 510–875 bp. The ITS region was amplified and digested using MspI restriction enzyme, creating two-band patterns for C. tropicalis (182,340), C. albicans (238,297), C. glabrata (316,559) and C. krusei (261,249). However, the ITS region of C. parapsilosis lacked a recognition site for this enzyme, and its PCR and digestion products were identical (520 bp) [Table 1]. Thus, this PCR-RFLP can identify the most commonly isolated Candida spp at the species level but fails to identify isolates of C. parapsilosis at the species level. Extremely small amounts of DNA can be detected by PCR techniques, which can also lead to early detection of harmful fungi and, as a result, earlier initiation of antifungal therapy, which may increase survival chances. These techniques have a high degree of specificity and sensitivity for detecting fungus directly. There have been reports of a number of research employing PCR methods along with restriction digesting enzymes for specific species identification. These studies also indicated a number of methods using universal primers for the identification of diverse fungi. The 5.8SrDNA gene is located around these ITS-1 and ITS-2 regions. These areas are suitable for the medically significant processes of fungus diagnosis, identification, taxonomy and phylogeny. The use of universal primers is typically a wise strategy for clinical microbiological diagnosis.[15] Williams et al. amplified the ITS region of rDNA using PCR, and three restriction enzymes were used to digest the amplified products. They concluded that eight different Candida spp. could be distinguished based on size and sequencing variance. Being rapid and easy to use, the PCR-RFLP approach has an advantage over other molecular techniques like RFLP with genomic DNA and electrophoretic karyotyping.[16] The study of Mousavi et al. applying the same method gave 100% agreement with the present study.[17]

C. albicans is the most prevalent species of Candida, according to a number of earlier research.[11],[12] However, numerous studies in recent years have noted a significant occurrence of other species as well. In the present study, C. albicans accounted for 28.57% and NAC species for 71.42% of all the Candida colonisation cases. Among the 71.42% NAC species isolates, the most common was C. tropicalis 25 (35.71%), followed by C. glabrata 10 (14.28%) C. krusei 8 (11.42%) and C. parapsilosis 7 (10%), respectively. Fatima et al. reported 12.22% of C. albicans and the rest being NAC species out of which C. krusei was the most common (53.33%), followed by C. parapsilosis (13.33%), C. lusitaniae (8.88%), C. tropicalis (5.55%), C. glabrata (4.44%) and C. guilliermondii (2.22%).[18]

In the Indian subcontinent, especially in rural areas, herbal remedies are widely utilised and frequently the sole alternative for treatment for more than 70% of the population. Numerous innovations using herbal molecules have been made over the years as a result of the potential chemotherapeutic properties of herbal drugs and their ability to combat antimicrobial resistance. P. niruri has been utilised to treat a variety of illnesses in India.[19]

The current in vitro investigation shows that according to the findings of our investigation, P. niruri herbs have antimicrobial inhibitory zones that range from 26 to 14 mm against clinical isolates [Table 4] suggesting that the herbs can prevent the growth of certain Candida species.

In the present study, the antifungal activities of the ethanol extracts of P. niruri leaves were tested against Candida ATCC strains and isolates taken from clinical samples of a patient with suspected and confirmed fungal Candida infection. In vitro antifungal activity was tested for the presence or absence of a zone of inhibition in diameter in contrast with the reference antifungal drug. Any agent's exhibits a zone of inhibition >5 mm is considered an effective antimicrobial agent.[20]

The findings of our investigation indicate that ethanolic extracts of P. niruri leaves have antifungal activity against all the tested Candida species to varying degrees at a concentration of 100 mg/ml. The maximum mean zone of inhibition was seen in C. albicans (23.05 ± 1.75), followed by C. tropicalis (21.36 ± 1.4), C. parapsilosis (19.85 ± 1.6), C. glabrata (18.2 ± 1.03) and C. krusei (14.62 ± 0.77) against P. niruri plant extract.

Fluconazole showed maximum activity against C. albicans (22.7 ± 2.2), followed by C. parasilosis (20.42 ± 1.39), C. tropicalis (21.76 ± 1.83), C. glabrata (17.1 ± 1.28), whereas no activity was shown against C. krusei. Thus, it is shown C. albicans, C. glabrata and C. krusei has better susceptibility towards P. niruri extract when compared with the standard drug. In the study of Nivetha and Roy, the maximum zone of inhibition was shown by C. albicans (18 mm), followed by C. tropicalis (15 mm) C. krusei (11 mm) and C. parasilosis (13 mm) at a concentration of 2500 μg/ml,[21] whereas in Shilpa et al.'s study, no antifungal activity of aqueous of P. niruri was seen against Candida.[6]

Our study findings agree with Njoroge et al.'s study which showed good activity of P. niruri extract against Candida.[22]

The most notable observation is the antifungal activity of the P. niruri extract against Candida species is comparable to or higher than fluconazole. Another noteworthy finding is that all strain of C. krusei was found to be 100% resistant to fluconazole, followed by C. glabrata (50%), and despite plant extract having excellent antifungal activity against all Candida species.

[Table 3] reveals that the ethanol extract of P. niruri leaves had significant activity against all ATCC strains. Among the standard strains, C. albicans ATCC 90028, followed by C. tropicalis, C. parasilosis and C. krusei isolates were the most susceptible, with a zone of inhibition comparable to fluconazole. There was no significant difference in the zone of inhibition between ATCC strain and clinical isolates tested by P. niruri extracts.

The antimicrobial action of P. niruri is due to the presence of secondary metabolites such as alkaloids, phyllanthin, carboxylic acids, phyllanthenol, phyllochrysine and saponin.[23]

According to Anusha et al., phyllanthin is one of the active lignans responsible for the anti-candidal properties of P. niruri.[24]

Limitation of the study

Only the leaves of the herbs were employed in this study; other components of the plant, such as the seeds, stems and fruits, should also be investigated for their anti-candidal potential. This was an in vitro study since in vivo environment is distinct, we cannot expect the same results when the herb is used in vivo settings.


  Conclusion Top


The identification of active and effective biocompounds is critical because most infections are developing resistance to a variety of medications. The study shows that P. niruri leaf extracts had significant activity against all tested Candida species recovered from clinical samples as well as standard ATCC strains. This study's findings may thus support the use of this plant against all Candida species, particularly against fluconazole-resistant Candida species.

Acknowledgements

We would like to thank Mr. Robin, Era's Lucknow Medical for the PCR-RFLP technique. We would also like to acknowledge Mr. Waseem, Era's Lucknow Medical College for the plant extraction method. We would also want to express our gratitude to Prof. (Col.) M.Azam, Director General Administration and Research, Career Institute of Medical College Lucknow, India for his enthusiastic support and guidance in assisting us in completing this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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