Academia Journal of Medicinal Plants 8(9): 107-123, September 2020 DOI: 10.15413/ajmp.2020.0123 ISSN: 2315-7720 ©2020 Academia Publishing
Research Paper
Evaluation of anti-candida activity of aqueous extracts of leaves, root and stem of Rhinacanthus nasutus against Candida albicans in vitro study
Accepted 3rd June, 2020
ABSTRACT
Search for naturally occurring compounds with anti-candida activity has become quite intense due to the side effects of synthetic fungicides and the development of pathogens against such fungicides. Therefore in recent years, considerable attention has been directed towards the identification of anti-fungal activity. Hence searching of varieties of plants for their antifungal activity against Candida albicans was considered worthwhile. The main goal of this research was to assess the anti-candida activity of the plant Rhinacanthus nasutus (L) Kurz against the C. albicans. For this study, fresh and dry plant materials (leaves, root and stem) were used at different concentration (100, 50 and 25%) and different time period (12, 24 and 48 h) using hot and cold extracts to test effectiveness. C. albicans sample were collected from university of Colombo and Sabouroud Dextrose Agar media were used to determine the anti-candida activity and the antifungal activities were assessed by the presence or absence of inhibition zones in disc diffusion method. Among the different plant extracts, the results showed fresh leaves are more effective than root and stem; the zone of inhibition against the C. albicans for leaves, root and stem are 14.14±0.12, 12.65±0.2 and 11.81±0.69 mm respectively. Jesintha.J1, Paheerathan.V2*, Piratheepkumar.R3 and Chanjugaa.U4 Fresh leaves extract (14.14±0.20 mm) were more efficient than dry extract (13.36±1.80 mm) and cold extract (14.14±0.20 mm) were more effective than hot 1Bandaranaike Memorial Ayurvedic extract (13.36±1.80 mm) and effective duration for the inhibition is 48h with Research Institute, Nawinna, Sri Lanka. 10mg/10ml of extract. Rhinacanthus nasutus (L) Kurz has better effective anti- 2Unit of Siddha Medicine, Trincomalee Campus, EUSL, Sri Lanka. candida activity than the positive control Fluconazole (10mg/10 ml) 3Unit of Siddha Medicine, Trincomalee (14.14±0.20>11.33±0.47). The findings suggest the usefulness of leaves, stem and Campus, EUSL, Sri Lanka. root aqueous extracts of Rhinacanthus nasutus (L) Kurz against pathogenic fungal 4Unit of Siddha Medicine, Trincomalee (C. albicans) strains. Campus, EUSL, Sri Lanka.
*Corresponding author. E-mail: Key words: Anti-candida activity, Rhinacanthus nasutus (L) Kurz, Candida [email protected]. albicans, Sabouroud Dextrose Agar, Concentration, zone of inhibition and effective.
INTRODUCTION
Plant-based medicines have a respectable position today, Medicine is of great antiquity. Traditional medicines have especially in developing countries where modern health evolved over centuries blessed with a plethora of services are not sufficient. Indigenous remedies are gaining traditional medicines and practices. Siddha science is an popularity in both rural and urban areas because they are ancient medical science encompassing variety of scientific effective, safe and inexpensive (Gul et al., 2012). A perusal formulations in scripts. There is a need for the firm of the literature of Siddha system of medicine revealed that scientific basis given to disperse the prevailing misbelieves there are several drugs available for the treatment of fungal and doubts among different section of people. With an diseases (Suresh et al., 1994). The Siddha system of increase in lifestyle-related disorders there is a worldwide Academia Journal of Medicinal Plants; Jesintha et al. 108
resurgence of interest in holistic system of health care Education (LIFE) portal has facilitated the estimation of the particularly with respect to the prevention and burden of serious fungal infections country by country for management of chronic non-communicable and systemic over 5.7 billion people (>80% of the world’s population). diseases (Patil Shailesh et al., 2014). Rhinacanthus nasutus These studies have shown differences in the global burden (L) Kurz is an antifungal (Poonjaamahatri) plant between countries, within regions of the same country and (Ramanathan, 2002) and also some investigations have between at risk populations and in 2018 around 138 shown that plant parts have antimicrobial activities million women were affected with Candidiasis (Figure 1) (Munavvar et al., 2004). The leaves have some chemical (Denning et al., 2018). Approximately 62-80% of the composition which is responsible for the antimicrobial world’s population still relies on traditional medicines for activity (Wu et al., 1988). But other texts mentioned root the treatment of common illness and traditional system of and stem of plant has antimicrobial activity medicine plays an important role in health care (Zhang, (Vaidyaratnam,1995) and the Rhinacanthin rich 2004). Plant-based medicines have a respectable position Rhinacanthus nasutus (L) Kurz extract also exhibited the today, especially in developing countries where modern antifungal activity against the C. albicans (Pruksakorn et al., health services are not sufficient. Indigenous remedies are 2018). Hence this study is solemnly designed to screen the gaining popularity in both. Candidiasis is a fungal infection antifungal activity in different parts of this plant (Leaves, caused by yeasts that belong to the genus Candida species root and stem). Fresh and dry aqueous extracts have been can infect humans; the most common is C. albicans used for this research purpose (Munavvar et al., 2004). (Mitchell, 1998). C. albicans is the most common human Despite the availability of several effective anti mycotics fungal pathogen causing diseases ranging from mucosal to for the treatment of candidiasis, failure of therapy is not systemic infections as a commensal, C. albicans uncommon due to the unique environment factors to asymptomatically colonizes mucosal surfaces (Christina et reduce the drug concentration to sub-therapeutic levels al., 2016). (Ellepola and Samaranayake, 2000). Many people are using Most infections occur in patients who are immune herbal based intervention in treatment of Candida infection compromised or debilitated in some other way such as HIV with lack of scientific validation. Therefore providing a or AIDS (Akpan and Morgan, 2002). Candidiasis is a unique drug with high effectiveness will help the patient common infection of the skin, oral cavity and esophagus, and upgrading the Siddha system. This problem will be gastrointestinal tract, vagina and vascular system of partly circumvented by the introduction of the different humans (Sudbery et al., 2004).It commonly occurs as form of plant extracts from Rhinacanthus nasutus. a superficial infection on mucous membranes in Sabouraud fungal culture helps to identify the anti-fungal the mouth or vagina, these patients predominantly develop (Candida) activity (Ramanathan, 2002). Hence, the purpose oropharyngeal or thrush candidiasis, which can lead to of this study is to determine the anti-fungal activity of malnutrition and interfere with the absorption of aqueous extracts on the plant Rhinacanthus nasutus (L) medication and methods of transmission include mother to Kurz. infant through childbirth, people-to-people acquired infections that most commonly occur in hospital settings where immune compromised patients acquire the yeast LITERATURE REVIEW from healthcare workers and has a 40% incident rate (McCullough et al., 1996). Micro pathogens cause many Candida species are ubiquitous organisms, an increasing diseases (Thurairasan, 1993). According to that ancient incidence of fungal infections with Candida albicans has siddhars stated about the Kaalaangal. Fungal diseases are been noted in immune compromised patients (Fidel et al., known as Poonjanoi or Kaalaan (Aanaimugan, 2003). This 1999). Nearly a billion people are estimated to have skin, poonjanam causes conditions are coming under the group nail and hair fungal infections, millions mucosal candidiasis of Kirumiroham or Nunpulukkal. Nunpulukal mean micro- and more than 150 million people have serious fungal organism (Sambasivam Pillai, 1936). Therefore fungus C. diseases, which have a major impact on their lives or are albican is a type of micro-organism (Timbury et al., 2002) fatal. However, severity ranges from asymptomatic-mild or kind of kirumi (Sambasivam Pillai, 1936). Nowadays mucocutaneous infections to potentially life-threatening some western medications are obtained from fungus- systemic infection (Bongomin et al., 2017). Although the poonjai. Vitamin B from Yeast (C. albicans) is good for tissue epidemiology of fungal diseases has greatly changed over (Thissu), brain (Moolai) and Nerves (Narambuvalarchi) the past few decades, Aspergillus, Candida, Cryptococcus (Somasundharam, 2003). According to the above literature species, Pneumocystis jirovecii, endemic dimorphic fungi citations, C. albicans is categorized under the Kirumi/ such as Histoplasma capsulatum and Mucormycetes remain Poonjanamor Nunpulukkal and it causes diseases such as the main fungal pathogens responsible for the majority of Kapha yoni rogam, Asayanoiogal and Vellaisaithal cases of serious fungal disease (Bongomin et al., 2017). C. (Sambasivam Pillai, 1936).The characters of, Kapha Yoni albicans is the main agent responsible for mucosal diseases. rohangalare similar to the vaginal candidiasis, In the last four years, the Leading International Fungal Vellaisaithalare similar to Leucorrhoea and characters of
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16 14 12 10 8 6 4 2 0 Stand Stand Test Test Test Test Test Test ard COLD ard 100% 50% 25% 100% 50% 25% 100% 100% 12h 11.33 8.245 5.818 5.715 0 11.33 13.86 10.60 7.37 24h 11.33 8.29 7.06 6.24 0 11.33 14.13 11.12 8.57 48h 11.33 8.41 7.25 6.87 0 11.33 13.88 11.71 9.03
12h 24h 48h
Figure 1: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutus fresh leaves.
Asayanoigalare similar to the Oral thrush. (Murugesa Muthaliyar, 2003) and seeds are used to Poonjaicauses Poonjaithaaku and known as Candida medicinal purpose (Viviyan, 2008). Poonjaithaaku (Oral thrush) (Rajeshwary and Jegatheesan, 2003) or Asayanoigal (Sambasivam, 1936). It is characterized by creamy plaques a red oozing surface on Action removal on mouth and also redness of mouth and gum Germicide, aphrodisiac, stimulant and alterative (Murugesa (Eerumattrumvaisivappagum), tongue enlargement Mthaliyar, 2003) anti-septic, anthelmintic, anti-parasitic, (Naakuthadikkum) (Vasutheva and Venkadarajan, 2003), anti-cancerous and depurative (Vaidyaratnam, 1995). bad odour in mouth (Vaayilthurnaatarm), muscle growth (Thasaivalrchi) and produce cracks (Vedipugal) (Aanaimugan, 2003). Gunapadam view
Gunam (Qualities):Laghu, (Lightness), Ruksha(Roughness) Review of plant Rhinacanthus nasutus Suvai(Taste): Thuvarpu, (Astringent),Kaippu (Bitter) Veeryam(Potency): Ushnam (Hot) (Murugesa Muthaliyar, Botanical name: Rhinacanthus nasutus or Rhinacanthus 2003) communis Vipakam(Post-digestive action): Thuvarpu(Astringent) Family: Acanthaceae (Devanarayana et al., 2015). Sub family: Acanthoideae Order: Lamiales Genus: Rhinacanthus MATERIALS AND METHODS Specific epithet: (Linn) kurz in journ, Nees Tamil name: Anicahi (Jayapriya and Gricilda, 2015) Plant materials and extraction processes Throughout India on road sides and in the forest glades up to 1,500m (Vaidyaratnam, 1995) Ceylon, Malay, Java, The Rhinacanthus nasutus (L) kurz plant was obtained from Philippine Islands and Madagascar. It is native to Sri Lanka, research medicinal plant garden, Pallekale, Kandy. After the India, Malaysia, Philippine Islands and Tropical Africa. In collection it was authenticated by Gunapadam section, Unit Sri Lanka, it is a very common road-side plant at the edges of Siddha medicine. The plant was thoroughly washed with of the jungle in dry region of Tissamaharama. (Jayaweera, running water for 3 times to move the dust and again 2006).Whole plant (Vaidyaratnam, 1995) Root and leaf rinsed with distilled water. The water removed by blotting Academia Journal of Medicinal Plants; Jesintha et al. 110
over a filter paper and the plant materials dried under the quantity of fungus from the stock culture tube and for other shade and powdered (Air dry 28°C for 2 h).Plant extract fungi, used a half-spear point needle to remove the block of made with hot and cold distilled water with dry powder by agar containing mycelium then placed the block of agar into triturating with motor and pestle (Rajasekaran et al., 2009). the sterile tube face down near the bottom of the slant and Then it both extracts were diluted in a simple dilution withdraw the loop or needle and flame the mouths of the manner to make concentration in the range of 10, 5.0, 2.5 tubes finally replaced the caps and flamed the loop (Juliana, mg/ml to final volume of 10ml (100, 50 and 25%) 2006). (Chowdhury et al., 2011).
Preparation of inoculum Source of organisms The fungal test strain of Candida albicans (MTCC 3017) The C. albicans stock culture was obtained from freeze-dried culture and the suspension was made as per Department of Microbiology, Faculty of Medicine University protocol, 5 ml sterilized water was taken in a micro of Colombo. centrifuge tube and freeze-dried culture was transferred into it and mixed well, the resulting suspensions were vigorously homogenized for 15s with vortex mixer Sterilization (Munavvar et al., 2004). The mixture was allowed to stand for 20 min before transferring it on solid media This was All apparatus and glass wares used were sterilized by heat compared against the 0.5 MacFarland standard to obtain a sterilization: moist heat at 121°C for 20 min in autoclave. fungal concentration 4x106 colony forming cells/ml, were Sterilization of agar medium, needle loop sterilized with inoculated the surface of the solidified media and incubated blue flame of the Bunsen burner. The work bench was at 37°C for 24-48h (Rodriguez-Tudela et al., 2003). sterilized with 70% ethanol, because 70% of solution of ethyl alcohol kills the microbes through degenerating proteins (Timbury et al., 2002). Growth method
A population of micro-organisms is picked up with a sterile Preparation of fungal culture wire inoculation loop. It is diluted by moving the loop back and forth on the surface of agar-solidified in a petri dish as Sabouraud Dextrose Agar was prepared from commercially the loop is streak back and forth and fewer organisms are available dehydrated base according to the manufacturer’s deposited on the surface. Incubation at 37°C for 48 h may instructions; autoclaving for 20 min at 121°C for 15lb/ be necessary petri plates containing sabouraud dextrose immediately after autoclaving, allowed to cool in a 45 to agar (SDA) (HiMedia, Mumbai, India, and Catalogue No. 50°C water bath. Pour the freshly prepared and cooled M063) medium and incubated for 24–48 h at 35°C to give medium into glass petri dishes (100x15mm) on a level, white round colonies against a yellowish background horizontal surface to give a uniform depth of approximately (Taschdjian, 2018). Micro organisms will be repeatedly sub 4mm (Scognamiglio et al., 2010). This corresponds to 60 ml cultured using single streaking method and maintained to of medium for plates with diameters of 150 mm and 25-30 obtain pure isolation on the SDA for further drug sensitivity ml for plates with a diameter of 100mm; used ascetic assay and fungal inoculation prepared by inoculating technique, preferably in a sterile cabinet (Laminar air flow loopful of test organisms and the loop sterilized in a flame cabinet). Final pH should be maintained at 5.6 +/- 0.2 at (Timbury et al., 2002). 25ºC (Basu et al., 2015). The agar medium was allowed to cool to room temperature and unless the plate was used the same day, stored in a refrigerator (2 to 8°C). A represented Determination of antifungal activity by disc diffusion sample of each batch of plates was examined for sterility by method incubating at 30 °C to 35°C for 24 h or longer (Das et al., 2010. Using Stokes Disc diffusion sensitivity testing technique, to each plate a reference antibiotic was applied. The reference antibiotic disc contained Chloramphenicol - 50.0 mg to Transferring slant tube culture of fungi prevent the growth of other moulds). The discs were made by cutting discs (5-6mm) from a filter paper with a Hold the stock culture tube and the sterile agar slant tube in perforator, placing 3 of these discs in a vial and adding the palm of one hand then sterilized the inoculating loop by 10mg/10ml of each extract solution with different flaming it after that removed the cap from each tube and concentration (100, 50 and 25%) diluted with distilled did flame the mouths of the tubes then picked up a small water for the extraction for different concentrations and Academia Journal of Medicinal Plants; Jesintha et al. 111
these were left to dry. Discs were also made for the to compare the mean difference among the controls, for positive control (Standard) Fluconazole was extracts (hot and cold) fresh leaves with the taken 10mg in 10ml and for negative control were used the different time period. distilled water. Each disc was impregnated with the anticipated antifungal plant extract at appropriate concentration. This was then placed on a plate of sensitivity Efficient extract according to independent t-test of fresh testing SDA which was then incubated with the test leaves extract organism C. albicans. Incubation was done at 37°C for 12, 24 and 48 h (Munavvar et al., 2004). The antifungal This is shown in Table 2. compound diffuses from the disc into the medium. Following overnight incubation, the culture was examined for areas of no growth around the disc (zone of inhibition). Test of one-way ANOVA for comparison of mean The radius of the inhibition zone was measured from the difference among the two extracts (hot and cold) dry edge of the disc to the edge of the zone with vernier caliper leaves with the different time period scale. The end point of inhibition is where growth starts. Larger the inhibition zone diameter, greater is the antifungal activities. It is anticipated through the antifungal This is shown in Table 3. activity of plant extract, no area of growth will be induced around the disc. Fungal strains sensitive to the antifungal are inhibited at a distance from the disc whereas resistant Efficiency of the extracts according to independent t-test strains grow up to the edge of the disc. Discs applied to the of dry leaves extract plates already streaked with the fungus (Odds, 1991). This is shown in Table 4.
Cleanup and disposal Comparison of fresh and dry leaves extracts of plant After transfer work is completed area cleaned with Rhinacanthus nasutus disinfectant solution (70% of ethanol) and autoclaved all the used glass materials. The comparison of fresh and dry leaves extracts of the plant
Rhinacanthus nasutus is shown in Figures 1 and 2. Statistical analysis
Used homogeneity, one-way ANOVA and independent t- Anti-fungal activity of stem Rhinacanthusnasutus test in IBM SPSS statistics version 21. Furthermore, t-test was used to compare to the zone of inhibition between hours at P = 0.05 and one-way ANOVA was used to compare A test of one way ANOVA to comparison of mean to the zone of inhibition between concentration at P=0.05. difference among the two extracts (hot and cold) of stem with the different time period
RESULTS Table 5 shows the test of one way ANOVA for comparison of the mean difference among the two extracts (hot and cold) Anti- fungal activity in relation to the concentration of hot of stem with the different time period. and cold extract was analyzed using Homogeneity test and one-way ANOVA for the different extracts (100, 50 and 25%) and different duration (12, 24 and 48 h). Extracts of High efficient extract according to independent t-test of different parts of Rhinacanthus nasutus showed moderate fresh stem extract to potent (Partial to complete zone of inhibition of fungus growth) anti-fungal activity against the C. albicans and This is shown in Table 6. leaves are more efficient than the stem and root.
A test of one way ANOVA comparison of mean difference Anti-fungal activity of leaves of Rhinacanthus nasutus among the two extracts (hot and cold) of stem with the different time period Test of one-way ANOVA
Table 1 shows the test of one – way ANOVA done This is shown in Table 7.
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Table 1: ANOVA test for comparison of mean different among the two extracts of fresh leaves.
Per (%) Sum of squares Df Mean square F Sig HOT-100 Between groups 24.591 2 12.296 141.326 0.000 Within groups 1.305 15 0.087 Total 25.896 17
COLD-100 Between groups 126.588 2 63.294 139.717 0.000 Within groups 6.795 15 0.453 Total 133.383 17
HOT-50 Between groups 12.716 2 6.358 219.747 0.000 Within groups Total 0.434 15 0.029 Total 13.150 17
COLD-50 Between groups 93.055 2 46.528 37.392 0.000 Within groups 18.665 15 1.244 Total 111.720 17
HOT-25 Between groups 7.596 2 3.798 98.638 0.000 Within groups 0.578 15 0.039 Total 8.174 17
COLD-25 Between groups 70.691 2 35.345 20.538 0.000 Within groups 25.815 15 1.721 Total 96.506 17
Table 2: Indicating independent t test of fresh leaves extract.
Concentration Duration Sig (2-tailed) Mean±SD (mm)
100% 12h Cold - 0.000 Cold>hot Hot - 0.000 13.86±0.31 > 8.24±0.23
24h Cold - 0.000 Cold>hot Hot - 0.000 14.14±0.20 > 8.2917±0.23 48h Cold - 0.000 Cold>hot Hot - 0.000 13.86±0.22 > 8.41±0.23 12h Cold - 0.000 Cold>hot Hot - 0.000 10.61±0.86 > 5.82±0.32
50% 24h Cold - 0.000 Cold>hot Hot - 0.000 11.12±1.12 > 7.10±0.10
48h Cold - 0.000 Cold>hot Hot - 0.001 11.71±1.12> 86±0.71 12h Cold - 0.000 Hot 25% 24h Cold - 0.05 Cold>hot Hot - 0.15 7.37±0.73 > 5.72±0.33 48h Cold - 0.06 Hot Table 3: ANOVA test for comparison of mean difference among the two extracts of dry leaves Per (%) Sum of squares Df Mean square F Sig HOT-100 Between groups 17.824 2 8.912 14.298 0.000 Within groups 9.350 15 0.623 Total 27.174 17 COLD-100 Between groups 7.167 2 3.584 7.324 0.006 Within groups 7.339 15 0.489 Total 14.507 17 HOT-50 Between groups 22.627 2 11.313 24.950 0.000 Within groups 6.802 15 0.453 Total 29.488 17 COLD-50 Between groups 30.431 2 15.215 29.185 0.000 Within groups 7.820 15 0.521 Total 38.251 17 HOT-25 Between groups 23.372 2 11.686 27.082 0.000 Within groups 6.473 15 0.432 Total 29.845 17 COLD-25 Between groups 9.125 2 4.562 17.014 0.000 Within groups 4.022 15 0.268 Total 13.147 17 Table 4: Indicating significant P values dry leaves extract. Concentration Duration Sig 2-tailed Mean±SD (mm) Cold -0.000 Hot Cold -0.000 Hot Hot - 0.03 Hot Hot - 0.06 Hot Hot - 0.04 Hot Hot - 1.013 Hot 16 14 12 10 8 6 4 2 0 Standa Standa Test Test Test25 Test Test Test rd rd 100% 50% % cold 100% 50% 25% 100% 100% 12h 11.33 7.8 7 5.41 0 11.33 13.41 8.21 6.24 24h 11.33 8.3 7.36 5.61 0 11.33 13.35 8.44 6.89 48h 11.33 8.4 7.39 5.62 0 11.33 13.35 8.51 7.16 Figure 2: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutus dry leaves. Table 5: ANOVA test for comparison of mean difference among the two extracts of fresh stem. Per (%) Sum of squares Df Mean square F Sig HOT-100 Between groups 2.376 2 1.188 6.436 0.001 Within groups 2.769 15 0.185 Total 5.145 17 COLD-100 Between groups 23.412 2 11.706 101.124 0.000 Within groups 1.736 15 0.116 Total 25.149 17 HOT-50 Between groups 37.077 2 18.539 88.880 0.000 Within groups 3.129 15 0.209 Total 40.206 17 COLD-50 Between groups 40.544 2 20.272 572.927 0.000 Within groups .531 15 0.035 Total 41.075 17 HOT-25 Between groups 29.646 2 14.823 68.942 0.000 Within groups 3.225 15 0.215 Total 32.871 17 COLD-25 Between groups 58.295 2 29.147 108.341 0.000 Within groups 4.036 15 0.269 Total 62.330 17 Academia Journal of Medicinal Plants; Jesintha et al. 115 Table 6: Indicating significant P values fresh stem extract Concentration Duration Sig 2-tailed Mean±SD (mm) Cold - 0.000 Hot>cold 12h Hot - 0.000 11.81±0.70 > 8.72±0.34 Cold -0.000 Hot>cold 24h 100% Hot - 0.000 11.81±0.70 > 8.60±0.34 Cold - 0.000 Hot>cold 48h Hot - 0.000 11.81±0.69 > 8.72±0.32 Hot – 0.033 Hot>cold 12h Cold - 0.054 10.24±2.23 > 7.96±0.33 Cold - 0.000 Hot>cold 24h 50% Hot - 0.000 11.81±0.70 > 8.59±0.34 Cold - 0.000 Hot>cold 48h Hot - 0.001 11.81±0.70 > 8.59±0.34 Hot – 0.362 Hot>cold 12h Cold - 0.377 3.31±1.50 > 2.70±0.46 Hot – 0.000 Hot>cold 24h 25% Cold – 0.001 4.99±0.39 > 3.59±0.53 Hot – 0.000 Hot>cold 48h Cold – 0.000 5.31±1.99 > 4.96±0.39 Table 7: ANOVA test for comparison of mean difference among the two extracts of dry stem Per (%) Sum of squares Df Mean square F Sig HOT-100 Between groups 5.398 2 2.699 6.644 0.009 Within groups 6.094 15 0.460 Total 11.492 17 COLD-100 Between groups 7.339 2 3.700 37.367 0.000 Within groups 1.485 15 0.099 Total 8.884 17 HOT -50 Between groups 5.761 2 2.881 7.166 0.000 Within groups 6.030 15 0.402 Total 11.791 17 COLD-50 Between groups 9.240 2 4.620 48.078 0.000 Within groups 1.441 15 0.096 Total 10.682 17 HOT-25 Between groups 9.115 2 4.558 15.212 0.000 Within groups 4.494 15 0.300 Total 13.609 17 COLD-25 Between groups 12.459 2 6.230 82.666 0.000 Within groups 1.130 15 0.075 Total 13.589 17 Academia Journal of Medicinal Plants; Jesintha et al. 116 Table 8: Indicating significant P values dry stem extract. Concentration Duration Sig 2-tailed Mean±SD (mm) Hot – 0.029 Hot Cold -0.001 Hot Cold - 0.001 Hot Hot – 0.001 Hot Cold - 0.000 Hot Cold -0.001 Hot Hot – 0.000 Cold>hot 12h Cold - 0.000 6.61±0.24>6.13±0.35 Hot – 0.001 Hot Hot – 0.136 Hot High efficient extract according to independent t-test of Comparison of mean difference among the two extracts dry stem extract (hot and cold) of dry root with the different time period This is shown in Table 8. Table 11 shows the ANOVA test for comparison of the mean difference among the two extracts of fresh root. Comparison of dry and fresh stem extracts of plant Rhinacanthus nasutus High efficient extract according to independent t-test of fresh root extract The comparison of dry and fresh stem extracts of plant Rhinacanthus nasutus is shown in Figures 3 and 4 This is shown in Table 12. Anti-fungal activity of root of Rhinacanthus nasutus Comparison of dry and fresh root extracts of plant Rhinacanthus nasutus Test of one-way ANOVA to Comparison of mean difference among the two extracts (hot and cold) dry The comparison of dry and fresh root extracts of plant root with the different time period Rhinacanthus nasutus is shown in Figures 5 and 6 This is shown in Table 9. DISSCUSSION The activity of plant extracts against the fungus has been High efficient extract according to independent t-test of studied for years, but more aggressively during the last dry root extract three decades (Bongomin et al., 2017). Few of the micro- organisms that abound in nature are diseases producing, or This is shown in Table 10. pathogenic, for human (Ramage et al., 2013). The Academia Journal of Medicinal Plants; Jesintha et al. 117 14 12 10 8 6 4 2 0 Stan Stan Test Test Test 1. CO Test Test Test dard dard 100% 50% 25% LD 100% 50% 25% 100% 100% 12h 11.3 11.9 10.6 10.4 0 11.3 11.2 10.2 9.13 24h 11.3 12.1 10.6 10.7 0 11.3 11.0 10.7 10.1 48h 11.3 12.6 10.7 11.0 0 11.3 12 11.4 10.6 Figure 3: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutusfresh stem. 14 12 10 8 6 4 2 0 Stan Stan Test 1. Test dard Test Test dard Test Test 100 CO 100 100 50% 25% 100 50% 25% % LD % % % 12h 11. 8.6 5.5 3.3 0 11. 7.9 7.1 2.6 24h 11. 11. 7.5 4.9 0 11. 8.5 6.0 3.5 Figure 4: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutus dry stem. role in the discovery of new products from plants as present study reveals that Rhinacanthus nasutus has chemotherapeutic agents (Samuelsson et al., 1991). The components that can exert significant anti-fungal activity Academia Journal of Medicinal Plants; Jesintha et al. 118 Table 9: Indicating ANOVA test for comparison of mean difference among the two extracts of dry root. Per (%) Sum of sq. Df Mean square F Sig HOT-100 Between groups 6.007 2 3.003 8.214 0.004 Within groups 5.485 15 0.366 Total 11.492 17 COLD-100 Between groups 7.386 2 3.693 36.969 0.000 Within groups 1.498 15 0.100 Total 8.884 17 HOT-50 Between groups 6.481 2 3.241 9.154 0.003 Within groups 5.310 15 0.354 Total 11.791 17 COLD-50 Between groups 9.171 2 4.585 45.510 0.000 Within groups 1.511 15 0.101 Total 10.682 17 HOT-25 Between groups 10.515 2 5.257 25.482 0.000 Within groups 3.095 15 0.206 Total 13..609 17 COLD-25 Between groups 12.506 2 6.253 86.608 0.000 Within groups 1.083 15 0.072 Total 13.589 17 Table 10: Indicating significant P values dry root extract Concentration Duration Sig 2-tailed Mean±SD (mm) 100% 12h Hot – 0.052 Both are not significant Cold - 0.053 11.51±0.46<11.98±0.20 24h Hot – 0.051 Both are not significant Cold – 0.072 11.74±0.50<12.20±0.05 48h Hot – 0.048 Hot 50% 12h Hot – 0.550 Both are not significant Cold - 0.556 10.86±0.90>10.60±0.39 24h Hot – 0.164 Both are not significant Cold – 0.177 11.22±0.89>10.62±0.40 48h Hot – 0.018 Hot 25% 12h Hot – 0.003 Hot 24h Hot – 0.013 Hot Table 11: Indicating ANOVA test for comparison of mean difference among the two extracts of fresh root. Per (%) Sum of sq. Df Mean square F Sig COLD-100 Between groups 13.370 2 6.934 212.262 0.000 Within groups 4.680 15 0.033 Total 37.050 17 HOT-100 Between groups 32.370 2 16.185 51.874 0.000 Within groups 4.680 15 0.312 Total 14.358 17 COLD -50 Between groups 3.152 2 1.576 10.144 0.000 Within groups 2.330 15 0.155 Total 5.482 17 HOT-50 Between groups 16.537 2 8.268 40.460 0.000 Within groups 3.065 15 0.204 Total 19.602 17 COLD-25 Between groups 5.229 2 2.614 16.577 0.000 Within groups 3.040 15 0.203 Total 8.269 17 HOT-25 Between groups 6.389 2 3.194 20.538 0.000 Within groups 2.890 15 0.193 Total 9.279 17 Table 12: Indicating significant P values fresh root extract Concentration Duration Sig 2-tailed Mean±SD 12h Hot – 0.187 Both are not significant 100% Cold - 0.209 10.83±0.31<11.28±0.75 24h Hot – 0.854 Both are not significant Cold – 0.854 11.07±0.59<11.13±0.55 48h Hot – 0.023 Hot 12h Hot – 0.001 Hot 24h Hot – 0.020 Hot 25% 12h Hot – 0.001 Hot 24h Hot – 0.001 Hot 14 12 10 8 6 4 2 0 Stand Stand Test Test Test 1. CO Test Test Test ard ard 100% 50% 25% LD 100% 50% 25% 100% 100% 12h 11.3 11.5 10.8 10.0 0 11.3 11.9 10.6 10.4 24h 11.3 11.7 11.2 10.2 0 11.3 12.1 10.6 10.7 48h 11.3 12.2 11.6 10.4 0 11.3 12.6 10.7 11.0 Figure 5: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutus dry root. 14 12 10 8 6 4 2 0 Stand Stand Test Test Test 1. Test Test Test ard ard 100% 50% 25% HOT 100% 50% 25% 100% 100% 12h 11.3 11.2 10.2 9.13 0 11.3 10.8 10 7.58 24h 11.3 11.0 10.7 10.1 0 11.3 11.0 10.3 8.76 48h 11.3 12 11.4 10.6 0 11.3 11.1 10.6 9.7 Figure 6: Zone of inhibition of hot and cold extracts of Rhinacanthus nasutus fresh root. against C. albicans. The results of anti-fungal activity of different concentration (100, 50 and 25%) and different plant Rhinacanthus nasutus revealed a maximum zone of time period (12, 24 and 48 h) with different parts (Leaves, inhibition of fungal growth (C. albicans) was observed with stem and root). According to the statistical analysis all the Academia Journal of Medicinal Plants; Jesintha et al. 121 parts (Leaves, root and stem) are having anti-fungal activity because the zone of inhibition of fluconazole is 11.33±0.47 against the C. albicans with different concentration (100, 50 (<16mm) (Mohd et al., 2012) but the leaves of plant and 25%) and duration (12, 24 and 48 h). However the Rhinacanthus nasutus showed maximum zone of inhibition results of crude extracts of Rhinacanthus nasutus where against the C. albicans with positive (Fluconizole) and investigated for their potential anti-fungal activity, revealed negative control.Still no actions proved in leaves, root and a complete zone of inhibition of fungal growth in case of all stem of plant Rhinacanthus nasutus fresh and dry extract, so fungi tested at the concentration of 5mg/ml (Munavvar et the process of zone of inhibition (anti-fungal activity) is al., 2004), hence Rhinacanthus nasutus extract was not compared to the effects of Trihumors, suvai, veeriyam and active against C. albicans at concentration up to 2000 µl/ml vibakam. Effect on Dosha: It pacifies vitiated Kapha and (Puttarak et al., 2010) hence the extracts of petroleum Vatadosha (Later stage it will be aggravated) due to its ether, ethyl acetate, chloroform are accordingly, 22, 9, 20 thuvarpu and kaippusuvai and Ushna Veeryam and 18 mm inhibit the zone, but does not inhibit to the (Uthamarayan, 2005). methanolic extract ofthe Rhinacanthus nasutus leaves, Some texts are mentioned, the poonajithaaku (C. albicans) therefore water extracts are more efficient then the occurs due to the “Sleshma” vitiation to the body petroleum ether and methanolic extract (Jayapriya and (Rajeshwary and Jegatheesan, 2003) and also these Gricilda Shoba, 2015) and also leaves extract contain AgNps “Sleshma” vitiation occur due to the kalanilaima atrangal showed potential activity against all fungal colonies on such as (pin panikaalm and elavenilkalam) (Shanmugavelu, invitro antimicrobial study (Rao et al., 2012). Filter paper 1988). In these periods vatha get aggravation due to the disc impregnated with various extracts (20 mg/ml) of kaphadhosha vitiation and some infections (Ramanathan, Rhinacanthus leaves individually with petroleum ether 2002) pain and ulcers (Thrush) are produce in the Candida extract and methanol extract and ethyl acetate extract are infections (Timbury et al., 2002) can arises to the body or accordingly showed 00mm, 10mm and 22mm (Rajasekaran body parts. The treatment should be done balancing the et al., 2009), the zone of inhibition high in ethyl acetate Vathadhosha and by eliminating the kaphadhosha therefore however, aqueous extract is much more efficient ushnaveeriyam balances the vatha and eliminates the kapha (14.1350±0.196) than the methanol extract. (Shanmugavelu, 1988). This plant Rhinacanthus So far no recent articles shows the anti-fungal activity nasutus(Anichai) is an anti-fungal plant (Ramanathan, stem and root of plant Rhinacanthus nasutus, but plant 2002) and also it has an ushnaveeriyam(Murugesa Rhinacanthus nasutus stem has same chemical composition Muthaliyar, 2003) so far the plant (Anichai) can pacify the of leaves which act against the anti-microbial activity (Tian Kaphadhosha and cure the poonjaithaaku (C. albicans) et al., 1995) and the continuing investigation of the root based diseases. Plant Rhinacanthus nasutus has the of Rhinacanthus nasutus afforded a 1,4-naphthoquinone character of destroying the Rasa thadhu based ester, rhinacanthin-Q, accompanied by twenty-four known kirumikootangal (viviyan, 2008). The pathogenesis of C. compounds (Wu et al., 1988), and the stem has the albican has the ability to slough off epithelial tissues and compounds of 3, 4-dihydro-, 3-dimethyl-2H-naphtho [2, 3- plasma cells (Tsui et al., 2016), plasma consists of rasa 3] pyran-5, 10-dione [1] these compounds also act as an dhadhu (Shanmugavelu, 1988) and according to this theory anti-fungal (Kodama et al., 1993). Furthermore, the present the plant Rhinacanthus nasutus has the capability to act study reveals, different extracts of this plant showed against to the C. albicans. Plant Rhinacanthus nasutus has statistically significant value (P<0.05), hence the leaves Kaippusuvai and Thuvarpusuvai and they reduce the (14.1350±0.196) have more effective anti-fungal activity vitiated Kaphadhosha. Kaippusuvai consists of vayu and than the root (12.6500±0.281) and stem (11.8050±0.69) visumbu so it pacifies the kapham and Thuvarppu consist of and the fresh extracts can exert consistently better anti- vayu and pirithivi hence it also pacifies the kapham. fungal activity than the dry extract Kaippusuvai acts as an anti-infectious and Thuvarpusuvai (14.1350±0.196>13.3550±1.80) (Figures 2 to 6). The acts as anti-inflammatory (Uthamarayan, 2005). Therefore aqueous (hot and cold) of Rhinacanthus nasutus used was both suvai could act against the fungal infection found to have potent anti-fungal effect at the different (poonjaithaaku). Each taste has an energetic effect on concentration and different time duration. According to digestion called “Veeriyam” or “Potency”. The plant that cold extract is more efficient than the hot extract Rhinacanthus nasutus possesses the ushnaveeriyam, the (14.1350±0.196>13.3550±1.80) refer Tables 5, 8 and 10. increased kapha can be reduce by characters such as laghu This difference in activity of the fresh and dry extract may (Shanmugavelu, 1988). Based on the ushnaveeriyam where be related to the polarity of anti-fungal compounds present it increases the pitham and reduces both the vatha and in Rhinacanthus nasutus (Kodama et al., 1993).Mostly 100% kapham (Uthamarayan, 2005). of concentration is more potent than other concentrations Pungent, bitter, and astringent plants have a pungent (50 and 25%) and 24h and 48h are more effective time vipaka. This plant owes to the action of pungent (kaarpu), period than the 12h, so accordingly, the plant Rhinacanthus also it pacifies the kapham. Vipakam is the outcome of nasutus has an effective anti-fungal activity than the digestion and metabolism occurs due to digestive enzymes. positive control Flucanizole (14.1350±0.196>11.33±0.47), The drugs made from the plant Anichai (Rhinacanthus Academia Journal of Medicinal Plants; Jesintha et al. 122 nasutus) can be cure the Nunpulukal. Fungal (C. albicans) its history, taxonomy, epidemiology, virulence attributes and methods coming under the Nunpulukal (Sambasivam Pillai, of strain differentiation. Int. J. Oral Maxillofac. Surg. 25(2): 136-144. Mitchell AP (1998). Dimorphism and virulence in Candida albicans. Curr. 1936).Therefore stanza for Rhinacanthus nasutus Opin. Microbiol. 1(6): 687-692. mentioned in Gunapadam text was scientifically proved Munavvar Z, Azmin MN, Nor Azizan A (2004). Evaluation of anti-fungal through this study. Therefore based on the (Statistical and Anti-bacterial activity of a local plant Rhinacanthus nasutus (L.). J. analysis) the different parts, concentration and effective Biol. Sci. 4(4): 498-500. Murugesa Muthaliyar, K., 2008. Siddha materia medica medicinal plants duration of plant Rhinacanthus nasutus have scientifically division. In: 2. edition, ed. Chennai 600106: India maruthuvam proved the anti-fungal activity against the C. albicans. homeopathy, pp. 115, 142 and 799. Murugesa Muthaliyar KS (2003). Siddha Materia Medica medicinal plants division. Chennai 600106: India maruthuvam homeopathy. Odds F (1991). Sabouraud('s) agar. J. Med. Vet. Mycol. 29(6): 355-359. CONCLUSION Patil SB, Patil MS, Chittam KP, Wagh RD (2014). A review on Ayurvedha and Siddha: Indian systems of medicine. Pharm. Sci. Monitor. 5(2): 40- As per this laboratory experimental study, the data analysis 49. reveals the plant (Extract) possessing the Anti-fungal Pruksakorn P, Jaima C, Panyajai P, Mekha N, et al (2018). Antifungal activity of Rhinacanthus nasutus (L.) Kurz extracts against activity with maximum zone of inhibition. The quotation for dermatophytes. J.Thai Tradit. Altern. Med. 16: 2. general character of Rhinacanthus nasutus is scientificially Puttarak P, Charoonratana T, Panichayupakaranant P (2010). proven from the above data analysis. Antimicrobial activity and stability of rhinacanthins-rich Rhinacanthus nasutus extract. Phytomedicine. 17(5): 323-327. Rajasekaran A, Sundaranadavalli S, Murugesan S (2009). Antibacterial and antifungal evaluation of the leaves of Rhinacanthus nasutus linn. Int. J. REFERENCES ChemTech Res. 1(3): 574-576. Rajeshwary G, Jegatheesan R (2003). Kulandhai Nala Maruthuvam- Aanaimugan S (2003). Mahapperum Mahalir Maruthuvamum. 1st ed. Thohuthi. 1st ed. Thanjavoor: Thamil Plagalaikalga veliyeedu. Palmerston, North Newzeland. Ramage G, Rajendran R, Sherry L, Williams C (2013). Fungal biofilm Akpan A, Morgan R (2002). Oral Candidiasis. Postgrad. Med. J. 78(922): resistance. Int. J. Microbiol. 4(2): 1266-1273. 455-459. Ramanathan P (2002). Maruthuva nokkil Siddharkalum Basu S, Bose C, Ojha N, Das N, et al (2015). Evolution of bacterial and thathuvasindhanaigalum. 1st ed. Chennai: Thamarai pathipagam. fungal growth media. Bioinformation. 11(4): 182-184. Rao PV, Sujana P, Vijayakanth T, Naidu MD (2012). Rhinacanthus nasutus - Bongomin F, Gago S, Oladele RO, Denning DW (2017) . Global and Multi- Its protective role in oxidative stress and antioxidant status in National Prevalence of fungal diseases-Estimate Precision. J. Fungi streptozotocin induced diabetic rats. Asian Pac. J. Trop. Dis. 2(4): 327- (Basel). 3(4): 57. 330. Chowdhury NS, Alam B, Zahan R, Sultana S, et al (2011). Antimicrobial and Rodriguez-Tudela JL, Chryssanthou E, Petrikkou E, Mosquera J, et al toxicity studies of different fractions of the aerial parts of the Mikania (2003). Interlaboratory evaluation of hematocytometer method of cordata. Int. J. Pharm. Life Sci. 2(3): 592-598. inoculum preparation for testing antifungal susceptibilities of Christina T, Kong EF, Jabra-Rizk MA (2016). Pathogenesis of Candida filamentous fungi. J. Clin. Microbiol. 41(11): 5236-5237. albicans biofilm. Pathog. Dis. 74(4): ftw018. Sambasivam Pillai TV (1936). Dictionary based on Siddha and indian Das S, Sharma S, Kar S, Sahu SK, et al (2010). Is inclusion of Sabouraud Medical Science. In: Siddha Medical Dictionary. Chennai-106: dextrose agar essential for the laboratory diagnosis of fungal keratitis. Department of Indian Medicine and Homeopathy. p. 123. Indian J. Ophthalmol. 58(4): 281-285. Samuelsson G, Rah F, Laeson C, Gos PH, et al (1991). Inventory of plants Denning DW, Kneale M, Sobel JD, Rautemaa-Richardson R (2018). Global used in traditional medicine in somalia.I. Plants of the families burden of recurrent vulvovaginal candidiasis: a systemic review. Lancet Acanthaceae-chenopodiaceae. J. Ethnopharmacol. 35(1): 25-63. Infect. Dis. 18(11): e339-e347 16. Scognamiglio T, Zinchuk R, Gumpeni P, Larone DH (2010). Comparison of Devanarayana, A, Ediriweere ERHSS, Soysa SSSBDP, Karunarathna N inhibitory mold agar to sabouraud dextrose agar as a primary medium (2015). Therapeutic usages of Rhinacanthus nasutus (L) Kurz (aniththa) for isolation of fungi. J. Clin. Mcrobiol. 48(5): 1924-1925. in srilanka traditional medicine. Unique J. Ayurvedha Herb. Med. Shanmugavelu V (1988). Siddha Marthuvam, noi nadal noi mudhal naadal 3(4):10-15. thirattu pakudhi 2. 2nd ed. Chennai: Indian Maruthuvam Homeopathi Ellepola AN, Samaranayake LP (2000). Oral candidal infections and thurai. Antimycotics. Crit. Rev. Oral Biol. Med. 11(2): 172-198. Somasundharam S (2003). Maruthuvaththavarviyal pahuthi 1. In: 6th ed. Fidel Jr PL, Vazquez JA, Sobel JD (1999). Candida Glabrata: Review of Chennai, India: Aalvin enterprises. p. 108. epidemiology, pathogenesis, and clinical disease with comparison to C. Sudbery P, Gow N, Berman J (2004). The distinct morphogenic states of Albicans. Clin. Microbiol. Rev. 12(1): 80-96. Candida albicans. Trends Microbiol. 12(7): 317-324. Gul F, Shinwari ZK, Afzal I (2012). Screening of indigenous knowledge of Suresh B, Kalyanaraman VR, Dhanashekaran S, Dhanraj SA, Dube R (1994). herbal remedies for skin diseases among Local Communities of North Evaluation of certain siddha drugs in the treatment of candidiasis. Anc. West Punjab, Pakistan. Pak. J. Bot. 44(5): 1609-1616. Sci. Life. 14(1-2): 16-20. Jayapriya G, Gricilda Shoba F (2015). Phytochemical analysis and Taschdjian CL (2018). Routine Identication of Candida albicans: Current antimicrobial efficacy of Rhinacanthus nasutus (l) Linn. J. Pharmacogn. Methods and a new medium. Mycologia. 49(3): 332-338. Phytochem. 3(6): 83-86. Thurairasan K (1993). Noiyila nerin-Siddha Hygiene and Preventive Jayaweera D (2006). Medicinal plants (Indigenous & exotic ) used in Medicine. 3rd ed. Kiindy, Chennai-106: Indhiya Maruthuvam Matrum ceylon part 1. In: W. t. u. b. s. LK, ed. Colombo, Sri lanka: The nation Omiyopathy Iyakkam. science foundation. p. 21. Timbury M, Cartiney CM, Thakker B, Ward K (2002). Notes on Medical Juliana TH (2006). Techniques for studying bacteria and Fungi. In: USA: Microbiology. 5th ed. United Kingdom. pp. 518-522 Microbiology department, Carolina Biological supply company. pp. 18- Tsui C, kong EF, Jabra-Rizk MA (2016). Pathogenesis of Candida albicans 26. biofilm. Pathog. Dis. 74(4): ftw018. Kodama O, Ichikawa H, Akatsuka T, Santisopasri V, Kato A, Hayashi Y Uthamarayan S (2005). A compendium of siddha doctrine - Fundamental (1993). Isolation and identification of an antifungal naphthpyron principles of sidha system of medcine. 1st ed. Chennai: Department of derivative from Rhinacanthus nasutus. J. Nat. Prod. 56(2): 292-294. Indian Medicine and Homeopathy, Chennai-106. McCullough MJ, Ross BC, Reade PC (1996). Candida albicans: a review of Vaidyaratnam P (1995). Indian medicinal plants. 1st ed. Madras India: Academia Journal of Medicinal Plants; Jesintha et al. 123 Longman Anna Salai. Zhang X (2004). The role of traditional Knowledge in Healthcare and Vasutheva SK, Venkadarajan S (2003). Sarabendhira Vaithiya Muraigal Agriculture. 1st ed. New York and Geneva: United Nation Publication. (Siroroga Sigitchai). 3rd ed. Thanjavoor: Saraswathi mahal noolagam. Viviyan S (2008). Materia Medica II. In: Colombo 12: pp. 9-10. Wu TS, Tien HJ, Yeh MY, Lee KH (1988). Isolation and cytotoxicity of rhinacanthin-A and -B, two; naphthoquinones, from Rhinacanthus nasutus. Phytochemistry. 27(12): 3787-3788.