Abstract: The present study describes the antifungal potential of fruit and powdered ingredients of triphala churna, i.e. Emblica officinalis (Garetn.) (Amla), Terminalia bellirica (Gaertn.) Roxb. (Baheda) and Terminalia chebula (Retz.) (Harada), collected from the market of Gwalior (M.P.), India. Water extracts of all the fruits and powdered samples were tested (in vitro) for their antifungal activities by poisoned food technique against different Aspergillus species (A. flavus, A. fumigatus, A. versicolor, A. terreus and A. niger) associated with them during storage. All extracts displayed varied levels i.e. very low to very high antifungal activities on four Aspergillus species. The aqueous extracts of fresh fruits (37.96±7.59%) was observed to be most effective than dry fruits (34.95±7.59%) and powder (25.07±6.05%). Terminalia chebula (fresh and dry) extracts were found most active against the four Aspergillus species with 49.15 and 40.8% inhibition, respectively. None of the extracts were found effective against the growth of A. niger. All fruits and powdered aqueous extracts were observed to be ineffective against the A. niger. The variability in antifungal activity of aqueous extracts in the present study may be useful to study the relationship between antifungal potential of herbal drugs and prevalence of fungal contaminant during their storage.
INTRODUCTION
Plants have been used to treat or prevent illness since before recorded history. “Vrikshayurveda”, compiled before the beginning of Christian era, “Rig Veda” in 2000 B. C. have mentioned the use of a number of plants as medicines (Tandon et al., 2004). Nearly 80% population of the globe in developing countries still rely on traditional medicine for their primary healthcare (Shrikumar and Ravi, 2007) and about 25% of all modern medicine are directly or indirectly derived from plants (Calixto, 2000). This medicinal potential of herbal drugs is due to the presence of numerous biologically active compounds, which have been also reported to exhibit antimicrobial and insecticidal properties (Sulieman et al., 2007; Mann et al., 2008; Mahesh and Satish, 2008; Gayathri and Kannabiran, 2009). Triphala churna a wonderful ancient drug of Ayurveda, considered as a perfect tonic for proper digestion (Pandey et al., 2008). Triphala, literally means “three fruits, is a combination of Emblica officinalis (Garetn.) (Amla), Terminalia bellirica (Gaertn.) Roxb. (Baheda) and Terminalia chebula (Retz.) (Harada), which are medicinally important in raw and powdered form. This wonderful ayurvedic drug aids recovery from bowel complaints, lowers blood and cholesterol levels primarily, which are all afflictions that many of us fall victim to at some point in our lives (Juss, 1997). But as the case with other herbal drugs, raw and powdered ingredients of triphala churna i.e. E. officinalis, T. bellirica and T. chebula are also subject to operations of contamination by various microorganisms during growth (while the fruits are on tree), after harvesting (when fruits are dried), processing and during storage. Post-harvest spoilage by filamentous fungi is also one of the most common threats associated with stored raw and processed herbal products. Fungal contamination of stored herbal drugs not only linked to discoloration, quality deterioration, and reduction in commercial values as well as in therapeutic potential but may produces secondary metabolites like mycotoxins (Roy et al., 1988). Presence of mycotoxins may degrade the product, leading to decrease in the phytochemical and medicinal properties of herbal drugs and their raw materials which may reduce the antimicrobial potential also (Truckesses and Scott, 2008). Contamination of triphala churna, its raw and powdered ingredients by different Aspergillus species has already been reported (Gautam and Bhadauria, 2008; 2009, 2011). Therefore, the main objective of the present study was to examine the antifungal efficacy of raw and powdered ingredients of triphala churna against different Aspergillus species, which are associated with them during their storage.
MATERIALS AND METHODS
Fresh fruits of all the three medicinal plants were collected at the time of experiment. Sun dried fruits and powder form of these fruits were collected from the market of Gwalior (M.P.), India, in clean labeled packets and stored in the laboratory at the room temperature.
Isolation of mycoflora: Isolation of mycoflora from stored fruit samples of E. officinalis, T. bellirica and T. chebula was done by using Potato Dextrose Agar (PDA) and Czapek Dox Agar culture media. Small pieces of fruits samples were inoculated in each plate in triplicates along with a control (without samples). After inoculation plates were incubated for seven days at 25±1°C. The plates were examined after 3 to 7 days of incubation period. Morphological and cultural characteristics of different fungal isolates were studied and were identified on the basis of color, colony shape and change in their shape during growth (Gilman, 2001). Frequently occurring Aspergillus isolates were further confirmed at IARI, New Delhi. Pure culture of different Aspergillus isolates isolated from stored fruit samples were prepared on Czapek Dox agar media for five days at the optimum temperature.
Test organisms: Isolates of frequently isolated Aspergillus spp. (A. flavus #7413.09; A. fumigates #7408.09; A. versicolor, A. terreus #7406.09 and A. niger #7414.09) were selected to test the effect of different fruit and powder extracts on their mycelial growth.
Preparation of aqueous extracts of fruit and powdered samples: The fresh, sun dried fruits and powders of E. officinalis, T. bellirica, and T. chebula were analysed in the present study. The detailed descriptions about the samples (NISCAIR, 2010) are listed in Table 1. The fresh and sun dried fruit samples were first surface sterilized with 0.01% HgCl2, washed with sterilized distilled water and then air dried. Hundred gram of grinded powder of each fruit sample were taken in a beaker containing water 100 mL sterilized distilled water. Similarly, 100 g of powdered samples of E. officinalis, T. bellirica and T. chebula (sold in the market) were taken in a beaker containing 100 mL sterilized distilled water and boiled at 80°C for 10 min water bath (Awuah, 1989). The material was homogenized for 5 min, filtered through muslin cloth and filtrate was centrifuged at 5000 rpm for 15 min. The clear supernatant was collected. This was considered as 100% basic stock (Kiran and Adiver, 2006).
Screening of antifungal activity: Antifungal screening was carried out using poisoned food technique. Different concentrations of aqueous extracts (5, 10, and 20%) of fruit and powdered samples were incorporated. Small disk of the fungus culture was cut with a sterile cork borer from seven days old culture of test fungus (five isolates of Aspergillus flavus, A. fumigatus, A. versicolor, A. terreus and A. niger) and transferred aseptically in the centre of a petri dish containing the medium with different concentrations of extract. Three plates of each sample were used for each concentration as replicates. Control was kept where the culture disk were grown under same conditions on Czapek agar media without any extract. Inoculated plates were incubated at 27°C until mycelial growth of fungus covered the whole surface of medium in control treatment.
| Table 1: | Ethnobotanical and phytochemical data of triphala ingredients |
The fungus colony diameter was measured every 24 h. The colony diameter was compared with control (New, 1971). The efficacy of fruit and powder extracts in terms of percentage inhibition of mycelial growth was calculated by using the following formula (Singh and Tripathi, 1999):
| Where: | ||
| dc | = | Average increase in mycelial growth in control |
| dt | = | Average increase in mycelial growth in treatment |
RESULTS
A total of six Aspergillus isolates namely, Aspergillus niger, A. flavus, A. fumigatus, A. terreus, A. versicolor and A. parasiticus were isolated from fruit samples along with species of Penicillium, Alternaria, Curvularia, Syncephalastrum and Rhizopus. Frequently occurring Aspergillus species were confirmed further and used as test organism. The efficacy of fresh and shade dried fruits and powder extracts of E. officinalis, T. bellirica and T. chebula were evaluated in the present study. All the fruit and powdered extracts had shown more or less inhibitory effect on mycelial growth of the test organisms. The antifungal potential of the aqueous extracts was assessed by zone diameter (mm) measurement. The effectiveness of the extracts increased with an increase in concentration and it was recorded maximum at 20% concentration. Strong antifungal activity was shown by fresh fruits as compared to their dry fruits and powder extracts. All the aqueous extracts showed antimicrobial activity against at least four of the types of Aspergillus isolates tested, i.e. A. terreus, A. flavus, A. versicolor, A. fumigatus. None of the aqueous extracts were found effective against A. niger (Table 2).
The percent inhibition of aqueous extracts of fruit and powdered samples on mycelial growth of Aspergillus isolates were summarized in Table 3. The results revealed that fresh fruits were found more inhibitory (37.96±11.52) than dry (34.95±7.59) and powdered (25.07±6.05) samples. Among fruits extracts maximum inhibition of mycelial growth of fungi was obtained with fresh fruit aqueous extract of T. chebula (49.15±9.73) followed by dry fruits of T. chebula (40.8±9.13), fresh fruits of T. bellirica (38.61±12.07), dry fruits of E. officinalis (37.7±8.40). Minimum percent inhibition in mycelial growth was observed in dry and fresh fruits of T. bellirica (26.37±4.80) and E. officinalis (26.12±15.44), respectively.
Among powdered samples of all the three fruits extracts maximum inhibition of mycelial growth of fungi was obtained with aqueous extract of T. chebula (31.69±5.69) followed by E. officinalis powder (23.95±2.58). Minimum percent inhibition in mycelial growth was observed in T. bellirica powder (19.66±2.41). The gradation of antifungal potential i.e. low (+), medium (++), high (+++), very high (+++++) and no inhibition (-) of fruit and powdered samples against Aspergillus spp. is summarized in Table 4.
The detailed results about inhibition of mycelial growth Aspergillus isolates by different concentrations of aqueous extracts of fruits and powdered samples are depicted in Table 3. Our data revealed that the percent inhibition of mycelial growth was observed in the range of 60 to 17.31%. Among all the Aspergillus isolates, highest inhibition was recorded in A. terreus (60.5%) and A. flavus (53.3%) from fresh fruits of T. chebula at 20% conc. The next best growth inhibition form aqueous extracts was observed in fresh fruit of T. bellirica on A. terreus (70%) at 20% conc.; fresh fruit of T. chebula on A. terreus (63.6%) at 10% conc; triphala churna on A. terreus (62.5%) and A. flavus (62.5%) at 20% ; dry fruit of T. chebula on A. flavus (62.5%) at 20% and at 10% on A. terreus (62.5%) fresh and dry fruit of E. officinalis on A. versicolor (60%) and A. terreus (60%) at 20% ; fresh fruit of T. bellirica on A. terreus (60%) at 10% conc. Percent inhibition ranging from 58 to 50% was recorded at 20% conc. in fresh and dry fruit of T. bellirica and T. chebula on A. terreus (58.3%) and A. versicolor (58.3%); dry fruit of E. officinalis on A. versicolor (55.5%); fresh fruit of T. chebula on A . fumigatus (53.8%); dry fruit of E. officinalis on A. flavus (50%); fresh fruit of T. bellirica on A. flavus (50%); while at 10 % conc. of dry fruit of T. chebula it was 50% in A. flavus. It was interesting to note that, effect of aqueous extract of dry fruits of E. officinalis was found more effective in reducing the mycelial growth as compared to fresh fruits and powder extracts. One another investigation observed during present study was that as concentration of extracts increased, increase in diameter of mycelial growth of A. niger was found, which means aqueous extracts of all the samples were not effective against A. niger.
| Table 2: | Antifungal effect (%) of fruit and powdered samples on mycelial growth of Aspergillus isolates |
| *Data given are means of three replicates±SD | |
| Table 3: | Percent inhibition of mycelial growth of Aspergillus isolates |
| Mean of three replicates±SD, NE: Not effective | |
| Table 4: | Antifungal activity of various concentrations of fruit and powder extracts on different fungal species |
| +, Positive antifungal activity low inhibition. ++, Positive antifungal activity medium inhibition. +++, Positive antifungal activity High inhibition. ++++, Positive antifungal activity very high inhibition. -, No inhibition | |
DISCUSSION
The isolation of Aspergillus spp. (A. flavus, A. terreus, A. versicolor, A. fumigates and A. niger) as dominating fungi from fruit samples of E. officinalis, T. bellirica and T. chebula leading us to select them as test organism in the present study. The efficacy of fruit and powdered ingredients of Triphala churna i.e. E. officinalis, T. bellirica and T. chebula was investigated against various Aspergillus species. Aspergillus was reported earlier as one of the dominating fungal genera isolated from stored herbal drugs (Dubey et al., 2004; Roy and Chourasia, 1990; Aziz et al., 1998; Bugno et al., 2006; Gautam and Bhadauria, 2008; Gautam and Bhadauria, 2011). This fungal contaminant associated with stored herbal drugs decreases their medicinal potential as well as antimicrobial potential (Bugno et al., 2006; Truckesses and Scott, 2008).
During this study, an increase in antifungal activity of the extracts was observed by increase in their concentrations. This finding agrees with the earlier reports (Banso et al., 1999) that higher concentration of antimicrobial substance showed appreciation in growth inhibition. The fact that results of this study showed that fruits and powder extracts of E. officinalis, T. bellirica, T. chebula have antifungal property that justify their use as traditional medicinal plants (Mann et al., 2008). Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs (Ogundipe et al., 1998; Ibrahim, 1997). All the plants, plant parts differ significantly in their antimicrobial properties due to presence of varied concentrations of active compounds which may be responsible for their antimicrobial activities. Strong antifungal activity was shown by fresh fruits as compared to their dry fruits and powder except in case of E. officinalis. In E. officinalis dry fruits shown more antifungal activity as compared to their dry fruits, as in the fresh fruits the main ingredient is moisture (about 80%) which is completely eliminated upon drying. The difference observed in fungicidal activity of these fresh, dry and powdered aqueous extract is likely due to in the presence of active principles and their solubility in water which is also agrees with the previous reports (Qasem and Aau-Blan, 1996; Amadioha 2001). The active principles present in plants are influenced by many factors which includes, age of plants, extracting solvents, method of extraction and time of harvesting (Calixto, 2000). It has been observed that active principles are destroyed by enzymatic processes that continue for long periods of time after plant collection (Okigbo and Nmeka, 2005). Aspergillus niger was reported earlier as a major Aspergillus isolate from stored herbal drugs (Gautam and Bhadauria, 2008; 2009; 2011), which may justifies the ineffectiveness of all fruit and powdered extracts against this fungi.
CONCLUSION
On the basis of antifungal activity of fruits and powder ingredients of triphala churna in present study, we can reveal that shelf life decrease during storage. Decreased shelf life along with prolonged storage can reduce the efficacy of stored herbal drugs. Therefore, the study is an important step towards quality control practices and management of storage fungi. Moreover, this parameter can be helpful to study the relationship between antifungal activity and frequently occurring fungal contaminants in stored herbal drugs.
ACKNOWLEDGMENTS
This work was financed by Madhya Pradesh council of Science and Technology (MPCST), Bhopal, India. The Head, School of Studies in Botany, Jiwaji University, Gwalior is gratefully acknowledged for providing necessary laboratory facilities.