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COMPARATIVE STUDY OF MYCOFLORA, ANTIBACTERIAL ACTIVITY AND PHYTOCHEMISTRY OF SELECTED FRESH AND STORED MEDICINAL FRUITS

Objective: To understand the mycoflora, phytochemical constituents and antibacterial potential of fresh and stored herbal fruits of selected plants, extensively used in herbal medicines. Methods : Mycoflora analysis of Terminalia bellerica, Phyllanthus emblica and Myristica fragrans fruits was done employing serial dilution method, colony forming unit (CFU) and relative density of each fungi was recorded. The diversity of fungi associated with test fruits was calculated using various diversity indices. Methanol extracts of test fruits was subjected to preliminary phytochemical analysis, presence or absence of alkaloids, flavonoids, tannins, saponins, terpenoids, quinones and cardiac glycosides was detected. Antibacterial potential of test fruits was studied by well diffusion method. Results : Fresh fruits were free of fungal contamination, whereas stored fruits were found in association with various fungi. A total of 64 isolates of 29 species belonging to 13 genera were identified from stored fruits. Aspergillus was predominant followed by Penicillium . T. bellerica was highly contaminated (1x10 5 Conclusion : Results suggests the need of proper training to the herbal material handler’s right from the harvest to retail selling, and also points out the need to assess the fungal contamination of herbal materials before using them for herbal drug manufacture. Stored fruits high fungal diversity with highest Simpson’s diversity index Shannon-Wiener Dominance (d’=0.418). Phytochemical analysis of fresh and stored fruits did not show a significant difference in the presence or absence of tested phyto-constituents. Stored fruit extract recorded up to 41% increase in antibacterial activity.


INTRODUCTION
Plants have been used in the prevention, treatment and cure of disorders and diseases since ancient times [1]. Medicinal plants are widely used as raw material for pharmaceutical preparations and as a supplement for dietetic products, specifically for self-medication [2]. In recent decades, the use of herbal preparations has increased in developing and developed countries, due to the belief that being natural, they are harmless [3]. With the ever-increasing use of herbal medicines worldwide and the rapid expansion of the global market for these herbal materials and preparations, the safety and quality of raw materials have become a major concern for health authorities, pharmaceutical industries and the public [4]. The quality of raw material used has a direct bearing on the safety and efficacy of the herbal drug.
Unfortunately, reports of people experiencing negative effects, caused by the use of herbal drugs, has been increasing. One of the reasons for such problem could be the poor quality of herbal drug raw material and insufficient attention being paid to the quality assurance and control of these herbal medicines. Although world health organisation (WHO) has developed guidelines for the quality control of herbal drugs which provide a detailed description of the techniques and measures required for the appropriate cultivation and collection of medicinal plants, there is still a lacuna between the available knowledge and implementation, because farmers and other relevant persons like producers, handlers and processors of herbal drugs are not much aware of WHO's guidelines. As a result, the quality control measures are ignored by the practitioners, resulting in an inferior quality of herbal drugs with lots of contaminants like heavy metals, pesticides and microbes [5].
Many of the herbal drugs are plant products, and they can be infested by bacteria and fungi, especially moulds. Unscientific method of cultivation and collection, inappropriate harvesting and cleaning, unsuitable transportation, inadequate drying and storage, poor hygiene of producer and congenial climatic conditions render the raw plant materials prone to microbial contamination in general and fungal infestation in particular [6]. Fungi are the predominant contaminants that survive during drying and storage. Mould contamination has been reported to alter the phytoconstituent of herbal drug raw materials [7] which in turn alters the therapeutic value of the herbs.
Considering these, three fruits widely used in herbal medicine viz., Terminalia bellerica (Gaertn.) Roxb. Phyllanthus emblica Linn. and Myristica fragrans Houtt. were selected for the study to understand the changes if any in the mycoflora, phytochemistry and antibacterial activity of fresh and stored fruits.

Collection of samples
Dried and stored fruit samples of Terminalia bellerica (Gaertn.) Roxb. Phyllanthus emblica Linn. and Myristica fragrans Houtt. were randomly collected from different retail shops of the Mysuru city (Geographical coordinates in decimal degrees: Latitude 12.2979100° and Longitude: 76.6392500°). Fresh fruits of P. emblica, T. bellerica and M. fragrans were collected from Biligiri Ranga hills (Geographical coordinates in decimal degree: Latitude 11.9° and Longitude: 77.233333°) and Chandravana herbal farm of Mysuru (Geographical coordinates in decimal degree: Latitude 12.2979100° and Longitude: 76.6392500°). The samples were collected in sterilized lock covers to avoid further contamination and stored in airtight containers at 4 °C until further analysis. All the specimens were deposited at Centre for Innovative Studies in Herbal Drug technology, DOS in Botany, University of Mysore, Mysuru and voucher number was obtained as MGB-CISHDT-RR-TB-SF-M-0008a, MGB-CISHDT-RR-PE-SF-M-0006a and MGB-CISHDT-RR-MF-SF-M-0005a for stored fruits of T. bellerica, P. emblica and M. fragrans respectively, and MGB-CISHDT-RR-TB-FF-M-0008b, MGB-CISHDT-RR-PE-FF-M-0006b and MGB-CISHDT-RR-MF-FF-M-0005b for fresh  fruits of T

Mycoflora analysis
Serial dilution method [8] was employed to determine the colony forming unit (CFU) of fungi. One gram of each sample transferred into 20 ml screw capped bottles containing 9 ml of sterile distilled water and was mechanically homogenized at a constant speed for 15 min on an electronic shaker. The sample-water suspension was allowed to stand for 10 min with intermittent shaking before being plated. Appropriate ten-fold serial dilutions (1:10) were prepared and 1 ml aliquot of each dilution was aseptically surface plated and distributed uniformly on culture medium with the help of sterilized L-shaped glass spreader. Freshly prepared czapek dox agar (CDA) medium served as the culture medium. Plates were incubated at 28±2 °C for 7 d and observed daily. Subsequently, the CFU, diversity and density of the fungi were recorded. Morphologically different mould colonies were individually sub-cultured by hypha tip method on CDA medium and their pure cultures were maintained.
Colony forming units of fungi per gram of fruit (CFU/g) was calculated using the formula CFU= N×10 -n All the isolated fungal species were identified on the basis of their cultural and morphological characteristics with the help of standard manuals.
where N=total no of colonies, n=dilution [9] The relative density of each fungi was calculated using the formula: Relative density of fungi The fresh and stored fruits of P. emblica, T. bellerica, and M. fragrans were powdered with the help of a waring blender. 100g of each of the powder was extracted with methanol by cold extraction method. The extract was concentrated by evaporation and preserved at 4 °C until subjected to antibacterial activity assay [10] and phytochemical analysis.

Preliminary phytochemical analysis
The methanol extracts of the fruits were subjected to the detection of alkaloids, flavonoids, tannins, saponins, terpenoids, quinones and cardiac glycosides following the methods of Trease et al., and Sofowara [11,12].

Test bacteria
Authentic pure cultures of phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria (MTCC 2286) and Xanthomonas campestris pv. campestris (NCIM 5028) were obtained from Microbial Type Culture Collection, IMTECH, Chandigarh and National Centre of Industrial Microorganism, NCL, Pune respectively. Xanthomonas oryzae pv. oryzae, isolated from the diseased plant was obtained from the culture collections of Centre for Innovative Studies in Herbal Drug Technology, Department of Studies in Botany, University of Mysore, Mysuru. All the test bacteria were sub-cultural on nutrient agar. Twoday old nutrient broth cultures of the test bacteria were used for assay.

Antibacterial activity assay
Antibacterial activity of methanol extract was determined by well diffusion method on nutrient agar medium. Wells (6 mm) were made in nutrient agar plates using sterile cork borer and 50µ1 of inoculums of test bacteria was spread on the solid plates with a sterile swab. Then 100µ1 of the extract was poured into the wells. The treatment also included 100µ1 of methanol separately which served as control. The plates were incubated for 24 h at 37 °C and zone of inhibition if any around the wells were measured in mm and recorded [13].

Mycoflora analysis
Following serial dilution method, a total of 64 isolates of fungi were recorded from the stored herbal fruits while fresh fruit samples were free from fungal contamination. A maximum number of 24 isolates belonging to 12 species viz., Aspergillus niger, A. tamarii, Penecillium citrinum, Penicillium oxalicum, P. commune, Penicillium sp., Cladosporium sp., Heterociphalum, Chaetomium sp., Pestalotiopsis sp., Mycoleptodiscus sp. and one sterile fungi were recorded from Phyllanthus emblica (

Fig. 1: Fungal contamination of fresh and stored herbal fruits
Stored fruits of P. emblica was found in association with 12 different fungal species belonging to 8 genera. The genus Penicillium was found to be predominant recording 4 species followed by Aspergillus with two species. Among 12 species, A. niger recorded the highest relative density (82.608) followed by P. commune with a density of 31.63% (table 1). In case of T. bellerica 10 species of 5 different genera were observed among these the genus Aspergillus was found to be predominant comprising 5 species, this was followed by Curvularia (2 species). A. niger and A. flavus recorded highest relative density of 61.90 and 52.38 respectively in the stored fruits of T. bellerica (table 2). Eleven different species belonging to 4 genera were isolated from stored fruits of M. fragrans, with 7 species Aspergillus being the predominant one, followed by Penicillium (2 species). A. versicolor was found to be predominant in stored fruits of M. fragrans with the highest relative density of 60 (table 3). Fig. 1 presents the CFU of fungi per gram of test fruits.
Highest CFU of 1X10 5 was recorded in T. bellerica followed by1X10 4 in P. emblica and 1X10 3 in M. fragrans. CFU of each fungal isolate is presented in the tables 1-3. A. niger was found to be most predominant fungi in all the test fruits.

Preliminary phytochemical analysis
The methanol extract of both stored and fresh fruits of P. emblica showed the presence of alkaloids, flavonoids, tannins, saponins, steroids, phenols and quinones. Fresh and stored sample of T. bellerica was found to possess flavonoids, tannins, steroids, terpenoids, phenols and quinones. Flavonoids, steroids and terpenoids were detected in M. fragrans extract (table 5).

Table 6: Antibacterial activity of methanol extracts of Terminalia bellerica, P. emblica and M. fragrans against test bacteria (Zone of inhibition in mm)
Test bacteria

DISCUSSION
The traditional system of herbal medicine has become a topic of global importance both as medicinal and economical [14] since they are considered as rich sources of lead compounds and quietly safe for both human use and environment-friendly [15]. Considering this in the present study, assessment of fungal contamination of selected fresh and stored herbal fruits was done by employing serial dilution method. Results revealed that the stored fruits were contaminated with various fungi whereas fresh fruits were found to be free of fungal contaminants.
The occurrence of the high diversity of fungi of 64 isolates of 29 species belonging to 13 genera indicates the association of broad spectrum of mycobiota in stored fruits. Among these P. emblica was found to be highly contaminated with 12 species of fungi belonging to 8 genera followed by M. fragrans with 11 species, belonging to 4 genera and T. bellerica with 10 species, belonging to 5 genera.
Fungal contamination of M. fragrans [14,16] P. emblica [18,19] and T. bellerica [20] has been reported. Results of the previous study correlates with the results obtained in the present investigation, but the diversity and density of fungal contaminants recovered varies. Akhund et al., [19] have recorded Aspergillus, Penicillium, Fusarium, Alternaria, Cladosporium and Curvularia from P. emblica stored fruits and reported that A. niger and A. flavus are the predominant fungi. Gautam and Bhadauria [18] have isolated Aspergillus, Penicillium, Helminthosporium, Rhizopus, Syncephalastrum, Alternaria, and Curvularaia from P. emblica and T. bellerica. Rajeshwari and Raveesha [21] have reported fungal contamination of A. calamus roots, M. fragrans mace, C. angustifolia and C. asiatica leaves, T. cordifolia and W. somnifera stem collected from the retail herbal shops of Mysuru. Mycoflora analysis of the herbal fruits is done in various parts of the world including India, but no reports are available on fungal contamination of M. fragrans, P. emblica and T. bellerica fruits of Mysuru, which is an important hub of the herbal drug raw materials markets. Thus the present work is the first to report on the mycoflora of the stored herbal drug fruits from this region.
Diversity indices calculation revealed the occurrence of highest fungal diversity on M. fragrans recording high Simpson's diversity index (D-1=0.840) and Shannon-Wiener index (H=2.888). Although M. fragrans did not possess a high number of fungal species, the highest diversity was recorded in M. fragrans, as the term diversity considers not only the species richness but also the even distribution of species. On the other hand, P. emblica recorded high species richness (12) During the collection of stored fruits from the market, it was observed that the samples were displayed on open metal/plastic containers/wooden boxes/gunny bags or on the bare ground in local general stores, causing direct exposure to airborne bio-pollutants which may have resulted in fungal contamination. Apparently, healthy fresh fruit samples were collected directly from the plant and brought to the laboratory in sterilized lock covers and analyzed and did not show any fungal contamination.
The genus Aspergillus was found to be predominant genus recording 12 species. Apart from this 6 species of Penicillium, 2 species of Curvularia and one species each of Alternaria, Helminthosporium, Cladosporium, Heterociphalum, Chaetomium, Pestalotiopsis, Mycodypteria and 2 sterile fungi were recorded. Species of Aspergillus and Penicillium has been reported as the dominant mycoflora in some herbal drug raw materials collected from Tokyo (Japan); Hunan, Hubei and Guangxi province (China); Gwalior (North India) [23][24][25] the results of the present study also revealed the same. The association of A. niger in high density from all fruit samples, which are used by consumers, should be taken seriously as some strains can produce mycotoxins like ochratoxin A [26].
Fungal contaminants have been reported to affect the chemical compositions of the herbal drug raw materials on which they grow and thereby alter the medicinal property [7]. In order to understand the effect of fungal contamination on phytochemical constituents of both fresh (free of fungal contamination) and stored fruits (contaminated with fungi), methanol extracts of the same were screened for the phytochemical constituents. The extracts of test samples were tested for the presence of alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, phenols, quinones and cardiac glycosides. Methanol was the preferred solvent for extraction since it is known to dissolve most of the secondary metabolites due to its polar nature.
In the present investigation methanol extracts of fresh and stored fruits of P. emblica showed positive results for all the tested phytochemicals. Fresh and stored fruit samples of T. bellerica was found to possess flavanoid, tannins, steroid, terpenoids, phenols and cardiac glycosides. M. fragrans fresh and stored samples showed the presence of flavonoids, steroids, terpenoids and cardiac glycosides. Results did not show any significant difference in the presence or absence of test phytochemical constituents between fresh and stored herbal fruits. However, there could be adding some new compounds which have not been tested. Further quantitative and qualitative analysis need to be done to draw clear inferences on the role of fungal contamination on phytochemical constituents of tested fruits.
To understand the antibacterial potential of methanol extracts of selected fresh and stored herbal fruits, antibacterial activity assay was carried out against three plant pathogenic bacteria. Methanol extracts of all the tested samples showed antibacterial activity against all the test bacteria with varying zones of inhibition ranging between 24 mm and 30 mm. The presence of more phytochemicals in an extract correlates with more potential activity exhibited by that extract as the M. fragrans extract which showed presence of 4 phytochemicals (flavonoids, steroids, terpenoids and cardio glycosides) out of 9 tested, exhibited least antibacterial activity zone between 17 and 26 mm whereas extracts of P. emblica and T. bellerica recording presence of all and 7 phytochemicals out of 9 tested exhibited zone of inhibition range between 25-28 mm and 21-30 mm respectively. Antimicrobial activity of P. emblica [29,32], M. fragrans [27,33] and T. bellerica [34][35][36] is reported by researchers against some human pathogenic bacteria but not against plant pathogenic bacteria. Results of the present investigation demonstrate the antimicrobial potential of test fruits on phytopathogenic bacteria.
Although stored fruits were found in association various fungi they exhibited a comparatively larger zone of inhibition i.e. upto41 % more than the fresh fruit extracts which were free of contamination. Stored fruit of M. fragrans showed an increase of 41% inhibition against Xanthomonas campestris pv. campestris, 8.33% against Xanthomonas campestris pv. vesicatoria and 5.55% against Xanthomonas oryzae pv. oryzae. An increase of 28% of zone of inhibition was observed in stored fruits of T. bellerica against Xanthomonas oryzae pv. oryzae. An increase of 12% and 3.8% zone of inhibition was recorded in stored fruits of P. emblica against Xanthomonas campestris pv. campestris and Xanthomonas campestris pv. vesicatoria respectively. This may be due to the predisposition of stored fruits to stress conditions during drying and fungal infestation that might have resulted in the production of more or new secondary metabolites, responsible for the antibacterial property. However, this needs further investigation on quantification and characterization of active principle present in fresh and stored fruits for the better understanding.

CONCLUSION
Analysis of fresh fruit samples showed that fruits were free of fungal contaminants while the stored fruits were contaminated with fungi, but it was within the maximum permitted limit prescribed by WHO. However the occurrence of a high diversity of fungi that are capable of causing bio-deterioration and producing fungi mycotoxins viz., Aspergillus, Penicillium and Alternaria is a cause of concern. Results clearly indicate the need for adaptation of appropriate methods for harvesting, collection, transportation, handling and storage of herbal drug raw materials. Maintenance of hygienic condition may be helpful in the prevention of fungal contamination. Though there was no significant difference in the phytochemical constituents as well as the antibacterial potential of both fresh and stored samples. Long term storage may affect the quality of the herbal fruits, due to increased growth of mycobiota, which in turn may affect the phytochemical composition and antibacterial potential. Hence proper handling and storage methodology is a prerequisite for the maintenance of herbal drug raw materials. The results suggest proper training need to be provided herbal material handlers right from harvesting to retail selling.